Category Archives: Embryonic Stem Cells


YAP1 is a potent driver of the onset and progression of oral squamous cell carcinoma – Science Advances

Abstract

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common group of cancers in the world, and patients have a poor prognosis. Here, we present data indicating that YAP1 may be a strong driver of the onset and progression of oral SCC (OSCC), a major subtype of HNSCC. Mice with tongue-specific deletion of Mob1a/b and thus endogenous YAP1 hyperactivation underwent surprisingly rapid and highly reproducible tumorigenesis, developing tongue carcinoma in situ within 2 weeks and invasive SCC within 4 weeks. In humans, precancerous tongue dysplasia displays YAP1 activation correlating with reduced patient survival. Combinations of molecules mutated in OSCC may increase and sustain YAP1 activation to the point of oncogenicity. Strikingly, siRNA or pharmacological inhibition of YAP1 blocks murine OSCC onset in vitro and in vivo. Our work justifies targeting YAP1 as therapy for OSCC and perhaps HNSCC, and our mouse model represents a powerful tool for evaluating these agents.

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common group of cancers in the world, affecting 600,000 people annually. About half of HNSCC patients die from their disease (1). The head and neck region of the body includes the oral cavity, larynx, and pharynx, all structures that are covered with squamous epithelium. Among HNSCC subtypes, oral SCC (OSCC) is the most frequent, and tongue cancers comprise a large proportion of OSCCs (2). Because 15% of HNSCC patients carry the human papillomavirus (HPV), HPV is considered to be one of the major causes of HNSCC. HPV (+) HNSCC usually occurs in the oropharynx, and patients with this malignancy have better prognoses or may even be cured (1). In contrast, the 85% of HNSCC that are HPV () are highly resistant to even intensified chemo/radiotherapy (3) as well as to currently available molecular targeting drugs (4). The fundamental molecular mechanisms underlying the onset and development of HPV () HNSCC have yet to be identified, hampering the generation of new therapeutic strategies.

The Cancer Genome Atlas (TCGA) project has revealed the presence of many altered gene exons in HNSCC (2). In HPV () HNSCC, TP53 was highly mutated in 84% of cases. In addition, mutation of FAT atypical cadherin1 (FAT1) was observed in 32%, epidermal growth factor receptor (EGFR) in 15%, and Ajuba LIM protein (AJUBA) in 7% of all HPV () HNSCC. Strikingly, these mutations were rare in HPV (+) HNSCC, with even TP53 mutation at only 3%. Notably, mutations of phosphoinositide 3 kinase, catalytic subunit alpha (PIK3CA)/phosphatase and tensin homolog (PTEN; 50 to 60%) and TP63 (20 to 30%) were commonly observed in both HPV (+) and HPV () HNSCC. Considering that HPV E6 strongly inactivates TP53 (5), TP53 inactivation must be a crucial and common oncogenic event in HNSCC. However, loss of TP53 alone in mice never induces spontaneous HNSCC in vivo (6), meaning that other genetic and/or epigenetic alterations are also essential for HNSCC generation.

The core components of the Hippo pathway are the mammalian STE20like (MST) kinases, large tumor suppressor homolog (LATS) kinases, nuclear Dbf2related (NDR) kinase, and the adaptor proteins Salvador homolog 1 (SAV1) and Mps one binder kinase activator 1 (MOB1) (7). MOB1A/B are the adaptor proteins for both the LATS1/2 and NDR1/2 kinases, and by binding to LATS/NDR, MOB1A/B strongly increase the enzymatic activities of these kinases (7). Activated LATS/NDR kinases, in turn, phosphorylate Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ; also known as WWTR1). YAP1/TAZ are key downstream transcriptional cofactors that act mainly on TEA domain transcription factors (TEADs) to regulate numerous target genes involved in cell growth and differentiation (7). After phosphorylation by LATS/NDR kinases, YAP1/TAZ are excluded from the nucleus and retained in the cytoplasm, where they are ubiquitylated by E3-ubiquitin ligase SCFTRCP (also known as BTRC) and subjected to proteasome-mediated degradation (7). Thus, in most cell types, YAP1/TAZ are essentially positive regulators of cell proliferation that are negatively controlled by upstream Hippo core components. In vitro, YAP1/TAZ can be regulated by cell density, external mechanical forces, polarization, rigidity of the extracellular matrix, stress stimuli (7), or engagement of a G proteincoupled receptor (GPCR) by a soluble mediator (7). In vivo, YAP1 activation in mice results in organomegaly and tumor formation (8).

Several lines of evidence suggest a role for YAP1 in HNSCC. (i) Location 11q22 in the human YAP1 locus is amplified in 8.6% of HNSCC (9); (ii) YAP1 activity is associated with malignant phenotypes and poor prognosis both in vitro and in vivo (9, 10); and (iii) mutations of TP53, PIK3CA/PTEN, EGFR, or FAT1, which are often observed in HNSCC, increase YAP1 activation in several cell types (1114). HNSCC also frequently shows amplification of TP63, a master regulator of squamous cells, but the effect of this alteration on YAP1 activity is controversial (15, 16).

We previously reported that Mob1a/b null mutant mice succumb to embryonic lethality at embryonic day 6.5 (17). We have also demonstrated that Mob1a/b loss induces extreme hyperactivation of endogenous YAP1/TAZ, resulting in the most severe phenotypes reported among mice mutated in Hippo core components in various tissues (17). Thus, MOB1A/B is a crucial hub in the Hippo signaling pathway. Because of the accumulating evidence in the literature on the importance of YAP1 in HNSCC progression, we generated tongue epitheliumspecific Mob1a/b double knockout (tgMob1DKO) mice and examined them to dissect the function of endogenous YAP1 in the onset and progression of the OSCC subtype of HNSCC. We demonstrate that hyperactivation of endogenous YAP1 induced by loss of Mob1a/b triggers surprisingly early onset and rapid progression of OSCC. Our data reveal that YAP1 is a powerful oncogenic driver of this malignancy.

To investigate the role of the Hippo-YAP1 pathway in mouse tongue epithelium in vivo, we used our previously generated strain of tamoxifen (TAM)inducible Mob1a/bDKO mice [Rosa26-CreERT; Mob1aflox/flox; Mob1b/ (tgMob1DKO) mice], which were created by mating Rosa26-CreERT transgenic (Tg) mice with Mob1aflox/flox and Mob1b/ mice (17). Intraperitoneal injection of TAM into these animals causes early death at about 3 weeks due to widespread organ dysfunction, including hepatic failure (17). To extend mouse survival, we applied TAM directly and only to the tongue epithelium for 5 days starting on postnatal day 21 (P21; Fig. 1A and fig. S1A). Cre-mediated deletion of the floxed Mob1a gene was substantially achieved by 3 days after the initiation of TAM application (fig. S1B), with the MOB1A and MOB1B proteins being essentially absent by day 7 after TAM (fig. S1C).

(A) Diagram of the protocol to generate tongue epithelial cellspecific Mob1a/b DKO mice (tgMob1DKO). TAM was applied by a soft brush daily for 5 days to the tongues of 3-week-old Mob1aflox/flox; Mob1b/ (control) and Rosa26-CreERT; Mob1aflox/flox; Mob1b/ (mutant) mice. Mice were sacrificed at 1, 2, or 4 weeks (red arrows) after starting TAM application, and their tongue tissues were removed for histological analyses. (B) Representative macroscopic (small panels) and microscopic (large panels) views of H&E-stained sections of control (top) and tgMob1DKO (bottom) tongue epithelial layers at the indicated weeks after starting TAM application. White arrow, deep ulcer formation. Scale bars, 1 mm (small panels) and 100 m (large panels). Photo credit: Hirofumi Omori, Kobe University. (C) Percentages of the indicated lesion types present in the tongues of the mutant mice (n = 10 per group) in (B) at the indicated weeks after TAM. (D) H&E-stained sections of control and tgMob1DKO tongue epithelium at 1 week after TAM. Moderate nuclear heterogeneity and loss of polarity are apparent in the mutants, indicating dysplasia. Scale bar, 5 m. (E) H&E-stained sections of mutant tongue epithelium at 2 weeks after TAM showing atypical mitotic figures (left), nuclear enlargement (middle), and strongly heteromorphic cells (right), indicating CIS. Scale bar, 5 m. (F) H&E-stained section of mutant tongue epithelium at 4 weeks after TAM revealing submucosal invasive SCC. White arrowheads, cancer cells penetrating beyond the basement membrane (yellow dashed line). Scale bar, 10 m.

Macroscopically, the epithelial surface of tgMob1DKO tongue showed mild roughness at 1 week after TAM, very rough mucosa accompanied by keratosis at 2 weeks after TAM, and deep ulceration at 4 weeks after TAM (Fig. 1B). To our surprise, by 1 week after TAM, histological examination revealed an increased number of polymorphic epithelial cells with hyperchromatic nuclei and loss of polarity, evidence of dysplasia (Fig. 1, C and D). Although Ki67 was expressed only in the basal cells of the tongue epithelium before TAM treatment, the percentage of Ki67-positive cells among polymorphic epithelial cells increased markedly by 1 week after TAM (fig. S1D), demonstrating the increased proliferative capacity of MOB1-deficient epithelial cells. Atypical mitotic figures (Fig. 1E, left), nuclear enlargement (Fig. 1E, middle), and strongly heteromorphic cells (Fig. 1E, right) indicative of carcinoma in situ (CIS) were observed in the tongue as early as 1 week after TAM (Fig. 1C and fig. S1E). All mice developed tongue CIS by 2 weeks after TAM (Fig. 1, B, C, and E), and all mice developed invasive SCC by 4 weeks after TAM (Fig. 1, B, C, and F). Almost all of these SCC-bearing mutants died by 8 weeks after TAM, most likely due to malnutrition caused by their dysphagia. Because there were no significant histological differences among Mob1a+/+; Mob1b+/+ mice treated with TAM, Rosa26-CreERT; Mob1a+/+; Mob1b+/+ mice with TAM, Rosa26-CreERT; Mob1aflox/flox; Mob1b/ mice without TAM, and Mob1aflox/flox; Mob1b/ mice with TAM (fig. S1F), we used Mob1aflox/flox; Mob1b/ mice with TAM as controls for subsequent experiments unless otherwise stated. These studies were designed to explore why altered Hippo signaling induced the extremely rapid onset of tongue cancers.

We established a TAM-inducible Mob1a/bDKO tongue epithelial cell line (iMob1DKO cells) and treated them in vitro with (+) or without () TAM. Compared to control iMob1DKOTAM cells, iMob1DKO+TAM cells showed increased cell proliferation and saturation density (Fig. 2A). When cultures of these overconfluent iMob1DKO+TAM cells were stained to detect the tight junction protein ZO-1, we found only weak staining of this protein in the tight junctions, indicating impaired cell polarity (fig. S2A). In contrast, cultures of iMob1DKOTAM cells showed normal ZO-1 staining in the tight junctions. Because there was no difference in cell size between iMob1DKO+TAM and iMob1DKOTAM cells (fig. S2B), we concluded that cell-cell contact inhibition was impaired in the absence of Mob1a/b. In addition, the number of apoptotic cells was decreased in the mutant culture compared to the control (Fig. 2B). Next, to determine how MOB1 inactivation affected the self-renewal of tongue epithelial stem cells, we quantified the capacity of control (TAM) and mutant (+TAM) iMob1DKO cells to form colonies in culture. A lack of Mob1a/b induced a 2.2-fold increase in colony-forming efficiency (Fig. 2C, left panels). When these primary colonies were replated to test their ability to form secondary colonies, a 2.8-fold increase in secondary colony-forming efficiency was observed in the absence of Mob1a/b (Fig. 2C, right panels). A comparison of cell cycle and cell ploidy in iMob1DKOTAM versus iMob1DKO+TAM cells revealed a decrease in G0-G1 phase cells and increases in S phase cells and aneuploid cells in the mutant culture (Fig. 2D). Indirect immunofluorescence (IF) analysis of control and mutant cells using anti-tubulin and anti-tubulin antibodies uncovered increases in multipolar spindle formation (Fig. 2E) and micronuclei (Fig. 2F) in mutant cells, indicating chromosomal instability. Thus, the increases in cell proliferation and stem cell self-renewal observed in the absence of Mob1a/b, coupled with chromosomal instability, resistance to apoptosis, and inadequate cell contact inhibition, may underlie the rapid onset and development of tongue cancer in TAM-treated tgMob1DKO mice.

(A) Absolute numbers of iMob1DKO tongue epithelial cells that were left untreated (control; iMob1DKOTAM cells) or treated with 0.5 M TAM for 3 days (iMob1DKO+TAM cells) and then grown for the indicated number of days in the absence of TAM. (B) Flow cytometry (left) and quantitation (right) of propidium iodide (PI)positive dead cells in the cultures in (A). (C) iMob1DKO cells were treated in vitro with TAM (0.5 M) for 3 days (+TAM) or left untreated (TAM) and serially plated to generate first primary colonies and then secondary colonies. Crystal violet staining (left) and colony counts (right) of primary (left side) and secondary (right side) colonies were performed on day 7 after plating. Photo credit: Hirofumi Omori, Kobe University. (D) Top left: DNA content frequency histograms of control (iMob1DKOTAM) and Mob1a/b mutant (iMob1DKO+TAM) tongue epithelial cells. Right: Percentage of cells from the top left panels in the G0-G1, S, and G2-M phases of the cell cycle as determined by fractional DNA content. Bottom left: Overlay of aneuploid and polyploid cell numbers for the cells in the right panel. (E and F) Top: Immunostaining to detect -tubulin (green) and -tubulin (red) in control (iMob1DKOTAM) and mutant (iMob1DKO+TAM) tongue epithelial cells. DAPI (blue), nuclei. Scale bars, 1 m. Multipolar spindles and micronuclei (white arrow) were detected in mutant cells. Bottom: Quantitation of the percentage of cells in the top panels showing multipolar spindles (E) and micronuclei (F). Data are shown as means SEM of triplicate samples. *P < 0.05, **P < 0.01, and ***P < 0.001, t test. ns, not significant; i.p., intraperitoneally.

We next investigated the biochemical effects of Mob1a/b loss on Hippo components in iMob1DKO cells that were left untreated or treated with TAM for 7 days. As expected, iMob1DKO+TAM cells showed a reduction in LATS1 protein and an increase in the total protein levels of YAP1. Protein levels of several representative direct transcriptional targets of YAP1, including connective tissue growth factor (CTGF), baculoviral IAP repeat-containing protein 5 (BIRC5), and topoisomerase II-alpha (TOP2A), were also significantly elevated. However, there was no effect on total TAZ protein (Fig. 3A). Furthermore, YAP1 was predominantly localized in the nuclei of iMob1DKO+TAM cells even when cultured under highcell density conditions (Fig. 3B). Thus, YAP1 hyperactivation is a prominent feature of mutant tongue epithelial cells prone to OSCC development.

(A) Top: Immunoblots to detect the indicated proteins in total extracts of iMob1DKO tongue epithelial cells that were left untreated (TAM; control) or treated with TAM (+TAM) for 7 days. GAPDH, loading control. Bottom: Densitometric relative quantitation of the indicated proteins in the blots in the top panels. (B) Left: Immunostaining to detect YAP1 in iMob1DKOTAM and iMob1DKO+TAM tongue epithelial cells that were plated at low or high cell density. Scale bar, 10 m. Right: Percentages of cells in the cultures in the left panels that showed higher YAP1 levels in the nucleus (nuc YAP1) than in the cytoplasm (cyto YAP1). (C) Left: Representative H&E-stained sections (top panels) and macroscopic views (bottom panels) of tongue epithelium from control, tgMob1DKO, tgYap1TKO (tgMob1DKO plus Yap1 KO), and tgTazTKO (tgMob1DKO plus Taz KO) mice at 4 weeks after TAM (n = 10 mice per group). Scale bars, 100 m (top panels) and 1 mm (bottom panels). Right: Percentages of mice in the left panels displaying the indicated lesions. Photo credit: Hirofumi Omori, Kobe University. (D) Quantitation of SCC invasion depth in tongue epithelium of the mice in (C). The depth of invasion was measured from the level of the nearest adjacent normal mucosa to the extent of the deepest tumor invasion into the tongue musculature. Data are shown as means SEM of triplicate samples. *P < 0.05, **P < 0.01, and ***P < 0.001, t test.

To clarify the role of YAP1 in OSCC-related phenotypes, we generated strains of triple KO mice lacking MOB1A/B plus YAP1 (tgYap1TKO), or lacking MOB1A/B plus TAZ (tgTazTKO). Unlike tgMob1DKO mice, which all develop invasive SCC at 4 weeks after TAM, MOB1A/B-deficient mice also lacking YAP1 showed only mild to moderate dysplasia in the tongue (Fig. 3C and fig.S2C). In contrast, MOB1A/B-deficient mice also lacking TAZ developed a highly aggressive form of invasive SCC, with some lesions penetrating from the tongue surface into the floor of mouth. The measured depth of invasion of malignant cells into the mouth floor was significantly increased in tgTazTKO mice compared to tgMob1DKO mice (Fig. 3D). In addition, immunohistochemical (IHC) staining to detect YAP1/TAZ revealed that tgMob1DKO mice showed increased frequency of nuclear YAP1 localization compared to controls, but no alteration in the frequency of nuclear TAZ (fig. S2C). These results were further confirmed by IHC staining to visualize YAP1 or TAZ in the tongues of tgYap1TKO and tgTazTKO mice (fig. S2C). Thus, the MOB1A/B-deficient phenotype is largely dependent on YAP1 rather than on TAZ.

We speculated that inhibition of YAP1 hyperexpression might prevent the development of tongue cancer in our tgMob1DKO mice. To choose a compound to exert YAP1 inhibition in vivo, we first tested the effects of the candidate compounds dasatinib, simvastatin, verteporfin, and the Rock inhibitor Y-27632 on YAP1 protein expression in the human OSCC cell line HSC4 (fig. S3A). We also evaluated the effects of these drugs on YAP1 activity in H1299-Luc cells in a reporter assay (fig. S3B). Dasatinib was the most effective YAP1 inhibitor in both of these assays, guiding us to choose dasatinib for our in vivo experiments. Biochemically, dasatinib is a multikinase inhibitor that efficiently blocks Src family kinases such as SRC, LCK, YES, and FYN (18). SRC directly and indirectly activates YAP1, and inhibition of SRC by dasatinib has been shown to efficiently suppress YAP1 activation (19).

To investigate the effect of pharmacological YAP1 inhibition on our TAM-inducible tgMob1DKO mice, we treated these animals with dasatinib or dimethyl sulfoxide (DMSO; vehicle control) 3 days before applying TAM ointment to the tongue (Fig. 4A). The mice were then sacrificed at 2 weeks after TAM. We found that dasatinib treatment strongly blocked both YAP1 protein expression (Fig. 4B) and the excessive cell proliferation associated with YAP1 hyperactivation (Fig. 4C). Macroscopically, the mucosal irregularity accompanied by keratinization obvious in tgMob1DKO+TAM mice had improved after dasatinib treatment (Fig. 4D). Histological examination of tongue epithelial cells revealed that, whereas DMSO-treated control mice all developed CIS at 2 weeks after TAM, the onset of CIS in dasatinib-treated mice was completely blocked, although a mild or moderate dysplasia was still present (Fig. 4D). Thus, dasatinib inhibits the onset of YAP1-induced tongue carcinomas. To confirm this effect of YAP1 inhibition in vivo, we treated tgMob1DKO+TAM mice with simvastatin in the same fashion and observed results similar to those achieved with dasatinib (fig. S3C). These data suggest that drug-mediated inactivation of YAP1 could be of therapeutic benefit in OSCC.

(A) Diagram of the protocol of the chemoprevention assay. tgMob1DKO mice received daily intraperitoneal injection of dasatinib or DMSO (control; n = 6 per group) for a total of 17 days starting 3 days (P18) before TAM application on P21. Mice were sacrificed at 2 weeks after TAM. (B) Representative images of IF detection of YAP1 in tongue epithelium from the dasatinib- or DMSO-treated mice in (A). Scale bar, 50 m. (C) Top: Representative Ki67 immunostaining of tongue epithelium from the mice in (A). Scale bar, 50 m. Bottom: Percentages of Ki67-positive cells in the sections in the top panels. (D) Top: Representative H&E staining of sections (left panels) and macroscopic views (right panels) of tongues from DMSO-treated (n = 6) or dasatinib-treated (n = 6) tgMob1DKO mice after 2 weeks of treatment. Scale bars, 100 m (left panels) and 1 mm (right panels). Normal, H&E-stained section of a tongue from a tgMob1DKO mouse treated with DMSO but not TAM (control). Bottom: Percentages of the DMSO- or dasatinib-treated mice in (A) showing the indicated lesions. Photo credit: Hirofumi Omori, Kobe University. (E) Growth in culture of SCC9 cells that Dox-inducibly overexpressed constitutively active YAP1 (YAP1-5SA) and were treated (+) or not () with Dox. (F and G) Growth in culture of HSC4 cells that were left untreated (parent) or treated with (F) si-scramble (siSC#1; control) or siYAP1#1 or (G) DMSO (control), dasatinib, verteporfin, or simvastatin. (H and I) Left: Volumes of tumors in nude mice (n = 12 per group) that were xenografted subcutaneously with (H) Dox-inducible shYAP1-expressing HSC4 cells or (I) unmodified HSC4 cells. Mice were supplied with (H) normal drinking water or water containing Dox (2 mg/ml), or (I) DMSO or dasatinib that was administered intraperitoneally on day 10 when tumors became visible. Right: Representative macroscopic view of the tumors evaluated in the left panels of (H) and (I) at 16 days after treatment. Scale bars, 10 mm. (J) Diagram of the protocol of the chemotherapy assay. tgMob1DKO mice received daily intraperitoneal injection of dasatinib or DMSO (n = 6 per group) for 2 weeks starting at 2 weeks after TAM. Mice were sacrificed at 7 weeks of age immediately at treatment end (right red arrow). (K) Top: Representative Ki67 immunostaining of tongue epithelium from the mice in (J) after 2-week treatment. Scale bar, 50 m. Bottom: Percentage of Ki67-positive cells in the sections in the top panels. (L) Left: H&E-stained sections (left panels) and macroscopic views (right panels) of the tongues of the mice in (J) after 2-week treatment. Scale bars, 100 m (left panels) and 1 mm (right panels). Right: Percentages of the mice in (J) whose tongues exhibited the indicated lesions after 2-week treatment. Photo credit: Hirofumi Omori, Kobe University. Data are shown as means SEM. *P < 0.05 and ***P < 0.001, t test.

Our findings that YAP1 activation causes very early OSCC onset, and that loss of YAP1 prevents the appearance of these tumors, prompted us to theorize that YAP1 must be a potent oncogenic initiator of OSCC. We next investigated whether YAP1 plays a crucial role in not only tumor initiation but also tumor progression. We engineered the human OSCC cell line SCC9, which features only low YAP1 expression (fig. S4, A and B), to overexpress YAP1 by transfecting it with a plasmid driving expression of the constitutively active YAP1-5SA mutant protein (fig. S4C). YAP1-overexpressing SCC9 cells showed greatly enhanced proliferation in vitro (Fig. 4E). We then transfected HSC4 cells, which naturally feature strong YAP1 expression (fig. S4, A and B), with YAP1 small interfering RNA (siRNA; fig. S4D) or treated them with a YAP1 inhibitor such as dasatinib, simvastatin, or verteporfin. In all these cases, YAP1 inhibition significantly suppressed HSC4 cell proliferation in vitro (Fig. 4, F and G). Moreover, siRNA-mediated YAP1 knockdown enhanced the sensitivity of HSC4 cells to the chemotherapeutic cisplatin (fig. S4E), implying that combining a YAP1 inhibitor with cisplatin might be an attractive new approach for OSCC therapy.

To examine the effects of YAP1 inhibition in vivo, we first xenografted doxycycline (Dox)inducible shYAP1-transfected HSC4 cells (fig. S4F) into nude mice, which were then supplied with normal drinking water (control) or water containing Dox. We found that Dox-induced inhibition of YAP1 expression efficiently suppressed the ability of these modified HSC4 cells to grow into tumors in vivo (Fig. 4H). We then xenografted unmodified HSC4 cells into nude mice and treated these animals with DMSO or dasatinib. Again, blocking YAP1 activity decreased OSCC development in these mice (Fig. 4I).

Last, we applied these findings to our TAM-induced tgMob1DKO mouse model of tongue cancer. We treated TAM-inducible tgMob1DKO mice with dasatinib soon after CIS onset at 2 weeks after TAM (Fig. 4J). Tumor cell proliferation was inhibited compared to DMSO-treated controls (Fig. 4K), and the progression of these lesions into invasive tongue cancer had slowed significantly at 4 weeks after TAM (Fig. 4L). Histological analysis revealed that there was no significant increase in TUNEL+ (terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labelingpositive) cells in the tongues of dasatinib-treated tgMob1DKO+TAM mice, indicating that dasatinib did not increase apoptosis but rather blocked cell proliferation (fig. S4G).

Together, these data indicate that endogenous YAP1 hyperactivation is involved in both OSCC onset and progression and is a driving force in tongue cancer in mice and humans. These results further strengthen our contention that YAP1 inhibitors may be promising novel agents for OSCC therapy.

Previous reports had suggested that nuclear localization of YAP1 was frequently observed at the precancerous stage of human OSCC (10). We obtained samples of nontumorous tongue tissue (NT-control) and tongue dysplasia, CIS, or invasive SCC from 86 patients at the National Hospital Organization Kyushu Cancer Center. These samples were immunostained to detect YAP1, and YAP1 levels were quantified using a grade scale (see Fig. 5A and Materials and Methods). As expected, NT-control epithelium showed weak YAP1 expression (mean grade = 0.7) only in the basal layer, with negligible YAP1 expression above the basal layer (Fig. 5, A and B). Most patients with tongue dysplasia showed enhanced YAP1 expression (mean grade = 3.5) in the nonbasal upper layer, indicating that YAP1 expression is higher than in controls from the early precancerous stage. Patients with CIS in the tongue displayed stronger nuclear staining of YAP1 than dysplastic patients, and patients with invasive SCC exhibited much more intense YAP1 staining than either of these (Fig. 5, A and B). We next tested for YAP1 activation using IHC evaluation of the expression of the YAP1 target genes CTGF, BIRC5, and TOP2A. In examining 14 OSCC specimens with high YAP1 protein levels and 14 specimens with low YAP1 protein levels, we found that CTGF protein tended to rise in the YAP1-high group (fig. S5A), and that the BIRC5 (fig. S5B) and TOP2A (fig. S5C) proteins were increased significantly in these same specimens. Thus, YAP1 activation appears to have a very important function in the onset and progression of tongue cancer not only in mice but also in humans.

(A) Left: Low-magnification (top panels; scale bar, 40 m) and high-magnification (bottom panels; scale bar, 10 m) views of representative YAP1-immunostained plus hematoxylin-counterstained sections of normal human tongue (mean YAP1 activity grade = 0.67; n = 56), dysplasia (mean grade = 3.5; n = 63), CIS (mean grade = 4.8, n = 26), and invasive SCC (mean grade = 6.4, n = 86). Right: Determination of YAP1 grade. Representative sections from the left panels were scored for YAP1 frequency and intensity as indicated. Scale bars, 10 m (frequency) and 3 m (intensity). The YAP1 grade was the product of these scores (see Materials and Methods). (B) Compilation of YAP1 grade scores in sections of human normal tongue, and tongues with dysplasia, CIS, or invasive SCC. Data are shown as means SEM. *P < 0.05 and ***P < 0.001, t test. (C) Kaplan-Meier curves showing overall survival (left) and relapse-free survival (right) of 86 tongue cancer patients who underwent surgical resection. The patients were divided into a high YAP1 expression group (n = 14) and a low YAP1 expression group (n = 72; see table S1). **P < 0.01 and ***P < 0.001, Wilcoxon test.

We then looked at the effect of high YAP1 expression on the overall survival and relapse-free survival of human tongue cancer cases. We examined the histories of our 86 selected tongue cancer patients, each of whom had undergone surgical resection at the National Hospital Organization Kyushu Cancer Center. We found that high YAP1 expression in human tongue cancer patients (n = 14) correlated with lymph node metastasis (table S1), decreased overall survival (Fig. 5C, left), and reduced relapse-free survival (Fig. 5C, right). Thus, elevated YAP1 activity in human tongue cancer is a negative prognostic indicator.

Human HNSCCs often bear mutations of TP53, elements of PI3K/PTEN signaling, FAT1, or elements of EGFR signaling (2). All of these entities have been previously reported to activate YAP1 in one or more cell types (1114). Mutation of TP63, a master regulator of squamous cells, is also frequently observed in human HNSCC, but its effects on YAP1 remain under debate (15, 16). We hypothesized that one or more of these mutations would activate YAP1 in transformed epithelial cells from an OSCC patient.

The WSU-HN30 human HNSCC cell line is HPV (), low in EGFR, and wild type for TP53, FAT1, and PTEN (20). We transfected these cells with siRNA against TP53, PTEN, or FAT1 (fig. S6A) or treated them with EGF (1 g/ml) for 24 hours. All of these cultures increased their expression and/or activation of YAP1 as determined by immunoblotting to measure YAP1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or YAP1/phosphorylated YAP1 (pYAP1) ratios (Fig. 6, A and B, and fig. S6B). Nuclear YAP1 protein was also enhanced in these manipulated cells as detected by IF staining (Fig. 6C and fig. S6C). We next investigated the expression of CTGF, BIRC5, and TOP2A, which are major downstream targets of YAP1, and found that their mRNA levels were increased either by the silencing of TP53, PTEN, or FAT1 or by EGF treatment (fig. S7A). To confirm our results in another cell line derived from HPV () OSCC, we subjected Cal27 cells (TP53mt, PTENwt, and FAT1wt) to PTEN or FAT1 knockdown, or EGF treatment, and again detected up-regulation of YAP1 activity (fig. S7, B and C). Moreover, we found a positive correlation between TP53mt and YAP1 expression in our human clinical specimens (table S1). Thus, it seems that mutation of any of these tumor suppressor genes and/or increased EGF signaling can up-regulate YAP1 expression to some degree in OSCC cells. Intriguingly, some combinations of these gene alterations resulted in YAP1 activation to a high level (Fig. 6D). Thus, an accumulation of these mutations may explain the increased and sustained activation of YAP1 in head-and-neck cancer epithelial cells.

(A and B) Left panels: Immunoblots to detect YAP1 and pYAP1 in WSU-HN30 cells that were either (A) transfected with control siRNA (siSC#1) or with one of three independent siRNAs (#1 to #3) targeting TP53, PTEN, or FAT1 or (B) left untreated (Parent) or treated with EGF (1 g/ml). siRNA- or EGF-treated cells were harvested at 24 or 96 hours after treatment, respectively. Right panels: Ratios of YAP1/GAPDH (protein) and YAP1/pYAP1 (activity) were calculated as described in Materials and Methods. Data are presented as means SEM of (A) seven independent siRNA-transfected samples for each gene or (B) three EGF-treated cultures per group. (C) Top: IF-based detection (see Supplementary Materials and Methods) of nuclear versus cytoplasmic YAP1 in siRNA-transfected or EGF-treated WSU-HN30 cells treated as in (A) and (B). DAPI (blue), nuclei. Bottom: Ratio of cells with nuclear YAP1/cytoplasmic YAP1 in the top panels. Data are means SEM of three independent siRNA-transfected samples for each gene (siRNA transfection) or three cultures treated with EGF. (D) Top: Immunoblot to detect YAP1 and pYAP1 in WSU-HN30 cells that were transfected with the indicated siRNAs. Middle and bottom: Ratios of YAP1/GAPDH and YAP1/pYAP1 were analyzed and quantified as in (A) and (B). Data are means SEM of four independent experiments involving at least triplicate samples. *P < 0.05 and **P < 0.01, t test.

With respect to TP63, Mob1 deletion enhanced TP63 protein (p63) levels in mouse tongue epithelial cells (fig. S8A), and siRNA-mediated inhibition of Tp63 expression in SCC9 cells expressing Dox-inducible YAP1-5SA blocked their proliferation (fig. S8B). In addition, overexpression of Np63 only mildly interfered with YAP1 activation in the WSU-HN30 human tongue epithelial cell line (fig. S8, C to E).

The above data collectively indicate that TP53, FAT1, PTEN, and EGFR are all upstream regulators of YAP1, whereas TP63 is a downstream effector of YAP1. These findings prompted us to devise a model in which the mutation (functional inactivation) of TP53 plus a subset of these genes, including PIK3CA/PTEN, EGFR, and/or FAT1, results in YAP1 hyperactivation that may exceed an oncogenic threshold (fig. S9A). This elevated YAP1 may then activate downstream genes such as Np63 and may thereby initiate OSCC onset and/or progression.

In this study, we have demonstrated for the first time that YAP1 may be a strikingly potent oncogenic driver of OSCC onset and progression. Cancers usually initiate because of the additive or synergistic effects of several mutated genes that exert their effects during multistep carcinogenesis (1). However, because the onset of tongue tumors in our tgMob1DKO mice was so quick, we propose a new concept positing that OSCC may be initiated when sustained YAP1 activity exceeds a particular oncogenic threshold (refer to fig. S9A). Many recent reports have linked YAP1 activation to either loss-of-function (LOF) mutations of key genes such as TP53 and FAT1, or the triggering of pathways related to PI3K/AKT or EGFR (1114). We have confirmed these findings in human OSCC cells (Fig. 6, A to C, and fig. S7) and have established that an accumulation of YAP1 activity can be driven by various combinations of these alterations (Fig. 6D). Depending on the strength of YAP1 activation attributed to one alteration, the oncogenic threshold may be exceeded with only a few additional mutations.

The role of TP53 in OSCC poses an interesting conundrum. LOF of TP53 inhibits expression of PTPN14 and 14-3-3, which are downstream transcriptional targets of TP53 (11, 21), and so is likely to promote YAP1 activity. However, TP53 gain-of-function (GOF) mutations (e.g., R248L, R175H, and R273H), which are observed in 9.5% of HNSCCs, reportedly bind directly to and stabilize YAP1 (22), also promoting YAP1 activity. The binding of GOF-mutated TP53 to YAP1 can activate transcription factors such as NF-Y, whose target genes act to increase cell proliferation (23). Thus, both LOF and GOF mutations of TP53 can enhance YAP1 activity and so may contribute to human OSCC carcinogenesis. Nevertheless, because mice solely lacking normal TP53 function do not develop OSCC (6), there must be other factors required for the onset and development of OSCC.

Repeated exposure to carcinogens (including tobacco and alcohol), irritation of the oral mucosa (especially tongue epithelium) due to the presence of tooth decay, or mechanical stimulation by ill-fitting dentures are the main causes of human OSCC (1, 24). These events may also directly activate YAP1 or induce oncogenic mutations in the abovementioned genes that activate YAP1. Cigarette smoke extract (25) and mechanical stimulation (26) have both been shown to activate YAP1 in various cell types, including in esophageal and cervical cells. These observations support our hypothesis that OSCC is caused by the boosting of YAP1 activity over a certain threshold. Furthermore, although YAP1 protein itself is frequently activated and accumulates in most tumors (8), the actual DNA mutation of Hippo-related genes, including YAP1, is relatively rare in cancers (27). Thus, our work erases many years of doubt as to how HNSCCs can arise in the absence of GOF mutations of major oncogenes (28).

One possible reason for the frequent and early onset of OSCC in our mutant mice is the activation of Np63, a master regulator of epidermal keratinocyte proliferation and differentiation (29). Activated YAP1 binds directly to Np63 protein and stabilizes it (30). A lack of TP63 in mice results in the absence of the epidermis and its related appendages (29), and Tp63-deficient embryonic stem cells exhibit up-regulation of mesodermal genes (31). Conversely, overexpression of Np63 in the presence of KLF4 induces the conversion of fibroblasts to cells of the keratinocyte lineage (32). We found that TP63 accumulated in tongue epithelial cells in our mouse model of OSCC (fig. S8A). Last, we demonstrated that inhibition of Np63 expression blocked the cell proliferation induced by YAP1 overexpression (fig. S8B). We speculate that hyperactivation of YAP1 leading to high levels of stabilized Np63 may both skew cells toward the keratinocyte lineage and boost keratinocyte proliferation and dedifferentiation, which may, in turn, increase the chance of OSCC development. A second reason for the early onset of invasive SCC in our mutant mice may be increased production of BMP4. BMP4 is a soluble growth factor that plays an essential role in epidermal development by regulating Np63 (33). High levels of BMP4 were detected in tgMob1DKO tongue epithelial cells compared to controls when examined by microarray analysis (fig. S9B). A third possible reason for our observations may be the existence of positive feedback between EGF signaling and YAP1. YAP1 increases the transcription of EGF receptors (EGFR and ERBB3) and EGF-like ligands (HBEGF, NRG1, and NRG2) (16). Conversely, both HBEGF and NRG1 have been shown to activate YAP1 in ovarian cancer (14). Although we did not observe a significant increase in EGFR, ERBB3, or HBEGF mRNAs when MOB1 was deleted (YAP1 activated) in mouse tongue epithelium, we did detect elevation of NRG1/2 mRNAs (fig. S9B), suggesting the existence of an NRG1/2-(ERBB3)-YAP1-NRG1/2 autocrine loop that controls OSCC tumorigenesis and progression. All three of these mechanisms may contribute to OSCC genesis, perhaps explaining why the phenotype is so strong, especially in epidermal cells.

An important finding emerging from our study is that mice lacking MOB1 plus TAZ developed more aggressive invasive SCC than did mice lacking MOB1 alone. This result indicates that YAP1 and TAZ may be activated independently in the SCC context and that the mechanism by which MOB1A/B regulates YAP1 differs from its effects on TAZ in these malignancies. Further study will be required to understand and distinguish between the underlying molecular mechanisms. Nevertheless, our data imply that selective targeting of YAP1 may be an effective new mode of OSCC treatment.

Two TAM-inducible epidermal SCC models have been previously described. In the first model, the mutant mice bear a K-Ras transgene and an inducible Tp53 KO gene (34). Half of these mutants develop skin SCCs by 35 weeks after TAM. In the second model, the mice bear an AKT transgene and an inducible Tp53 KO gene, leading to HNSCC development in 50% of animals by 35 weeks after TAM (35). Thus, we were greatly surprised to observe CIS in the tongue as early as 1 week after TAM in our tgMob1DKO mutants, followed by the inevitable development of invasive SCC by 4 weeks after TAM. Considering that it takes more than 7 days to completely inhibit MOB1 protein expression (fig. S1C), it seems that Mob1a/b-deficient keratinocytes (which bear disruption of a single pathway) may become cancerous immediately without undergoing any other molecular alterations. Our mutant mice thus currently constitute the worlds fastest spontaneous cancer onset model. Moreover, cancer progression is synchronized in all these mutants, and the tumors are easily visualized on the mouse exterior. These characteristics make our model a particularly attractive tool for cancer research and the development of new anticancer drugs. This latter point is a pressing issue because, in the past, several dose-intensified chemo/radiotherapy trials were conducted for HNSCC treatment but quickly reached the limit of human tolerance, showing positive results for only a few select patients (3). Furthermore, recurrent and metastatic HNSCCs are refractory to both conventional chemotherapies and currently available molecular targeting drugs such as EGFR inhibitor (cetuximab) or anti-PD1 antibody (nivolumab), which only marginally improve patient survival (4). Our work has shown that inhibition of YAP1 not only prevents the onset of OSCC but also slows its progression. YAP1 may thus be an appealing molecular target for therapy of this devastating disease. We expect to use our mutant mice to identify new drugs targeting the Hippo pathway in epidermal cancers, including in HNSCCs, with the goal of bringing concrete benefits to patients.

Previously established mouse strains used in this study were Mob1aflox/flox; Mob1b/ (17), Rosa26-CreERT (The Jackson Laboratory), and Tazflox/flox (provided by J. Wrana). Yap1flox/flox mice were generated using Yap1flox/flox embryonic stem cells from the Knockout Mouse Project Repository (36). All mice were kept in specific pathogenfree facilities at Kobe and Kyushu Universities.

Human tongue SCC cell lines HSC3, HSC4, and SCC4 (all from the Japanese Collection of Research Bioresources); SCC9 and Cal27 (both from the American Type Culture Collection); WSU-HN30 (provided by S. Gutkind, University of California); and H1299-Luc (established by H.H.) were cultured in Eagles minimum essential medium, Dulbeccos modified Eagles medium (DMEM), DMEM/Hams F12 medium, or RPMI medium, respectively, supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37C in a 5% CO2/95% air incubator. Hydrocortisone (400 ng/ml) was added to the medium of SCC4 and SCC9 cultures, in line with a standard protocol.

Mob1a/b homozygous double-mutant mice (Rosa26-CreERT; Mob1aflox/flox; Mob1b/) were generated by mating Rosa26-CreERT Tg mice with Mob1aflox/flox; Mob1b/ mice. Rosa26-CreERT Tg mice were in a C57BL/6 background, and Mob1aflox/flox; Mob1b/ mice were backcrossed to C57BL/6 for more than six generations. To delete the floxed Mob1a gene, TAM (Sigma-Aldrich) diluted in 100% ethanol (10 mg/ml) was applied daily directly to the mouse tongue for 5 days by brush. The area of application is indicated in fig. S1A. Before TAM application, mice were anesthetized with a mixture of medetomidine hydrochloride, midazolam, and butorphanol. Mob1aflox/flox; Mob1b/ mice treated with TAM were used as controls unless otherwise stated. Rosa26-CreERT; Mob1aflox/flox; Mob1b/; Yap1flox/flox and Rosa26-CreERT; Mob1aflox/flox; Mob1b/; Tazflox/flox mice were generated by mating Rosa26-CreERT; Mob1aflox/flox; Mob1b/ mice with Yap1flox/flox or Tazflox/flox mice, respectively.

The primers used for mouse genotyping polymerase chain reaction were as follows: Mob1awt/flox, GTCTCGTGAAGGGTCTTGAGG/CCTGGTTGGGGTGGAGAATCAA [wt, 319 base pairs (bp); flox, 450 bp]; Mob1a, GTAATGTGTTCAGCTATGCTTTGAC/CCTGGTTGGGGTGGAGAATCAA (551 bp); Mob1bwt, CTTCAGGATCCTTGGTGGTTATCAG/AGAGCAAGGGGAAAAGAAGCTCAATG (586 bp); Mob1bmutant, CTTCAGGATCCTTGGTGGTTATCAG/TCAGGGTCACAAGGTTCATATGGTG (673 bp); Rosa26-CreERT Tg, AAAGTCGCTCTGAGTTGTTAT/CCTGATCCTGGCAATTTCG (825 bp); Yap1wt/flox, GCCCAAACATACCCACGTAAT/CAGTCCAGTCAAGACAAGAT (wt, 192 bp; flox, 336 bp); Tazwt/flox, AAGCAGTTTCCACTTCATGAAAC/AGTCAAGAGGGGCAAAGTTGTGA (wt, 250 bp; flox, 330 bp).

Tumor tissues were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and embedded in paraffin. Sections (4 m) of tumors were cut for hematoxylin and eosin (H&E) staining. Diagnoses of tongue epithelial dysplasia, CIS, or invasive SCC were confirmed by two pathologists.

Primary tongue epithelium from 3-week-old Rosa26-CreERT; Mob1aflox/flox; Mob1b/ mice without TAM was obtained using a dermal keratinocyte isolation protocol (37). Briefly, resected tongue tissues were placed into ice-cold dispase digestion buffer [250 U of dispase (Godo Shusei) in PBS] and incubated overnight at 4C. The epidermis was slowly separated from the dermis using forceps and floated on trypsin solution (Gibco) at room temperature for 40 min to create a primary tongue epithelial cell suspension. Tongue epithelial cells were cultured in CnT-PR medium (CELLnTEC) and passaged more than 40 times to generate the iMob1DKO tongue epithelial cell line. Loss of Mob1a/b in these cells in vitro was induced by treating them with TAM (0.5 M; Toronto Research Chemicals) for 3 days.

Immunoblotting was carried out using a standard protocol and primary antibodies recognizing MOB1A (#E1N9D; Cell Signaling Technology), MST1 (#3682S; Cell Signaling Technology), LATS1 (C66B5; Cell Signaling Technology), YAP1 (#4912S; Cell Signaling Technology), pYAP1(S127) (#4911S; Cell Signaling Technology), TAZ (V386; Cell Signaling Technology), pTAZ (S89) (#75275; Cell Signaling Technology), CTGF (L-20; Santa Cruz Biotechnology), BIRC5 (71G4B7; Cell Signaling Technology), TOP2A (EP1102Y; Abcam), or TP63 (4A4; Abcam). Primary antibodies were detected using horseradish peroxidase (HRP)conjugated secondary rabbit antibody (#7074; Cell Signaling Technology). Endogenous GAPDH (FL-355; Santa Cruz Biotechnology) was used as the internal control. Quantification of signal intensity was performed using Fujifilm Multi Gauge software.

Mice tongue tissues were fixed in 4% PFA, embedded in paraffin, and sectioned (4 m) using standard procedures. IHC or IF staining was performed using an indirect method using primary antibodies recognizing YAP1 (WH0010413M1; Sigma), Ki67 (ab15580; Abcam), or TP63 (4A4; Abcam). Primary antibodies were detected using REAL EnVision HRP-rabbit/mouse (Dako) or Alexa Fluor 568 (Molecular Probes). Some slides were counterstained with Mayers hematoxylin (Muto) or 4,6-diamidino-2-phenylindole (DAPI; Dojindo) before mounting using PermaFluor (Thermo Scientific). For Ki67 positivity studies, 200 cells per mouse were examined.

From the population of patients who were treated at the National Hospital Organization Kyushu Cancer Center in Japan from 2008 to 2013, we selected 86 patients who had received surgical resection of tongue SCC as their first line of therapy and performed a retrospective review of their medical charts. Their resected cancer tissues (n = 86), which had been fixed in formalin, were stained with antibodies recognizing: YAP1 (WH0010413M1; Sigma), TP53 (DO7; Sigma), CTGF (ab6992; Abcam), BIRC5 (EP2880Y; Abcam), or TOP2A (TOP2A/1362; Abcam). Within these stained resected tissues, areas of NT epithelium, dysplasia, CIS, or invasive SCC were determined and levels of YAP1 activity (grade) were scored. YAP1 grade was defined by multiplying the YAP1 frequency score by the YAP1 intensity score, as previously described (38). A score of >8 classified a sample into the YAP1-high group.

To compare overall survival and relapse-free survival rates between groups of patients with high (n = 14) or low (n = 72) YAP1 expression, Kaplan-Meier curves were generated and a Wilcoxon test was used to analyze statistical differences. Overall survival was calculated on the basis of the length of time between date of surgery and date of death. Follow-up duration was 68.1 months on average (range of 3 to 128 months).

To investigate factors influencing YAP1 activity, the following clinicopathological factors were included in the univariate analyses: age, sex, history of smoking, history of alcohol, T stage (which describes the primary tumor size and site), N stage (which describes the degree of regional lymph node involvement), clinical stage, recurrence, degree of tumor differentiation, presence of multiple cancers, and TP53 mutation status [defined as previously described (39)] (see table S1). Univariate analyses were performed using the chi-square test.

siRNA targeting of YAP1, TP53, PTEN, FAT1, or NF2 expression was performed using siRNA oligonucleotides of the following sequences: si-scramble #1, CGUACGCGGAAUACUUCGA; si-scramble #2, UUCUCCGAACGUGUGUCACGU; si-scramble #3, siNC1 (Ambion); si-YAP1 #1, GGCCCUUUGAUUUAGUAUA; si-TP53 #1, GUAAUCUACUGGGACGGAA; si-TP53 #2, GAAAUUUGCGUGUGGAGUA; si-TP53 #3, GGUGAACCUUAGUACCUAA; si-PTEN #1, GCAUACGAUUUUAAGCGGA; si-PTEN #2, CACCGCAUAUUAAAACGUA; si-PTEN #3, CAAGAAAUCGAUAGCAUUU; si-FAT1 #1, GGACCGAAAUUCCUUCGAA; si-FAT1 #2, CGGAAGUUAUCGUUCCGAU; si-FAT1 #3, GACCGAAAUUCCUUCGAA; si-NF2 #1, CAAGCACAAUACCAUUAAA; si-NF2 #2, CCCAAGACGACGUUCACCGUGA; si-NF2 #3, AGAAGCAGAUUUUAGAUGA; si-TP63#1, GAACCGCCGUCCAAUUUUA; and si-TP63#2, UGAUGAACUGUUAUACUUA.

Transfection of siRNA oligonucleotides (10 nM) into exponentially growing WSU-HN30 tongue cancer cells was performed using Lipofectamine RNAiMAX (Invitrogen) following the manufacturers protocol. At 96 hours after transfection, protein lysates were subjected to immunoblotting and cells were IF-stained to detect YAP1 as described above.

WSU-HN30 cells (2 105) were seeded in six-well plates. After 48 hours, EGF (1 g/ml; PeproTech) was added to the culture medium and cells were incubated for 24 hours before harvesting. Immunoblotting and IF staining to detect YAP1 were conducted as described above.

To determine the in vitro effects of drugs known to target YAP1, HSC4 cells (1 104 per well in 24-well plates) were cultured for 1 to 3 days in DMEM/Hams F12 medium containing 5 M dasatinib (Abcam), 5 M verteporfin (USP), 5 M simvastatin (TCI), or vehicle (DMSO; negative control). Inhibition of cell growth was assessed by counting cell numbers per well.

To determine the in vivo effects of dasatinib and simvastatin on the initiation and progression of tongue cancer in tgMob1DKO mice, dasatinib (5 mg/kg, intraperitoneally), simvastatin (50 mg/kg, intraperitoneally), or vehicle (DMSO; negative control) was administered daily for 14 to 17 days starting either 3 days before TAM application (for Fig. 4A) or after CIS onset starting at 2 weeks after TAM (for Fig. 4J).

To determine the effect of YAP1 silencing in vivo, human tongue SCC cells (HSC4; 1 107) that had been transfected with Dox-dependent shYAP1 were injected subcutaneously into the flanks of 9-week-old female BALB/cAJcl-nu/nu mice (CLEA Japan). After visual detection of tumors (usually at 10 days after injection), mice were supplied with normal drinking water or water containing Dox (2 mg/ml). To determine the in vivo effects of dasatinib on human tongue SCC cells, nontransfected HSC4 cells (1 107) were injected subcutaneously into nude mice as above. After visual detection of tumors (usually at 10 days after injection), mice were treated daily with dasatinib (5 mg/kg, intraperitoneally) or vehicle (DMSO; negative control). In both cases, tumor volumes were measured every 4 days using calipers.

Unless otherwise indicated, all results represent the mean SEM. Statistical comparisons between different groups were performed using the two-tailed Students t test. For all statistical analyses, differences of P < 0.05 were considered statistically significant. All experiments were repeated at least three times.

Animal experiments were approved by the Kobe University (#P170604) and Kyushu University (#28-156) Animal Experiment Committees, and the care of the animals was in accordance with institutional guidelines. All clinical samples were approved for analysis by the Ethics Committee at the National Hospital Organization Kyushu Cancer Center (#2015-43). Written informed consent was obtained from all patients whose cancers were analyzed in this study.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaay3324/DC1

Supplementary Materials and Methods

Table S1. Clinicopathological features of 86 cases of human tongue squamous cell carcinoma.

Fig. S1. Induction of Mob1a/b deletion in tgMob1DKO mice by postnatal application of TAM.

Fig. S2. Cell-cell junction collapse and retained cell size of iMob1DKO cells and YAP1/TAZ expression and localization in tongue epithelium of tgMob1DKO, tgYap1TKO, and tgTazTKO mice.

Fig. S3. Effects of dasatinib, simvastatin, verteporfin, and Y-27632 on YAP1 protein expression and activation and tumor-suppressive effect of simvastatin.

Fig. S4. YAP1 expression in OSCC cell lines, the effect on an OSCC cell line of YAP1 depletion combined with cisplatin, and the effect of dasatinib on cell death in tgMob1DKO mice.

Fig. S5. YAP1 target gene expression correlates with YAP1 nuclear expression in human clinical OSCC specimens.

Fig. S6. Evaluation of gene knockdown and ectopic gene expression in the WSU-HN30 HNSCC cell line and activation of YAP1 by knockdown of TP53, PTEN, or FAT1.

Fig. S7. Activation of YAP1 target gene expression by molecules that are frequently altered in human OSCC.

Fig. S8. Positive correlation of Np63 protein expression with YAP1 protein expression.

Fig. S9. Graphical abstract and microarray analysis of growth factors and receptors whose mRNAs are up-regulated in tgMob1DKO tongue epithelial cells.

References (40, 41)

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We thank H. Togashi, Y. Shimono, and K. Okada (all of Kobe University) for expert technical assistance and critical discussions. We thank J. Wrana (Lunenfeld-Tanenbaum Research Institute) for Tazflox/flox mice and J. S. Gutkind (University of California) for WSU-HN30 cells. Funding: We are grateful for the funding provided by the Japanese Society for the Promotion of Science (JSPS; grants 17H01400, 26114005, and 26640081 to A.S.); the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University; Nanken-Kyoten, Tokyo Medical and Dental University (TMDU); the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT; grant 11088019 to A.S.); the Japanese Agency for Medical Research and Development [P-CREATE (AMED); grant JP19cm0106114 to A.S.]; the Uehara Memorial Foundation (to A.S.); the Shinnihon Advanced Medical Research Foundation (to A.S.); the Daiichi-Sankyo Scholarship Donation Program (to A.S.). Author contributions: Conceptualization: H.O., M.N., T.M., and A.S.; analysis: H.O., M.N., T.M., Y.M., F.U., T. Nakano, and K.T.; resources: H.H., H.N., T.K., and M.M.; data curation: T. Nakano, K.S., K.M., and H.T.; writing of the original draft: H.O., T.M., and A.S.; supervision: M.M., K.M., T.W.M., K.N., and T. Nakagawa; project administration: T.M. and A.S.; funding acquisition: A.S. Competing interests: The authors declare that they do not have competing interest. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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YAP1 is a potent driver of the onset and progression of oral squamous cell carcinoma - Science Advances

Can hybrid embryos save the white rhinos from extinction? – Science 101

The northern white rhino population is in jeopardy

The northern white rhino is one of the animal kingdoms many majestic giants, but years of poaching has taken a toll on their population. From 1970 to 1980, their numbers plummeted from 500 to 15 as illegal hunters pursued white rhinos for the ivory of their horns.

Things started to turn around during the 1990s and 2000s, groups and individuals began to crack down on poachers within the white rhinos range. As a result, the population of white rhinos in the wild recovered slightly, peaking at around 32 individuals.

Since 2003, the rate of white rhino poaching has been on the rise and has affected the animals numbers. As of 2008, northern white rhinos have been declared extinct in the wild, and in 2018, the last male northern white rhino died. Now, there are only two of these magnificent beasts left on Earth. Both of them are females.

Najin and Fatu are the last two northern white rhinos in existence. They live at the Ol Pejeta Conservancy in Kenya, and they could be the species last hope for the future. In 2014, keepers in the Czech Republic collected sperm samples from a male northern white rhino living in their care.

Those samples were frozen and stored, and later, they were used in an attempt to breed Najin and Fatu. Both attempts at inducing pregnancies in the two female rhinos were unsuccessful, forcing scientists to consider new methods of approach for saving the white rhinos from extinction.

Typically, when a species is placed on the endangered list, a recovery plan is established by whatever local conservancy group oversees the population. From there, breeding programs of captive individuals are used to begin bolstering the number of individuals on the planet.

When healthy breeding populations have been established, in most cases, reintroduction begins. Small populations of the species are released into the wild to begin repopulation. However, in the case of the northern white rhinos, scientists and conservationists alike have been stuck at step two for decades.

Unwillingness and inability to breed arent uncommon among captive species and individuals, and in most cases, zoos can jockey animals around until a pair matches and produces offspring. In the case of Najin and Fatu, the options for procreation are far more limited. Even the fallback of artificial insemination isnt working for them, so what are scientists to do?

Weve revived entire species from the dead before, but it has never been an easy task. Fortunately, the world of reproductive sciences has been evolving quickly, and conservationists and animal experts now have myriad options to choose from when it comes to creating new life.

Neither surviving female is healthy enough to birth live young. Aside from that, there is the added challenge of finding an option that preserves the northern white rhino genome while maintaining high enough levels of viability.

One possible route to repopulation involves approaching conventional methods from a new and enlightened angle. Although neither Najin nor Fatu can bear young, they both still produce viable egg cells, which can be harvested, frozen, and kept in a lab.

Much like humans undergoing fertility therapy or other conception aids, the grandmother-granddaughter pair or northern white rhinos can hope for success through in-vitro fertilization. This method of conception combines sperm and multiple egg cells in an external environment before implanting them in a host mother.

By using multiple eggs during the in-vitro process, the chances for success, even in females with fertility issues, is significantly increased. In some fortunate cases, the method is so effective, and it results in multiple pregnancies. Once the sperm has fertilized the eggs, the cells are transferred to a living host.

While Najin and Fatu may not be the physical mothers of any of their calves, modern reproductive science has made it possible for their genes to be passed on to another generation.

How? with modern science, a surrogate mother from the thriving population of southern white rhinos could become the mother to their children.The two types of animals have similar enough reproductive organs and their eggs could be used in place of Najin or Fatus.

While the animals are compatible, gathering eggs from them is a far more complicated procedure.

Researchers working on bringing back the northern white rhinos have managed to gather a few eggs so far, but not nearly enough to repopulate an entire species.

Its no secret that rhinoceroses are large animals. Just as cattle and horses have significantly larger hearts than we humans do, rhinos have much larger reproductive organs. Locating and withdrawing eggs from a rhinos ovaries is a far greater ordeal than it is for humans.

To complicate matters further, the ovaries of a southern white rhino are located three to four feet from her rump, and the veterinarian seeking to collect the eggs must guide a probe that distance up her rectum and into an ovary before using a catheter to remove the eggs.

The procedure is anything but easy. In addition to the difficulty involved in the process of extracting eggs, the success rate of current methods is hardly ideal. Researchers working on bringing back the northern white rhinos have managed to gather a few eggs so far, but not nearly enough to repopulate an entire species.

The odds of reestablishing a sustainable population of northern white rhinos through in-vitro fertilization and surrogacy currently seem pretty slim. Fortunately for the rhinos, science has a few other methods up its sleeve.

In the last decade, stem cell research has gone from a thing of whimsy to an advanced field of study that continues to improve by leaps and bounds with every passing year. Its applications are seemingly endless, and they just might be the answer that the northern white rhino conservationists have been looking for.

Stem cells are sort of like biological canvases. They come in different varieties: Totipotent, pluripotent, multipotent, oligopotent, and unipotent. Each of these types has unique limitations and can be found in various sources from embryonic tissue to adult bone marrow.

To make baby rhinos, scientists have been focused on induced pluripotent stem cells, which are gathered and grown from the skin of adult white rhinos

A cell from your bicep and a cell from your gametes (sperm or egg) both hold the same blueprints; they just come in different packaging.

Pluripotent cells behave similarly to embryonic stem cells, which can be coaxed into becoming just about any other type of cell. In this case, even though the original cells were taken from the skin of adult rhinos, they can be trained to become something different, such as egg cells.

Using what knowledge we currently have of stem cells and their manipulation, scientists can tell a northern white rhinos skin cell to become a viable egg or sperm cell. From there, they can attempt in-vitro fertilization and implantation into a surrogate, even without fertile parents.

The method is still in its infancy, but it has been successfully carried out more than once.

With stem cells as a backup and surrogates abound, Najin and Fatu have plenty of options. In late 2019, conservationists and rhinos alike received promising news. Eggs gathered from the two northern white rhinos had been fertilized and resulted in successful embryos. Those embryos were frozen in liquid nitrogen and prepared for a long journey.

Waiting down in southern Africa are the lucky mamas who will become the surrogates for the next generation of northern white rhinos. The embryos have quite a ways to travel before they can be implanted. After that, they can grow within their new mother for the 16 to 18-month gestation period typical of white rhinos.

Although the methods of creating viable embryos are currently long, challenging, and not terribly efficient, these babies-to-be are incredibly promising first steps. In addition to the two successful in-vitro attempts in September, December of 2019 saw the creation of a third viable embryo.

2020 will undoubtedly see further attempts at creating more embryos. With luck, we can soon hope to hear news of successful implantations in surrogate moms. In 2021, we can throw a worldwide baby shower for some bouncing baby northern white rhinos, whose births will serve as a beacon of hope for a dying species.

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Can hybrid embryos save the white rhinos from extinction? - Science 101

Autologous Stem Cell Based Therapies Market 2020: Potential Growth, Challenges, Attractive Valuation | Key Players: Anterogen, Holostem Advanced…

Global Autologous Stem Cell Based Therapies Market Report is a professional and in-depth research report on the worlds major regional market conditions of the Autologous Stem Cell Based Therapies industry, focusing on the main regions and the main countries (United States, Europe, Japan and China).

Market Segmentations: Global Autologous Stem Cell Based Therapies market competition by top manufacturers, with production, price, revenue (value) and market share for each manufacturer.

Based on type, report split into Embryonic Stem Cell, Resident Cardiac Stem Cells, Umbilical Cord Blood Stem Cells.

Based on the end users/applications, this report focuses on the status and outlook for major applications/end users, consumption (sales), market share and growth rate for each application, including Neurodegenerative Disorders, Autoimmune Diseases, Cardiovascular Diseases.

The report introduces Autologous Stem Cell Based Therapies basic information including definition, classification, application, industry chain structure, industry overview, policy analysis, and news analysis. Insightful predictions for the Autologous Stem Cell Based Therapies Market for the coming few years have also been included in the report.

Autologous Stem Cell Based Therapies Market landscape and market scenario includes:

The Autologous Stem Cell Based Therapies industry development trends and marketing channels are analyzed. Finally, the feasibility of new investment projects is assessed, and overall research conclusions offered.

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Autologous Stem Cell Based Therapies Market 2020: Potential Growth, Challenges, Attractive Valuation | Key Players: Anterogen, Holostem Advanced...

22.5% Growth Rate for Synthetic Stem Cells Market by 2028 | Overview, Top Technologies, Key Insights and Company Profiles – News Times

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According to Market Study Report, Synthetic Stem Cells Market provides a comprehensive analysis of the Synthetic Stem Cells Market segments, including their dynamics, size, growth, regulatory requirements, competitive landscape, and emerging opportunities of global industry. This report also provides market landscape and market share information in the Synthetic Stem Cells Market. An exclusive data offered in this report is collected by research and industry experts team.

Top Key Players Profiled in the Synthetic Stem Cells Market include are North Carolina State University (NCSU) (US) and Zhengzhou University (China).

Synthetic Stem Cells Market is expected to grow from US$ 14 Million in 2023 to US$ 37 Million by 2028, at a CAGR of 22.5% during the forecast period. The synthetic stem cells market is driven by various factors such as ethical concerns regarding embryonic stem cells and the risk of tumor formation and immune rejection of natural stem cells.This report spread across 55 Pages, profiling 02 companies and supported with tables and figures are now available in this research.

The neurological disorders segment is expected to witness the highest CAGR during the forecast period.

The neurological disorders application is the faster-growing segment in the overall synthetic stem cells market. Based on application, the synthetic stem cells market is segmented into cardiovascular diseases, neurological disorders, and other diseases that include various cancers, wounds and injuries, musculoskeletal disorders, and blood disorders that require regenerative therapies.

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North America is expected to record the highest growth rate during the forecast period.

Countries in North America are constantly modernizing their healthcare infrastructure by investing in advanced therapies. The increasing prevalence of target diseases, focus on development of for regenerative medicines, fast adoption of advanced therapies, and regulatory support for stem cell therapies will result in the high rate of adoption of synthetic stem cell therapies in this region by 2023.

The Study Objectives of this report are:

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The report will provide the market leaders/new entrants in this market with information on the closest approximations of the revenue of the overall synthetic stem cells market and its sub segments. This report will help stakeholders understand the competitive landscape and gain insights to better position their businesses and plan suitable go-to-market strategies. The report will also help stakeholders to understand the pulse of the market and provide them with information on key market drivers, restraints, challenges, and opportunities.

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22.5% Growth Rate for Synthetic Stem Cells Market by 2028 | Overview, Top Technologies, Key Insights and Company Profiles - News Times

Worldwide Cell Therapy Market Projections to 2028 – The Largest Expansion Will Be in Diseases of the Central Nervous System, Cancer and Cardiovascular…

DUBLIN, March 12, 2020 /PRNewswire/ -- The "Cell Therapy - Technologies, Markets and Companies" report from Jain PharmaBiotech has been added to ResearchAndMarkets.com's offering.

The cell-based markets was analyzed for 2018, and projected to 2028. The markets are analyzed according to therapeutic categories, technologies and geographical areas. The largest expansion will be in diseases of the central nervous system, cancer and cardiovascular disorders. Skin and soft tissue repair as well as diabetes mellitus will be other major markets.

The number of companies involved in cell therapy has increased remarkably during the past few years. More than 500 companies have been identified to be involved in cell therapy and 309 of these are profiled in part II of the report along with tabulation of 302 alliances. Of these companies, 170 are involved in stem cells.

Profiles of 72 academic institutions in the US involved in cell therapy are also included in part II along with their commercial collaborations. The text is supplemented with 67 Tables and 25 Figures. The bibliography contains 1,200 selected references, which are cited in the text.

This report contains information on the following:

The report describes and evaluates cell therapy technologies and methods, which have already started to play an important role in the practice of medicine. Hematopoietic stem cell transplantation is replacing the old fashioned bone marrow transplants. Role of cells in drug discovery is also described. Cell therapy is bound to become a part of medical practice.

Stem cells are discussed in detail in one chapter. Some light is thrown on the current controversy of embryonic sources of stem cells and comparison with adult sources. Other sources of stem cells such as the placenta, cord blood and fat removed by liposuction are also discussed. Stem cells can also be genetically modified prior to transplantation.

Cell therapy technologies overlap with those of gene therapy, cancer vaccines, drug delivery, tissue engineering and regenerative medicine. Pharmaceutical applications of stem cells including those in drug discovery are also described. Various types of cells used, methods of preparation and culture, encapsulation and genetic engineering of cells are discussed. Sources of cells, both human and animal (xenotransplantation) are discussed. Methods of delivery of cell therapy range from injections to surgical implantation using special devices.

Cell therapy has applications in a large number of disorders. The most important are diseases of the nervous system and cancer which are the topics for separate chapters. Other applications include cardiac disorders (myocardial infarction and heart failure), diabetes mellitus, diseases of bones and joints, genetic disorders, and wounds of the skin and soft tissues.

Regulatory and ethical issues involving cell therapy are important and are discussed. Current political debate on the use of stem cells from embryonic sources (hESCs) is also presented. Safety is an essential consideration of any new therapy and regulations for cell therapy are those for biological preparations.

Key Topics Covered

Part I: Technologies, Ethics & RegulationsExecutive Summary 1. Introduction to Cell Therapy2. Cell Therapy Technologies3. Stem Cells4. Clinical Applications of Cell Therapy5. Cell Therapy for Cardiovascular Disorders6. Cell Therapy for Cancer7. Cell Therapy for Neurological Disorders8. Ethical, Legal and Political Aspects of Cell therapy9. Safety and Regulatory Aspects of Cell Therapy

Part II: Markets, Companies & Academic Institutions10. Markets and Future Prospects for Cell Therapy11. Companies Involved in Cell Therapy12. Academic Institutions13. References

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Worldwide Cell Therapy Market Projections to 2028 - The Largest Expansion Will Be in Diseases of the Central Nervous System, Cancer and Cardiovascular...

Single-Cell Analysis of Ovarian Cortex Fails to Find Stem Cells – The Scientist

The first single-cell analysis of the human ovarian cortex revealed six main types of cells, but none of the oogonial stem cells that other researchers say they have isolated, according to a study published earlier this week (March 2) in Nature Communications. These findings are backed by the most advanced technologies, the authors say, and could put to rest a heated debate about the properties of the adult ovary that has raged for more than a decade.

The results of the experiment dont leave a lot of space for different interpretations, says Susana Chuva de Sousa Lopes, a developmental biologist at Leiden University Medical Center in the Netherlands who served on the PhD dissertation committee of coauthor Sarita Panula but was not involved in the research. It seems, she says, that cells previously identified as ovarian stem cells are in fact perivascular cells, which support blood vessel structure and help regulate blood flow.

But the discoverers of ovarian stem cells in adult mammals and other proponents of the cells existence are not convinced, citing methodological weaknesses of the new study.

Until relatively recently, scientific consensus was that a female mammals oocyte pool is fixed at birth. Adult ovaries, it was assumed, are simply unable to generate new eggs. But in 2004, Northeastern University reproductive biologist Jonathan Tilly and colleagues published findings that appeared to upend this understanding of oocyctes by presenting evidence of ovarian stem cells in adult mice.

A few years later, scientists in China claimed to have also found such germ line stem cells in the ovaries of adult mice, and showed that these cells could differentiate into functional eggs that gave rise to viable mouse pups. And in 2012, Tillys group reported the existence of germ cells in samples of human ovarian tissue, claiming that these cells could similarly generate oocytes in vitro and in vivo when injected into mice.

These findings generated a lot of publicity because they suggested that human fertility wasnt fixed after all. But the data has always been criticized, says Fredrik Lanner, an embryonic stem cell researcher at the Karolinska Institute and a coauthor on the newly published study that failed to find such stem cells.

We quite feel certain to say that in the human adult ovary in this cortex region, there is no cell that would be the oogonial stem cell.

Pauliina Damdimopoulou, Karolinska Institute

While some groups have been able to reproduce the results, others have tried and failed. Debates have erupted over methods, techniques, and protocols, and Tilly and his colleagues have published lengthy replies to those who have challenged their work. Today, the field is more or less divided into two camps regarding the existence of ovarian stem cells, says Chuva de Sousa Lopes.

To try to get to the bottom of the issue, Lanner and his collaborators harvested high-quality ovarian tissue samples from 21 healthy patients of reproductive age and isolated the ovarian cortex, the outer layer of the ovary where researchers claim to have found the elusive stem cells. The team used enzymes to break down the ovarian tissues, yielding 24,000 individual cells in total, then performed single-cell transcriptome and cell surface marker profiling, revealing six main cell types: oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. None of the single-cell profiles matched those of reported ovarian stem cells.

When Lanner and colleagues stained the cells with an antibody against DDX4, a germ cell marker that is reported to select for oogonial stem cells, they found that they had instead isolated perivascular cells. The team then stained intact ovarian tissue and saw that the antibody similarly identifies perivascular cells. A comparison of the 24,000 cells to existing transcriptome data from both human fetal ovaries and the ovarian medulla, the inner region of the ovary, also failed to reveal any oogonial stem cells.

We quite feel certain to say that in the human adult ovary in this cortex region, there is no cell that would be the oogonial stem cell, says coauthor Pauliina Damdimopoulou, a cell biologist at the Karolinska Institute. She believes that other researchers have succeeded in using the DDX4 isolation technique to select and culture cells, but that what they have found are in fact perivascular cells and not oogonial stem cells.

This study again highlights that the DDX4 isolation technique is not something that can be used to isolate oogonial stem cells, University of Adelaide cell biologist Keith Jones, who was not involved in the work but coauthored a 2016 papersuggesting that the same antibody does not isolate DDX4 positive cells, writes in an email to The Scientist. It brings into question the existence of such stem cells, and leads us back to the dogma that prevailed previously in the fieldthe adult ovary does not contain oogonial stem cells.

Damdimopoulou also notes that she and her colleagues found that small, mature oocytes can slip through the filtration process, and when cultured, may appear as if they had been generated from stem cells. We think [the oocytes] were there all along from the beginning, she says. The formation of new vasculature by perivascular cells surrounding these oocytes, Chuva de Sousa Lopes suspects, could trigger dormant egg cells to become active and then mature, which might explain the results published by other labs.

Perivascular cells dont undergo meiosis, perivascular cells dont express meiotic genes, perivascular cells dont express germ cell genes.

Jonathan Tilly, Northeastern University

Others are not ready to give up on the idea of ovarian stem cells just yet. Deepa Bhartiya, a stem cell biologist at the National Institute for Research in Reproductive Health in India who was not involved with the research, has been working with ovarian stem cells since 2010 and says that they can be easily detected. Research with sheep ovarian tissues has shown that simple scraping of [the] ovary surface can show the presence of stem cells amongst the ovary surface epithelial cells, she writes in an email to The Scientist. The problem with the new study out of Sweden, Bhartiya says, is the speed at which the researchers spun their cellsmuch too slow to isolate the stem cells, which due to their small size do not pellet down at lower speeds and are therefore unknowingly discarded. Bhartiya writes that the study used novel techniques, but revealed nothing new: if sample preparation is not properone will get negative data.

Tilly argues that there are numerous methodological problems with the study. He says that at this point four independent groups have reported on the existence of oogonial stem cells, showing that the cells can generate new oocytes in both somatic ovarian tissue and outside the body in culture, and that they can undergo complete meiosis, a germ cell-specific event. Perivascular cells dont undergo meiosis, perivascular cells dont express meiotic genes, perivascular cells dont express germ cell genes, he says.

What the field really needs, says Chuva de Sousa Lopes, is more communication among researchers. The scientists that claim there are stem cells in the ovary and the scientists that are against that are somehow not really talking to each other, she says. I wish there would be more open dialogue, because sooner or later all these populations [of cells] will be clarified . . . and things will be more clear.

M. Wagner et al., Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells,Nat Commun,doi:10.1038/s41467-020-14936-3, 2020.

Amy Schleunes is an intern atThe Scientist. Email her ataschleunes@the-scientist.com.

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Single-Cell Analysis of Ovarian Cortex Fails to Find Stem Cells - The Scientist

Organoids, iPSCs, and advanced cell models: Advancing discovery from basic research to drug discovery – Science Magazine

Various in vitro cell culture assays have been used for decades to evaluate disease pathology and uncover potential therapeutic treatments. Despite many successes with these models, they have critical shortcomings. Growing evidence suggests that models providing more predictive and translational observations are desperately needed. Researchers are now moving from reductionist, 2D monoculture assay models to more complex 3D cell models, such as organoids and induced pluripotent stem cell (iPSC) cultures, in order to better evaluate the dynamic interactions between cells in an environment more closely emulating that of the in vivo milieu, and to assess patient-specific phenotypic effects following drug treatment. Effective, well-characterized, advanced cell models hold promise for improving our understanding of disease pathology and progression, and are critical for the identification of novel therapeutic targets.

During this roundtable webinar, the speakers will:

This webinar will last for approximately 60 minutes.

STEMCELL TechnologiesCambridge, UK

Dr. Simmini is an R&D scientist in the gastrointestinal biology group at STEMCELL Technologies. His group focuses on developing products that support the generation of 3D gastrointestinal organoid cultures both from human primary tissue and human induced pluripotent stem cells. Prior to joining STEMCELL Technologies in 2016, he obtained his Ph.D. in stem cells, developmental biology, and cancer at the University of Utrecht in The Netherlands. During that time, he conducted research with the group of Jacqueline Deschamps at the Hubrecht Institute in Utrecht, where he investigated the molecular mechanisms controlled by transcription factor CDX2 in adult mouse intestinal stem cells and during embryonic development. In 2015, he began postdoctoral research, joining the group of Jan Paul Medema and Louis Vermeulen at the Amsterdam Medical Centre in Amsterdam, where he investigated mechanisms regulating intestinal stem cell proliferation and differentiation in colorectal cancer. He is currently involved in several Horizon 2020 European Research Council projects in different roles: researcher within the INTENS (INtestinal Tissue ENgineering Solution) consortium; partner in the SINERGIA (Advanced technologieS for drug dIscovery and precisioN mEdicine: in vitRo modellinG human physiology and diseAse) project; and supervisor and member of the executive board of the Organovir-ETN (Organoids for Virus Research-European Training Network) grant.

Wellcome Sanger InstituteCambridge, UK

As a staff scientist at the Wellcome Sanger Institute, Dr.Hale undertakes basic research projects into hostbacterial interactions while also teaching relevant skills to students and visiting scientists. Her projects include growing and differentiating human induced pluripotent stem cells to either a macrophage-like lineage or as intestinal 3D organoids, then utilizing them to investigate pathogen interactions. The main techniques used are flow cytometry, confocal imaging, high-throughput Cellomics assays, Luminex cytokine assays, and cell culture. The pathogens have varied over the years, but have included Salmonella, Klebsiella, enteropathogenic Escherichia coli (EPEC), Chlamydia, and Leishmania.

UK Dementia Research InstituteCambridge, UK

Dr. Avezov received his Ph.D. in cell research and immunology from the George S. Wise Faculty of Life Sciences at Tel Aviv University in 2010. He conducted his postdoctoral work at the University of Cambridge Wellcome-MRC Institute of Metabolic Science and the Cambridge Institute for Medical Research until 2017 with David Ron, FRS. Quantitative cell biology in the context of human disease has been at the core of Dr. Avezovs research. Working at the interface of biomedical research, physics, and mathematical sciences, he developed the cross-disciplinary expertise for probing intracellular chemical and physical processes in real time. This enabled discoveries of unexpected features of the endoplasmic reticulum (ER), such as an active ER luminal transport mechanism. These findings provide insights into the roles of the ER and its morpho-regulation in neuronal (patho)physiology. Dr. Avezov is currently a UK Dementia Research Institute Group Leader running an interdisciplinary program that seeks to understand early contributions of fundamental cellular processes ranging from ER transport to neurodegeneration.

Science/AAASWashington, D.C.

Dr. Oberst did her undergraduate training at the University of Maryland, College Park, and her Ph.D. in Tumor Biology at Georgetown University, Washington D.C. She combined her interests in science and writing by pursuing an M.A. in Journalism from the Philip Merrill College of Journalism at the University of Maryland, College Park. Dr. Oberst joined Science/AAAS in 2016 as the Assistant Editor for Custom Publishing. Before then she worked at Nature magazine, the Howard Hughes Medical Institute, The Endocrine Society, and the National Institutes of Mental Health.

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Organoids, iPSCs, and advanced cell models: Advancing discovery from basic research to drug discovery - Science Magazine

Stem Cell Therapy Market Opportunity Analysis and Industry Forecast up to 2017 2025 – Jewish Life News

TMRR, in its recent market report, suggests that the Stem Cell Therapy market report is set to exceed US$ xx Mn/Bn by 2029. The report finds that the Stem Cell Therapy market registered ~US$ xx Mn/Bn in 2018 and is spectated to grow at a healthy CAGR over the foreseeable period.

The Stem Cell Therapy market research focuses on the market structure and various factors (positive and negative) affecting the growth of the market. The study encloses a precise evaluation of the Stem Cell Therapy market, including growth rate, current scenario, and volume inflation prospects, on the basis of DROT and Porters Five Forces analyses. In addition, the Stem Cell Therapy market study provides reliable and authentic projections regarding the technical jargon.

In this Stem Cell Therapy market study, the following years are considered to project the market footprint:

The content of the Stem Cell Therapy market report includes the following insights:

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On the basis of solution, the global Stem Cell Therapy market report covers the following solutions:

Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

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The Stem Cell Therapy market study answers critical questions including:

All the players running in the global Stem Cell Therapy market are elaborated thoroughly in the Stem Cell Therapy market report on the basis of R&D developments, distribution channels, industrial penetration, manufacturing processes, and revenue. In addition, the report examines, legal policies, and comparative analysis between the leading and emerging Stem Cell Therapy market players.

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Stem Cell Therapy Market Opportunity Analysis and Industry Forecast up to 2017 2025 - Jewish Life News

The very interesting life of the Hydractinia – Jill Lopez

A little-known ocean-dwelling creature most commonly found growing on dead hermit crab shells may sound like an unlikely study subject for researchers, but this animal has a rare ability -- it can make eggs and sperm for the duration of its lifetime. This animal, calledHydractinia, does so because it produces germ cells, which are precursors to eggs and sperm, nonstop throughout its life. Studying this unique ability could provide insight into the development of human reproductive system and the formation of reproductive-based conditions and diseases in humans.

"By sequencing and studying the genomes of simpler organisms that are easier to manipulate in the lab, we have been able to tease out important insights regarding the biology underlying germ cell fate determination -- knowledge that may ultimately help us better understand the processes underlying reproductive disorders in humans," Dr. Andy Baxevanis, director of the National Human Genome Research Institute's (NHGRI) Computational Genomics Unit and co-author of the paper. NHGRI is part of the National Institutes of Health.

In a study published in the journalScience, collaborators at NHGRI, the National University of Ireland, Galway, and the Whitney Laboratory for Marine Bioscience at the University of Florida, Augustine, reported that activation of the geneTfap2in adult stem cells inHydractiniacan turn those cells into germ cells in a cycle that can repeat endlessly.

In comparison, humans and most other mammals generate a specific number of germ cells only once in their lifetime. Therefore, for such species, eggs and sperm from the predetermined number of germ cells may be formed over a long period of time, but their amount is restricted. An international team of researchers have been studyingHydractinia's genome to understand how it comes by this special reproductive ability.

Hydractinialives in colonies and is closely related to jellyfish and corals. AlthoughHydractiniais dissimilar to humans physiologically, its genome contains a surprisingly large number of genes that are like human disease genes, making it a useful animal model for studying questions related to human biology and health.

Hydractiniacolonies possess feeding polyps and sexual polyps as a part of their anatomy. The specialized sexual polyps produce eggs and sperm, making them functionally similar to gonads in species like humans.

During human embryonic development, a small pool of germ cells that will eventually become gametes is set aside, and all sperm or eggs that humans produce during their lives are the descendants of those original few germ cells. Loss of these germ cells for any reason results in sterility, as humans do not have the ability to replenish their original pool of germ cells.

In a separate study, Dr. Baxevanis at NHGRI and Dr. Christine Schnitzler at the Whitney Lab have completed the first-ever sequencing of theHydractiniagenome. In this study, researchers used this information to scrutinize the organism's genome for clues as to why there are such marked differences in reproductive capacity between one of our most distant animal relatives and ourselves.

"Having this kind of high-quality, whole-genome sequence data in hand allowed us to quickly narrow down the search for the specific gene or genes that tellHydractinia's stem cells to become germ cells," said Dr. Baxevanis.

The researchers compared the behavior of genes in the feeding and sexual structures ofHydractinia. They found that theTfap2gene was much more active in the sexual polyps than in the feeding polyps in both males and females. This was a clue that the gene might be important in generating germ cells.

The scientists next confirmed thatTfap2was indeed the switch that controls the process of perpetual germ cell production. The researchers used the CRISPR-Cas9 gene-editing technique to removeTfap2fromHydractiniaand measured the resulting effects on germ cell production. They found that removingTfap2fromHydractiniastops germ cells from forming, bolstering the theory thatTfap2controls the process.

The researchers also wanted to know ifTfap2was influencing specific cells to turn into germ cells. Their analysis revealed thatTfap2only causes adult stem cells inHydractiniato turn into germ cells.

Interestingly, theTfap2gene also regulates germ cell production in humans, in addition to its involvement in myriad other processes. However, in humans, the germ cells are separated from non-germ cells early in development. Still, despite the vast evolutionary distance betweenHydractiniaand humans, both share a key gene that changes stem cells into germ cells.

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The very interesting life of the Hydractinia - Jill Lopez

Intrinsic disorder controls two functionally distinct dimers of the master transcription factor PU.1 – Science Advances

INTRODUCTION

Eukaryotic transcription factors are highly enriched in intrinsically disordered regions (IDR), which are sequences that do not adopt a stably structured conformation but are nevertheless essential for activity. Compared with only ~5% in prokaryotes and archaea, more than 80% of eukaryotic transcription factors have extended IDRs (1). In the unicellular bakers yeast (Saccharomyces), transcription factors comprise the most prodigious functional category of disorder-encoding proteins (2). In multicellular organisms, ~50% of all residues in eukaryotic factors from model animals (humans, Drosophila) and plants (Arabidopsis) map to disordered regions (3). Clearly, IDRs constitute a major component in eukaryotic gene regulation, and it is therefore important to define their contributions to the molecular properties of transcriptional factors.

While IDRs are generally diverse in sequence, charge characteristics confer specific properties to transcription factor IDRs. For example, positively charged tails mediate diffusion along DNA (4) and ubiquitination by E3 ligases of several transcription factors, notably p53 (5). More common, however, are negatively charged (acidic) IDRs such as transactivation domains, which recruit basal factors such as TFIIB and TATA binding protein to the promoter (6, 7), and signaling moieties such as PEST domains that are rich in Glu and Asp residues (8, 9). While IDRs exhibit sequence-dependent conformational preferences on their own, these preferences are also modified by folded domains to which they are tethered (10). In transcription factors, IDRs are highly enriched around DNA binding domains (DBDs) (11), which display electrostatically biased surfaces to their surroundings. Their DNA contact surfaces are typically rich in positively charged residues while exposing neutral or even negatively charged residues elsewhere. Because DBDs alone represent an incomplete context in functional regulation, our aim is to elaborate the mechanism by which charged, particularly acidic IDRs regulate the recognition of tethered DBDs with each other as well as with target DNA.

As a model system for understanding the impact of intrinsically disordered tethers in transcription factors, the ETS family protein PU.1 exemplifies the most common (known as type I) configuration (3), in which its eponymous DBD of ~90 residues comprises the only well-folded structure. The remaining ~170 and 12 residues that flank N- and C-terminally, respectively, are intrinsically disordered sequences. The extended N-terminal IDR consists of an acidic transactivation domain (human residues 1 to 80), a Q-rich domain (residues 81 to 116), and a highly negatively charged PEST domain (residues 117 to 165), all of which are characteristic disordered regions in eukaryotic factors (9). This domain architecture, conserved among PU.1 orthologs, commends PU.1 as an ideal model from which more complex transcription factor architectures may be approached.

In addition to the canonical attributes representative of eukaryotic transcription factors, PU.1 is also specifically required for life. During hematopoiesis, all circulating blood cells are ultimately derived from a small population of self-renewing stem cells. PU.1 is a master regulator that is required for the renewal of the hematopoietic stem cells (12) and, in collaboration with other factors, directs their differentiation to every major myeloid and lymphoid lineage. Aberrant PU.1 activity is associated with lymphomas (13), myeloma (14), leukemias (15), and Alzheimers disease (16). Most recently, PU.1 was also identified as the key trigger for tissue fibrosis (17). Genetic and pharmacologic interventions targeted at PU.1 have established its therapeutic potential in acute myeloid leukemia (18, 19) and fibrotic diseases (17). Mechanisms that govern the molecular interactions of PU.1 are therefore relevant to developmental genetics and multiple therapeutic areas including hematology/oncology, immunology, neurology, and rheumatology.

Despite its biological significance, detailed knowledge of the molecular properties of PU.1 has been limited to its structured ETS domain. PU.1 therefore exemplifies the incomplete context problem in structural biology, which we have now tackled by addressing the role of the N- and C-terminal IDRs in the behavior of the ETS domain. The data reveal that these tethered IDRs critically control the propensities of the ETS domain to form discrete dimers with and without cognate DNA. These dimeric states, which are conformationally distinct, establish a novel regulatory mechanism that enables negative feedback in PU.1 transactivation. In addition to implications on PU.1 autoregulation in vivo, these results address a general class of problems in which negatively charged IDRs, which are abundant in transcription factors as transactivation and other functional domains, exert direct functional control at the protein/DNA level.

The DNA binding (ETS) domain of PU.1 represents its only structured domain, whose 1:1 complex with cognate DNA (Fig. 1A) is structurally conserved in this family of transcription factors. However, in contrast with other ETS members, the ETS domain of PU.1 (N165) forms 2:1 complexes at single DNA cognate sites in biophysical assays (20, 21). Measurements of self-diffusion by protein-observed diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) showed that inclusion of the N-terminal PEST domain (N117) maintained the DNA binding modes accessible to the ETS domain (Fig. 1B). Specifically, DOSY titrations of both N117 and N165 with DNA oligomers harboring a single cognate binding site showed two distinct bound states, with the minima in diffusion coefficient occurring sharply at a DNA:protein ratio of 0.5, corresponding to a 2:1 complex. The single minima at DNA:protein = 0.5 excluded the formal possibility of a 2:2 complex or nonspecific binding. If PU.1 were binding DNA nonspecifically beyond the 1:1 complex at equilibrium (i.e., in an unsaturable manner proportional to the concentration of free protein), then the minima in diffusion coefficient would occur at the lowest DNA:protein ratios where protein would be at greatest excess relative to DNA. Independently, protein-into-DNA titrations showed that N117 enhanced the affinity of the 1:1 complex (KD1) by more than twofold and reduced the affinity of the 2:1 complex (KD2) relative to N165 by about fourfold (Fig. 1B). Taking the ratio KD2/KD1 as an index of cooperativity in DNA binding, 2:1 complex formation by N117 was therefore more negatively cooperative than N165. The PEST domain, therefore, preserved the intrinsic binding modes of the ETS domain of PU.1, namely, a 1:1 and 2:1 complex with cognate DNA, while modulating their affinities in solution.

(A) The ETS domain (N165) is the only structured and minimal DNA binding unit (PDB: 1PUE). (B) Left: DOSY NMR titrations of N117 and N165 with cognate DNA yielding equivalence points at DNA:protein = 0.5 and 1.0, corresponding to 2:1 and 1:1 complexes, respectively. The absence of single global minima at DNA:protein = 1:1 formally excludes the possibility of a 2:2 complex. Right: Fluorescence anisotropy titrations with labeled cognate DNA. Both N117 and N165 form a 2:1 complex at a single DNA site with different negative cooperativity as defined by the ratio of the two sequential dissociation constants KD1 and KD2 (dashed lines; see Materials and Methods). Parametric values are given in table S1. (C) Scheme of negative feedback in PU.1 trans-regulation. A mechanistic link between dimerization and negative feedback predicts a reduction in PU.1 activity under conditions permissive of an inactive 2:1 complex. (D) Synthetic PU.1-dependent EGFP reporters. A minimal TATA box was driven by enhancers composed only of tandem EBS (yellow blocks) spaced 20 bp apart. Hatched blocks represent mutated sites. (E) Representative flow cytometric data of untreated HEK293 cells and upon transfection with a constant dose of the 5EBS reporter and/or up to 25 ng of an expression plasmid encoding full-length PU.1 (see Materials and Methods). Quadrant Q2 contained the EGFP-positive cells to be counted out of all PU.1-expressing cells (Q2 + Q3). (F) EGFP fluorescence in Q2 taken over the summed fluorescence in Q2 + Q3 at 24 hours after cotransfection of the EGFP reporter plasmid and the indicated dose of PU.1 expression plasmid. Each data point represents the means SE of triplicate or more samples. (G) RT-PCR measurements of pu.1, csf1ra, and e2f1 mRNA abundance (relative to gapdh) in THP-1 cells induced with PMA following exposure to either doses of a PU.1 inhibitor for 2 hours (left) or a fixed dose of 20 M inhibitor for various periods (right). Cells were visualized at 40 magnification after Giemsa staining.

In tissues that natively express PU.1, such as macrophages, PU.1 activity is highly inducible (22). The 1:1 complex formed by ETS domains represents the established trans-regulatory complex for ETS transcription factors. Little is understood about the functional nature of the 2:1 complex, for which no ETS analog is known, although its negative cooperative relationship with the 1:1 complex suggests an inactive species (Fig. 1C). To solve this puzzle, we measured PU.1 transactivation in cells using an enhanced green fluorescent protein (EGFP) reporter gene under the control of various synthetic enhancer elements consisting only of tandem copies of the B motif (Fig. 1D), a PU.1-specific ETS binding site (EBS) derived from the lymphoid Ig2-4 enhancer (GenBank X54550). Each consecutive site was spaced by 20 base pairs (bp), or two helical turns, such that bound proteins were arrayed on the same helical face to facilitate the recruitment of the transcriptional machinery. In addition, as the 2:1 complex was known to require an extended site size relative to the monomer (20), presenting the bound protein along one helical face would amplify site-site interactions and DNA perturbations, thus rendering most manifest the functional effects of the 2:1 complex.

When transiently transfected into PU.1-negative human embryonic kidney (HEK) 293 cells, the reporters were negligibly activated by endogenous transcription factors, including other ETS family proteins (Fig. 1E). Cotransfection of an expression plasmid encoding full-length PU.1, which was independently tracked by a cotranslating infrared fluorescent protein (iRFP) marker, yielded EGFP fluorescence in a dose-dependent manner. We established a dosing range for the PU.1 expression plasmid that gave a linear variation in PU.1 abundance in HEK293 cells within the physiologically inducible range found in PU.1-expressing myeloid cells (fig. S1). In this configuration, PU.1-dependent transactivation was quantified as the fraction of iRFP-positive cells that were also EGFP positive (Fig. 1E). The functional outcome of an inactive, negatively cooperative 2:1 complex would be a bell-shaped reporter dose-response as the enhancer, which varied in density and spacing of EBS (i.e., cis-regulatory syntax), became saturated with nonproductively (2:1) bound PU.1. In the alternative, the reporter signal would dose-dependently settle to a saturable level, depending on the level at which the 2:1 complex retained activity relative to the 1:1 complex. The synthetic B reporters were therefore well suited to interrogate cellular PU.1 activity, free from the requirement or interference from other promoter-specific cofactors, at the protein/DNA level.

At equivalent PU.1 doses, all enhancer configurations showed graded reporter expression in step with the density of EBS at each enhancer (Fig. 1F). This was consistent with an expected multivalent effect with respect to PU.1 binding sites. However, EGFP expression increased monotonically only with enhancers harboring tandem 3 and 5 EBS. Upon peaking at intermediate PU.1 doses, the 1 and 2 enhancers were repressed by further increases in PU.1. To determine whether the reversal in transactivation involved PU.1 interactions at the enhancer, we mutated the even-numbered sites in the 5EBS reporter to generate a 3EBS variant in which the cognate sites doubled in spacing (Fig. 1D). The resultant 3-alt-EBS reporter exhibited lower transactivation than the more densely spaced 3EBS, and its reporter signal also no longer increased monotonically (Fig. 1F). The spacing effect, therefore, demonstrated that the functional reversal could not be due solely to PU.1 interactions away from the DNA, which would be inert to syntax changes at the DNA. The observation of bell-shaped dose response for the 1 and 2 enhancers, but not the 3 or 5EBS enhancers, suggested additive perturbations of the local DNA structure, which were amplified by the helical spacing of the sites. This interpretation was supported by previous DNA footprinting of the PU.1 ETS domain, which showed strong differences between the singly and doubly PU.1-bound DNA (20). Alternatively, binding at the higher-density sites might exhaust a required co-repressing factor for the 2:1 complex. However, this possibility was discounted by the different dose responses exhibited by the 3EBS and 3-alt-EBS, which had the same site density, and the occurrence in a cell line (HEK293) that does not natively use PU.1 in gene regulation. Because net transactivation activity was reduced under conditions corresponding to population of the 2:1 complex, the evidence suggested that the 2:1 complex lost activity relative to the 1:1 complex. Thus, manipulation of enhancer syntax (density and spacing) demonstrated negative feedback in PU.1 transactivation in a manner consistent with self-titration of the transcriptionally active 1:1 complex by an inactive dimer bound to DNA.

To extend our functional results to a more physiologic context, we evaluated the impact of graded PU.1 inhibition on three PU.1 target genes in THP-1 cells, a widely used human monocyte/macrophage model. Cells were treated with a PU.1 inhibitor (fig. S2), as a function of dose or incubation period, before stimulation with phorbol 12-myristate 13-acetate (PMA) to mimic PU.1 induction during myeloid differentiation. As PU.1 targets, we examined the pu.1 (Spi-1) gene itself, which is autoregulated (23); csf1ra, a PU.1 target that encodes the subunit of the colony-stimulating factor receptor; and e2f1, which is negatively regulated by PU.1 (24). We first tested the effect of dose-dependent inhibition of PU.1 for a fixed period of 2 hours on the transcription of these genes by reverse transcription polymerase chain reaction (RT-PCR) (table S2). Expression of pu.1 and csf1ra, both positively regulated PU.1 targets, was increased by lower doses of inhibitor before marked reduction to ~50% at higher doses, yielding bell-shaped profiles (Fig. 1G). In the case of negatively regulated e2f1, expression was further inhibited across the dosage range of inhibitor tested. Trans-regulation of all three genes upon dose-dependent inhibition of PU.1 was consistent with an increase in PU.1 activity associated with the relief of negative feedback.

To assess the impact of PU.1 inhibition temporally, we tested a fixed dose of inhibitor (20 M) over time, up to 16 hours before PMA induction. While PU.1 expression gave a bell-shaped dose response at 2 hours of inhibitor exposure, continued exposure at an intermediate (derepressing) dose became strictly inhibitory (Fig. 1G). In contrast, derepression in csf1ra expression continued for 8 hours. Expression of the negative-regulated e2f1 gene, which was dose-dependently reduced at 2 hours of PU.1 inhibition, began to increase by 8 hours of inhibitor exposure. These results thus demonstrated a dynamic nature to the negative feedback that corresponded to the specific effect of PU.1 on the target gene (peaks in activated genes or troughs in repressed genes). The opposing behavior of csf1ra and e2f1 expression, in accordance to their opposite dependence on PU.1, supported the physiologic relevance of PU.1 negative feedback. Last, the latency exhibited by the two target genes relative to the autoregulated pu.1 gene suggested a combined effect between changes in PU.1 availability at the expression level and competition for binding at the DNA level.

In summary, the expression profiles of pu.1, csf1ra, and e2f1 showed that graded PU.1 inhibition led to nonmonotonic changes in trans-regulatory activity in a manner consistent with derepression of negative feedback. Together with the dependence of the synthetic B reporter on PU.1 dose and enhancer syntax (site density and spacing), the data support the biophysically observed 2:1 complex as a functionally relevant species in the cell and motivate specific interest in how the ETS domain dimerizes in its native structural context.

Comparison of DNA binding by N117 and N165 shows that the N-terminally tethered PEST domain enhanced the affinity of the 1:1 complex but reduced the affinity of the 2:1 complex (Fig. 1B). To better understand the influence of the PEST domain on DNA recognition by PU.1, we first established whether the PEST domain was disordered in the cognate complex by comparing the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) fingerprint region of DNA-bound N165 and N117 (Fig. 2A). As with N165 (20), the unbound and 1:1 complex gave well-dispersed spectra, while >80% of the cross peaks for 2:1-bound N117 were broadened out (fig. S3). The similar behavior by the two constructs indicated that broadening was not due to the larger size of the 2:1 complex, in which case broadening would be exacerbated for N117. In 1:1-bound N165, whose resonances were well resolved, 88 of the 95 assigned residues overlapped with N117, with all PEST residues clustered around 8.2 0.2 parts per million (ppm) on the 1H dimension, a chemical shift characteristic of disordered structures. Because this region also represented the residues that were detected in HSQC of the 2:1 complex (fig. S3), the evidence suggested similar changes in the chemical environment for the structured ETS domain (represented by the dispersed resonances in intermediate exchange) between free and DNA-bound states of N117 and N165. Thus, the local structure of the ETS domain was not altered upon DNA binding by the flanking residues, and the PEST domain behaved as a disordered tether in the ETS/DNA complex.

(A) 1H-15N HSQC of N117 and N165 in the 1:1 complex with cognate DNA. Assignment of the N165 spectrum was 90% complete. (B) DNA binding by N165 and N117 in 0.1 M and 0.05 M NaCl, showing the impact of the PEST domain on the cooperativity of 2:1 complex formation. Parametric values of the equilibrium dissociation constants are given in table S1.

Ligand binding to DNA is generally sensitive to electrostatic interactions. To better understand the impact of the disordered PEST domain on 2:1 complex formation, we probed the electrostatic contribution to site-specific binding by N165 and N117 (Fig. 2B). Reducing Na+ concentration from 0.15 M (as shown in Fig. 1B) to 0.10 M did not affect 2:1 binding by N117. However, the biphasic binding indicative of strongly negatively cooperative formation of the 2:1 complex for N165 was abolished as the anisotropy values showed. A further reduction to 0.05 M salt resulted in monophasic transitions to the 2:1 complex by both constructs. Notably, binding weakened with decreasing Na+ concentration and therefore could not reflect simple electrostatic effects on DNA binding. These observations indicated that additional unbound species must regulate DNA recognition by PU.1 and that these species were salt sensitive and controlled by the disordered PEST domain.

The isolated ETS domain, N165, forms a feeble dimer without DNA, as judged by heteronuclear NMR (25) as well as static and dynamic light scattering (21). To determine the role of the disordered PEST domain in PU.1 dimerization without DNA, we examined several hydrodynamic parameters, which are highly sensitive to self-association, of N117 as a function of concentration (Fig. 3A). DOSY NMR spectroscopy revealed a marked concentration dependence for the apparent diffusion coefficient. The profile was described by a two-state monomer-dimer equilibrium (detailed in Materials and Methods) with a dissociation constant below 10 M (table S1). To assess concentrations below 50 M, which was limiting for NMR, we performed intrinsic Trp fluorescence anisotropy measurements, which is sensitive to rotational diffusion. N117 exhibited a substantial change in steady-state anisotropy that was also described by a two-state dimer with a dissociation constant at below 10 M. In contrast, N165 showed no change. The localization of the three Trp residues in the structured ETS domain of both constructs represented further evidence that the concentration-dependent changes in N117 involved the ETS domain. Last, high-precision densimetry showed a concentration-dependent transition by N117 that was again described by two-state dimerization. (Because density varies directly with concentration, density-detected transitions sit on sloped baselines as opposed to the flat baselines in spectrometric titrations.) Relative to the diffusion probes, the densimetric titration gave a higher dissociation constant, 35 15 M. As a control, N165 gave a concentration-independent partial specific volume (from the slope, see Materials and Methods) of 0.77 0.01 ml/g, a value characteristic of structured globular proteins. Multiple orthogonal probes therefore described a reversible N117 dimer that was considerably more avid than N165.

(A) Concentration-dependent changes in hydrodynamic and volumetric properties by DOSY NMR, intrinsic Trp fluorescence anisotropy, and high-precision densimetry of N117 in 0.15 M Na+ at 25C. Red curves represent fits of the data to a two-state monomer-dimer transition. (B) Representative zero-charge ESI mass spectra of N117 at 13 and 840 M total concentration, normalized to the height of the monomer (17 kDa) peak. The ratios of the integrated dimer-to-monomer intensities (molecular weight shown) were French-curved to guide the eye. (C) Far-UV CD spectra of N117 and N165 at 25 M, plotted on a per-molecule basis to highlight the contribution of the N-terminal residues. (D) Concentration-dependent, per-residue spectra of N117 and N165 (left). Dimerization as revealed by singular value decomposition of the N117 spectra and fitted to a two-state transition. (E) 1H-15N HSQC of 400 M N117 and N165 at 0.15 NaCl. Under these conditions, N117 was predominantly dimeric and N165 was monomeric. The assignments shown are for N117. Inset: {1H}15N-NOE for N117 and N165.

We pause to note that concentration dependence of the equilibrium constant (and melting temperatures) rules out monomolecular interactions, such as conformational changes without association. Local conformational changes can and do produce changes in diffusion and volumetric parameters, but this behavior without an intermolecular component cannot depend on total concentration at thermodynamic equilibrium. Artefacts such as aggregation during the experiments are unlikely based on the linear posttransition baselines for all three probes (DOSY NMR, fluorescence anisotropy, and density). Independent evaluation of purified N117 by SDSpolyacrylamide gel electrophoresis (PAGE) and mass spectrometry (MS) (fig. S4) also confirmed the absence of detectable contamination and aggregation. At a deeper level of analysis, the two-state self-association model, given by Eq. 7 in Materials and Methods that fitted the titration data in Fig. 3A, is an nth-order polynomial, where n is the stoichiometry of the oligomer. The value of n (= 2 for dimer), which is fixed in the fitting, imposes a severe constraint on the shape of the titration to which the model may adequately fit. As detailed elsewhere (26), oligomers n 3 invariably show sigmoidal (S-shaped) transitions. Only a two-state dimer exhibits nonsigmoidal profiles on linear concentration scales, precisely as constructed in Fig. 3A and observed in the data. On this basis, the biophysical evidence is unambiguous in showing homodimerization of PU.1 without DNA, and the range of dissociation constants yielded by the different probes reflected the distinct molecular properties they sampled.

To further strengthen this evidence, we resolved N117 by electrospray ionization (ESI)MS up to a concentration of 840 M. Using an established maximum entropy procedure (27), peaks corresponding to both monomeric and dimeric PU.1 were observed in deconvoluted zero-charge mass spectra (Fig. 3B). The integrated intensities of the two species were quantitative, but they did not correspond to solution conditions in the other experiments. This was due to the techniques requirement for a volatile buffer (NH4HCO3), species-dependent ionization efficiency, and the potential for ionization-induced dissociation of the complex. Notwithstanding, the ratio of dimer-to-monomer intensities varied in favor of the dimeric species with increasing total protein concentration (Fig. 3B, bottom). The concentration dependence excluded the possibility that either species could represent a static contaminant but rather corresponded to a N117 monomer and dimer at dynamic equilibrium.

To gain insight into the conformational structure of the free PU.1 dimer, we interrogated N165 and N117 by circular dichroism (CD) and NMR spectroscopy. At an identically low concentration (25 M), a net contribution of coil content due to the PEST domain was apparent (Fig. 3C). With increasing concentration, N165 showed a spectral shift but without an endpoint at 300 M. In contrast, the corresponding spectra for N117 (weighted by contributions from the disordered PEST domain) underwent a nonsigmoidal transition that, unlike N165, was substantially completed at 300 M (Fig. 3D). Model fitting of the farultraviolet (UV) CD spectra, which are sensitive to secondary structure content, to a two-state dimer yielded a dissociation constant of K2 = 46 19 M. As an analysis of full CD spectra by singular value decomposition rendered more structural information than the other titration probes in Fig. 3A, we will use the CD-fitted K2 for comparison with other PU.1 constructs and solution conditions.

To probe the local structure of the PU.1 dimer, we compared the 1H-15N HSQC fingerprint of 400 M N117 and N165, concentrations at which the preceding experiments showed that N117 was predominantly dimeric, while N165 remained monomeric (Fig. 3E; compare to Fig. 3A). Dispersed cross-peaks for the two constructs mostly overlapped within experimental uncertainty (inset). PEST domain residues were clustered at 8.2 0.2 ppm. {1H}15N-NOE (nuclear Overhauser effect) measurements confirmed that the ETS residues in N117 remained well ordered throughout, similarly as N165, while PEST residues exhibited much lower values as a group (Fig. 3E, inset). Thus, the N117 dimer was a fuzzy complex in which the PEST domain did not deviate from a tethered IDR to the structured ETS domain.

The 2:1 complex formed by PU.1 at a single cognate site suggested that the PU.1 dimer was asymmetric, as a symmetric dimer that exposes the DNA contact surfaces would logically yield a 2:2 complex. However, this stoichiometry was excluded by the DOSY titration data, which showed two inflections with the least diffusive species at a DNA:protein ratio of 1:2, corresponding to the 2:1 complex (Fig. 1B). Unbound PU.1 also formed a homodimer, which could logically arise only if the complex was symmetric. Experimentally, a symmetric dimer was strongly inferred by a single set of 1H-15N signals for unbound N117 at high concentrations (Fig. 3E). Moreover, the CD-detected structure of PU.1 showed negligible changes upon titration by DNA (Fig. 4A), in contrast with the self-titration in the absence of DNA (Fig. 3D). These clues suggested that DNA-bound and free PU.1 dimerized into distinct conformers.

(A) Far-UV CD spectra of the DNA-bound N165 upon subtracting the spectrum of the cognate DNA acquired under identical conditions (75 M and 0.15 M Na+). (B) Residues involved in the DKCDK mutant and in the binding-deficient mutant (R230A/R233A). The structure is homology-modeled against the cocrystal 1PUE. (C) Purification of the DKCDK mutant by ion exchange chromatography under nonreducing conditions. Lysate was loaded at 0.5 M NaCl and extensively washed before elution over a linear gradient to 2 M NaCl. SDS-PAGE of purified fractions is shown. Fractions containing primarily monomer (e.g., 1 and 2) or dimers (e.g., 5 onwards) were concentrated and dialyzed separately into buffer containing 0.15 M NaCl with or without 5 mM DTT, respectively. (D) CD spectra of the DKCDK monomer (top) and dimer (bottom) under various conditions with wild-type N165 as reference. The spectrum for the DKCDK monomer was less well resolved due to the presence of DTT, which contributed to the total absorption of the sample at 50 M protein. See text for details. (E) Fluorescence anisotropy measurements of cognate DNA binding by monomeric and dimeric DKCDK with wild-type N165 as reference. (F) CD spectrum of 25 to 100 M of the R230A/R233A mutant, with N165 at 25 M as reference. (G) DNA loading by the R230A/R233A mutant in the presence of wild-type N165 (solid symbols). Concentrations of the mutant and wild-type protein that individually failed to bind DNA collaborated to bind DNA as a heterocomplex. (H) Proposed model for the formation of two nonequivalent PU.1 dimers: an asymmetric one in the 2:1 DNA complex and a symmetric one without DNA.

To test these notions, we constructed a constitutive ETS dimer via insertion of a single Cys residue into N165, which did not harbor this amino acid, between residues 194 and 195 (Fig. 4B). We targeted this position given its turn conformation in the known structures of the ETS monomer [Protein Data Bank (PDB): 5W3G] and the 1:1 complex (1PUE), and its reported involvement in 2:1 complex formation by heteronuclear NMR (20). Purification of this mutant, termed DKCDK, by ion exchange chromatography under nonreducing conditions eluted monomer and its cystine-linked dimer at >1 M NaCl (Fig. 4C). Fractions containing predominantly monomer or dimer were separately dialyzed into a buffer containing 0.15 M NaCl with or without 5 mM dithiothreitol (DTT), respectively. In the absence of DNA, the far-CD spectrum of the DKCDK monomer (maintained with 5 mM DTT) overlapped closely with the spectrum of N165 (Fig. 4D) and formed the 1:1 complex with cognate DNA similarly as wild-type N165, indicating that the Cys insertion was nonperturbative in the DKCDK monomer (Fig. 4E). In stark contrast, the cystine-linked DKCDK dimer exhibited a CD spectrum that was altogether unlike PU.1 at equivalent molar concentrations (400 M). It bore some similarity to a spectrum for N165 at the highest concentration available (800 M), which contained a greater fraction of dimeric PU.1 (dashed spectrum in Fig. 4D). However, the dimeric DKCDK spectrum was further redshifted by ~7 nm and ~15% more intense. Moreover, the DKCDK dimer bound cognate DNA >100-fold more poorly than wild-type N165 (Fig. 4E). Thus, the DKCDK mutant showed that a symmetric configuration was severely perturbed in conformation without DNA and unlike DNA-bound wild-type N165 (compare to Fig. 4A). Together with a deficiency in DNA binding, the DKCDK mutant demonstrated that the wild-type DNA-bound dimer was not a symmetric species in contrast with the unbound PU.1 dimer.

To assess the feasibility of an alternative, asymmetric configuration in forming the 2:1 complex, which would involve the DNA contact surface, we then examined an R230A/R233A mutant in the DNA-recognition helix H3 of PU.1 (Fig. 4B). The double RA mutant retained an indistinguishable CD spectrum as wild-type N165 (Fig. 4F). At a subsaturating concentration of wild-type N165, the addition of the mutant at a concentration that showed no DNA binding on its own nevertheless produced strong DNA loading (Fig. 4G). Such a result would most simply arise if the RA mutant associated with the wild-type 1:1 complex to drive the 2:1 heterocomplex. The data thus pointed to an asymmetric PU.1 dimer in the 2:1 complex, in which the secondary structure content of PU.1 did not change significantly. Both features contrast sharply with the symmetric conformation required by the DNA-free dimer.

A synthesis of the evidence leads us to propose a model for PU.1 dimerization in the presence and absence of DNA (Fig. 4H). In terms of affinity, the 1:1 active complex is strongly favored (>102-fold) over either the 2:1 complex or the unbound dimer. Excess PU.1 drives one or the other dimeric state depending on the presence of DNA. The key cornerstone of this model is the nonequivalence of the two dimeric states. Specifically, the incompatibility of the free dimer with DNA binding means that a preexisting dimer cannot serve as an intermediate for the 2:1 complex. Thermodynamic insulation of the two dimeric species leads to a mutually antagonistic relationship, in which the formation of one species is favored at the expense of the other. N117 illustrates this antagonism, as relative to N165, the N-terminal PEST domain promotes dimerization without DNA and reduces the affinity of 2:1 complex formation (Fig. 1B). Together with enhancing the apparent affinity for 1:1 binding, the result is a widened concentration window for the 1:1 complex for N117.

The ETS domain as embodied by N165 is highly enriched in Lys and Arg residues, with an isoelectric point (pI) of 10.5. Dimerization should, therefore, be highly sensitive to salt concentration. Contrary to the expectation that the dimer would be stabilized at high salt, which would screen electrostatic repulsion, the opposite was observed. CD-detected self-titration of N165 at 50 mM Na+ showed a nearly complete two-state transition (Fig. 5A) but not at 150 mM Na+ (compare to Fig. 3A). The low-salt spectra, extended in wavelength to 190 nm and protein concentration to 800 M because of the reduced Cl level, showed the same transition characteristics as acquired at 150 mM Na+ (fig. S5), indicating that the same transition was inspected at both salt concentrations. Although the transition at 50 mM Na+ corresponded to a dissociation constant of ~200 M, it was still fivefold higher than that for N117 in 150 mM Na+ (Fig. 5B). The data, therefore, reaffirmed the stimulatory role of the disordered PEST domain in dimerization of the ETS domain while revealing an electrostatic basis in the unbound PU.1 dimer.

(A) CD-detected titration of N167 at 50 mM NaCl from 25 to 800 M. (B) Analysis of the titration by singular value decomposition yields a two-state transition with a dissociation constant K2 of 202 72 M. (C) 1H-15N HSQC as a function of salt from 25 to 500 mM NaCl. Residues with the strongest 1H-15N CSPs (Y173, M223, G239, V244, and L248) are boxed. Inset: Salt dependence of the CSPs of these residues. (D) Summary of the residue CSPs with the average %SASA from the unbound NMR monomer, 5W3G. Residues above a 0.5-ppm cutoff are colored in dark blue, and the subset of internal residues (<35% SASA, based on the termini) is marked with yellow circles. Residues implicated in the DNA-bound dimer are marked with green circles. (E) Mapping of the high-CSP residues to 5W3G. Green residues mark known residues involved in the DNA-bound dimer (20). (F) Chemical shiftderived secondary structure prediction via the CSI using 1H and 15N signals. The color scheme follows the HSQC in (C). Regions with significant changes in secondary structure are marked by arrows. (G) Near-UV CD-detected thermal melting of N117 and N165. Two salt concentrations were evaluated for N165 (blue and gray). Inset: Representative near-UV CD spectra. (H) DSC thermograms (solid) for N165 under conditions (salt and concentrations) in which the protein was primarily monomeric or dimeric. The Cp values are given in kJ (mol monomer)1 K1. Dashed curves represent the two-state transition for a monomer (black) and dimer (blue). (I) Trp fluorescence-detected denaturation by urea of N117 and N165 at two monomer concentrations. Curves represent fit to the linear extrapolation model for a two-state dimer. The marked concentrations represent urea concentration at 50% unfolding.

The sensitivity of the ETS dimer to salt allowed us to access the local structural changes in the DNA-free ETS dimer by NMR spectroscopy. 1H-15N HSQC spectra of N165 with 0.5 to 0.025 M NaCl (Fig. 5C) revealed a panel of residues with significant chemical shift perturbations (CSPs). Taking the spectrum acquired in 0.5 M NaCl as the reference for monomeric PU.1, the CSPs exhibited a well-ordered salt dependence (Fig. 5C, inset). The salt-induced CSPs were plotted as a function of residues (Fig. 5D), and a cutoff of 0.05 was applied to identify the residues most affected by electrostatic interactions. These perturbed residues were spatially diffuse, as a formal mapping to the unbound PU.1 structure demonstrated (Fig. 5E) and did not overlap with the known residues involved in 2:1 complex formation (20). We also examined the transverse spin relaxation (T2) properties of the methyl proton peaks in the 1H spectra as a global representation of the tumbling of N165 at different NaCl concentrations (fig. S6). The effective T2* relaxation values for the three characteristic methyl 1H peaks at 0.025 M NaCl were up to ~25% lower than at 0.5 M NaCl and well beyond experimental error. This result indicated that the salt-induced CSPs reflected the formation of a slower tumbling dimer.

To correlate the NMR data with the CD-detected changes, we used the heteronuclear chemical shifts to infer secondary structure via the chemical shift index (CSI) (28). The CSI results corroborated the CD-detected loss of -helical and gain in /coil content and furthermore localized these changes to helix 1 (H1) and the loop between sheet 3 (S3) and sheet 4 (S4) (Fig. 5F). Local H1 unwinding accounted for the CSPs observed near the N terminus of N165, including the particularly strong CSP at Y173, while the loop between S3 and S4 gained -sheet structure.

The N-terminal IDR promotes a structurally perturbative PU.1 dimer in the absence of DNA. To reveal the underlying conformational thermodynamics of the PU.1 dimer, we performed thermal melting experiments over a range of protein concentrations, using the near-UV CD spectrum from 250 to 300 nm as a probe. The thermal transition was analyzed from a singular value decomposition of the full spectra at each concentration and fitted to a two-state model. The apparent melting temperature (Tm) dropped with increasing concentration in step with the propensity for dimer formation (Fig. 5G). N117 suffered a larger drop than N165 over a ~10-fold increase in concentration. A reduction in salt concentration, which drove dimerization, similarly caused a larger drop in Tm for N165 (0.15 versus 0.05 M Na+; Fig. 5G, dashed line).

The presentation of Fig. 5G as Tm1 versus the logarithm of concentration implies that steeper slopes correspond to lower enthalpies (heats) of dissociation/unfolding, which relate to the quality of conformational interactions. To rigorously define the conformational thermodynamics of the PU.1 dimer, we performed differential scanning calorimetry (DSC) experiments on N165 under conditions (salt and protein concentrations) where the quantitatively major population was either monomer or dimer (Fig. 5H, all values on a per-mole monomer basis). The thermograms showed a much greater calorimetric molar enthalpy (area under the curve) for the monomer (300 M at 0.15 M Na+) than dimer (500 M at 0.05 M Na+). In addition to enthalpy, DSC yields heat capacity changes (Cp, difference in the pre- and posttransition baselines) that inform on changes in water-accessible surface area. The N165 monomer exhibited a Cp of 3.1 0.3 kJ/(mol K), in good agreement with the structure-based value of 3.3 kJ/(mol K) from the NMR structure of the PU.1 monomer (29). In contrast, the N165 dimer exhibited a significantly reduced Cp of 0.97 0.27 kJ/(mol K). Assuming identical thermally unfolded states, the differences in heat capacity changes indicated that N165 was less well folded than the monomer.

To probe the effect of the N-terminal IDR on the conformational stability of the PU.1 ETS domain, we performed chemical denaturation experiments with urea, which could be reported by intrinsic tryptophan fluorescence at much lower protein concentrations than DSC. At a strictly monomeric concentration (1 M) at 0.15 M Na+, N117 was only slightly more sensitive to urea, as judged by the urea concentration at 50% unfolding, than N165 (Fig. 5I). At 100 M concentration, at which N117 is mostly dimeric but N165 remains monomeric, N117 became significantly more sensitive to urea, suggesting highly perturbative interactions between the PEST and ETS domains. A conformationally perturbed N117 dimer was also implied by its volumetric properties. The posttransition density slope in Fig. 3A yields a partial specific volume of N117 of 0.85 0.01 ml/g, which is atypically high for structured globular proteins and suggests altered molecular packing and hydration properties. N165 was more stable at 1 M at 0.05 M Na+ than at 0.15 M Na+, an observation consistent with the ~2C higher apparent Tm for 10 M N165 over the same Na+ concentrations (Fig. 5G). The N165 monomer and dimer were therefore opposite in their conformational stabilities with respect to salt, underlining the structure perturbation by salt or the anionic PEST domain.

(A) Schematic of the phosphorylated Ser residues in human PU.1, marked by green pins. The SerAsp substituted positions in D2N117 and D4N117 are shown. (B) D2N117 and D4N117 exhibit enhanced dimeric propensities without DNA relative to N117 (compare to Fig. 3D). (C) D2N117 and D4N117 are progressively impaired in 2:1 DNA complex formation. (D) The anionic crowders ovalbumin and BSA modulate DNA binding by N117 in a similar way as the phosphomimetic substitutions on N117 to an extent that correlates with their sizes and low pIs. Inset: Stoichiometric determination using 1 M DNA (first binding transition in the case of ovalbumin). The spacing of the ordinates is identical to main plots. The surface potentials of the structures were computed using the Adaptive Poisson-Boltzmann Solver (APBS) at 0.15 NaCl.

In summary, spectroscopic and calorimetric measurements showed that the PU.1 ETS dimer was destabilized with respect to unfolding relative to its monomeric constituents. Structural considerations aside, conformational destabilization contributes to the DNA binding deficiency of the apo ETS dimer. A destabilized dimeric state implied favorable concentration-dependent interactions within the unfolded ensemble over the folded state. The ability of the anionic PEST domain to promote formation of the unbound dimer in N117 therefore further suggests a basis in mitigating the electrostatic repulsion among the cationic ETS domains.

In addition to the N-terminal IDR, the structured ETS domain of PU.1 is also tethered at the C terminus to a shorter, 12-residue disordered segment (residues 259 to 270), as apparent in the unbound PU.1 monomer structure (fig. S7A) (29). Far-UV CD spectra at 0.15 M Na+ showed that hPU.1(117-258) and hPU.1(165-258), termed sN117 and sN165, respectively (fig. S7B), lacked the secondary structure changes characteristic of N117 and 165 across comparable concentrations (fig. S7C; compare to Fig. 3D). sN117 was also much less sensitive to urea over the same protein concentration range as N117, and sN165 showed no change relative to N165. In contrast to their dimeric deficiency without DNA, sN117 and sN165 were both intact with respect to dimerization with cognate DNA (fig. S7D). While sN117 formed 1:1 and 2:1 DNA complexes with the same affinities as N117 in 0.15 M NaCl within experimental error, sN165 was a significantly poorer DNA binder than N165 (table S1). In particular, 2:1 complex formation was less negatively cooperative for sN165, with the concentration window (KD2/KD1) for the 1:1 complex only ~65% that for N165 (fig. S7D). Last, unlike N165 at 0.05 M Na+, sN165 showed a negligible propensity to dimerize and exhibited biphasic binding with cognate DNA (fig. S7E; compare to Fig. 2B). The divergent impact of removing the C-terminal IDR on dimerization with and without DNA stood in clear agreement with our concept of nonequivalent dimeric states for PU.1 and the structural distinctiveness of the two states.

Characteristic of many IDRs flanking DBDs, the N-terminal PEST domain in PU.1 is highly enriched in Glu and Asp residues (pI 3.5), in sharp contrast with the positively charged DBD (pI 10.5) to which it is tethered. The foregoing structural and thermodynamic evidence strongly suggests that the acidic IDR interacts with the ETS domain and shifts it toward dimer formation. Functional studies have established a panel of Ser residues in the PEST domain, including residues 130, 131, 140, and 146 (human numbering), which are multiply phosphorylated in cells (30, 31). Phosphoserines at these positions would enhance the anionic charge density by a substantial amount from 11 (3 Asp + 8 Glu) to 17 (~1.5 per phosphoserine). Because these residues are disordered, we made phosphomimetic substitutions of these residues to Asp, generating a di-substituted (140 and 146, termed D2N117) and tetra-substituted mutant (termed D4N117), to probe their general charge-dependent effects (Fig. 6A). Far-UV CD spectra showed that the phosphomimetic substitutions progressively drove the affinity of the DNA-free dimer, and the resultant dimers appeared to harbor greater random coil content than their wild-type counterpart (Fig. 6B; compare to Fig. 3D). In DNA binding experiments, the di-substituted mutant D2N117 behaved approximately as wild-type N117, while the affinity of the 2:1 complex for the tetra-substituted mutant D4N117 was ~15-fold lower than that for wild-type N117 (table S1). Stimulation of the unbound dimer was therefore associated with a marked reduction in the affinity of the 2:1 complex by D4N117 (Fig. 6C). As a result, the selective effect on the 2:1 complex in D4N117 resulted in greater negative cooperativity (i.e., increasing KD2/KD1) in the dimerization of DNA-bound PU.1. In turn, the concentration window for the 1:1 complex widened more than fourfold for D4N117 relative to wild-type N117.

The reinforcing effects of multiple phosphomimetic substitutions in the disordered PEST domain strongly suggest that it influences the behavior of the ordered ETS domain via a generally electrostatic, nonstructurally specific mechanism. To further establish this notion, specifically the absence of dependence on structurally specific interactions, we tested the effect of crowding concentrations (in the range of 102 g/liter) of ovalbumin or bovine serum albumin (BSA) on DNA binding by N165 (Fig. 6D). These two anionic proteins share pIs (pI = 5.2 and 4.7 for albumin and BSA, respectively) that are close to the PEST domain (pI = 3.5) but present well-formed globular structures. If PEST/ETS interactions involved structurally specific interactions between the two domains, the anionic crowders should differ significantly from the PEST domain in their effects on DNA recognition by the ETS domain. DNA binding in the presence of up to 20% (w/v) ovalbumin showed little effect on the 1:1 complex (Fig. 6D, inset) while progressively decreasing the affinity of 2:1 binding. This behavior mirrored closely the phosphomimetic mutants, and similarly, the more pronounced biphasic appearance in the presence of ovalbumin was a result of the increased negative cooperativity and widening concentration window for the 1:1 complex. With BSA, an even more anionic crowder, the effect was correspondingly more pronounced. A concentration of 5% suppressed 2:1 binding at 105 M, an almost 105-fold molar excess of N165 over DNA. Only the 1:1 complex was formed (inset). In contrast, the neutral crowder PEG 8K preserved biphasic DNA binding (fig. S8A), showing that the effects of BSA and ovalbumin were not due to volume exclusion from crowding alone and highlighting the importance of charge. To test our models prediction that BSA would, therefore, promote the PU.1 dimer, we evaluated N165 labeled with 5-fluoroTrp by 19F NMR in the presence of BSA. The three tryptophan residues in N165 underwent distinct CSPs with 5% BSA under conditions that gave monomers in dilute solution (fig. S8B). These changes reflected conformational perturbations consistent with unbound dimer formation. Thus, phosphomimetic substitutions and acidic crowding supported nonmicrostructural electrostatic field interactions on the ETS domain as the basis of the PEST-stimulated dimerization in the absence of DNA.

PU.1 is a markedly inducible transcription factor during hematopoiesis and immune stimulation (22). Open-source repositories such as the Human Protein Atlas show that the expression of PU.1 varies among a panel of resting cell lines by ~25-fold. Independently, single-cell cytometry shows that the abundance of PU.1 transcript in unstimulated murine bone marrow cells ranges from less to 5% to ~50% that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (32), a housekeeping glycolytic enzyme that is present at ~70 M in the cell (33). Induction by ligands such as retinoic acid (34) or bacterial endotoxins (35) increases PU.1 expression another 10-fold or more. Depending on the combination of cell line, physiology, and the presence of stimulatory ligands, cellular PU.1 abundance varies by a multiplier comparable to the ratio of the two dissociation constants KD2/KD1 (102- to 103-fold) of the 1:1 and 2:1 PU.1/DNA complexes.

In patients and animal models, PU.1 dosage is well established as critical to hematopoietic physiology and dysfunction in vivo (15, 36). Dosage effects have been extensively defined in terms of expression, but relatively little is understood about direct dosage effects on transactivation at the protein/DNA level. In this study, manipulation of enhancer syntax in HEK293 cells, which do not use PU.1, demonstrated negative feedback in ectopic PU.1 trans-regulation, independent of modifying interactions with tissue-specific coactivators. The recapitulation of negative feedback, manifested by dose-dependent derepression of endogenous PU.1 in myeloid THP-1 cells, strongly supports functional relevance in native PU.1-dependent gene regulation. Characterization of the attributable species, a 2:1 DNA complex, revealed two nonequivalent dimeric states that are reciprocally controlled by DNA and the IDRs tethered to the structured DBD. Under physiologic salt conditions, structural alterations that bias unbound PU.1 toward dimerization (e.g., full phosphomimetic substitutions of the N-terminal IDR) oppose dimerization of DNA-bound PU.1. Conversely, alterations that abrogate PU.1 dimerization (e.g., truncation of the C-terminal IDR) promote the formation of the 2:1 DNA complex. Only at low salt conditions (e.g., 50 mM Na+) are the DNA-free and DNA-bound dimers both favored, and the 1:1 complex is not populated. The tethered IDRs do not appear to become part of the structured dimer but determine preference between the two dimeric states such that the DNA-free dimer remains essentially cryptic without both terminal IDRs.

If the 2:1 complex represents the structural basis of negative feedback, what functional role does the unbound PU.1 dimer play? The thermodynamic relationships among the various states accessible to PU.1 (Fig. 4H), critically the nonequivalent free and DNA-bound dimer, suggest a novel push-pull mechanism of PU.1 autoregulation by distinct pools of dimeric protein. By antagonizing the 2:1 complex, we postulate that the unbound dimer suppresses negative feedback and dynamically increases the circulating dose of transcriptionally active PU.1. This model affords for the first time a unifying basis for the PU.1-activating effects of PEST phosphorylation by casein kinase II and protein kinase C (31, 37, 38), as well as the PU.1-inactivating effects of phosphorylation inhibition by oncogenic transcription factors (39), by biasing PU.1 conformations toward or away from the unbound dimer.

Earlier in vitro studies reporting on dimers of the ETS domain (20, 21, 25), including our own, did not appreciate their functional significance. The solution NMR structure of the unbound monomer (5W3G) reflects the incomplete context afforded by the ETS domain alone (i.e., N165) at physiologic ionic strength (0.15 M Na+/K+). The dissociation constant for the PU.1 dimer in dilute solution (105 M) should not be misinterpreted as denoting a physiologically irrelevant interaction. The complex formed by PU.1 and its partner GATA-1 is functionally critical in cell lineage specification during myeloid differentiation in vivo (40), but its equilibrium dissociation constant in dilute solution was 104 M as determined by NMR spectroscopy (41). This and other examples show how volume exclusion and other crowding effects favor interactions in vivo relative to dilute solution. Facilitated diffusion along genomic DNA is also expected to promote cognate occupancy beyond the affinity for oligomeric targets from free solution.

As the tethered IDRs remain disordered in fuzzy PU.1 dimers with and without DNA, their formation is therefore unrelated to paradigms such as induced fit, conformational selection, or fly-casting mechanisms that involve order-disorder transitions by the IDRs (42, 43). Instead, the charged intrinsic disorder in PU.1 is involved in through-space electrostatic interactions. In the absence of the N-terminal IDR, dimerization of the highly cationic ETS domain (N165) is favored by low salt. Conformational destabilization of the resultant dimer under these conditions suggests an electrostatic penalty arising from charge-charge repulsion of the DBDs. The stimulatory effect of the negatively charged IDR on the DNA-free dimer, therefore, arises from attenuation of this repulsive penalty of association. A nonstructurally specific basis is borne out by the similarly favorable effects of reinforcing negative charges via phosphomimetic substitutions in N117 as anionic crowders on N165. As phosphoserines confer higher charge density (~1.5) than carboxylates, phosphorylation is expected to exert an even greater effect than the phosphomimetic substitutions. Notably, despite its nominal designation as a proteasome-recruiting signal, the PEST domain does not target PU.1 for metabolic turnover, but it is associated with a local role in dimerization and protein-protein partnerships such as with the lymphoid-specific factor IRF4 (44).

The properties of the flanking IDRs on DNA binding as revealed in this study highlight the divergent roles played by intrinsic disorder within the ETS transcription factor family, which is united by eponymous, their structurally homologous DBDs. Many ETS members are controlled by autoinhibition, a mechanism that specifically involves short flanking helices in the unbound state that unfolds and disrupts DNA binding allosterically or at the DNA contact interface (45). High-affinity binding to native promoters requires coactivators or homodimerization at tandem sites to displace autoinhibitory helices, forming with positive cooperativity 2:2 complexes (46). The regulatory strategy is activation through recruitment by other coactivators. In Ets-1, the paradigm autoinhibited ETS member, disordered elements in the serine-rich region (SRR) domain upstream of the autoinhibitory helices further modulate the regulatory potency of the autoinhibitory helices. Progressive phosphorylation of the SRR domain reinforces autoinhibition (47). PU.1, and likely its proximal ETS relatives, upends this paradigm. Lacking autoinhibitory domains, high-affinity DNA binding is the default behavior. The disordered elements flanking its structured ETS domain regulate DNA binding by modifying negative feedback. PU.1 is the recruiter in protein-protein partnerships such as IRF4 (44). Phosphorylation of the disordered PEST domain promotes the persistence of the active 1:1 complex and has been established as broadly stimulatory (30, 31, 37, 39). These contrasting features help frame in molecular and functional terms the evolutionary divergence in the ETS family, one of the most ancient families of transcription regulators in metazoan evolution.

As a final remark, both dimeric forms of PU.1 represent highly novel structures. Asymmetric DNA-bound dimers are known in the case of the zinc finger protein HAP1 (48). Zinc fingers are obligate dimers with a 2:2 subunit-to-DNA subsite configuration. Asymmetry in the HAP1 dimer is directed by the polarity of the DNA subsites bound to the subunits. The asymmetric 2:1 complex with PU.1 involves only a single DNA site without significant change in conformation. Functional deficiency of the 2:1 complex as evidenced by the cellular experiments in Fig. 1, therefore, suggests perturbation of DNA structure relative to the singly bound state or denial of specific surfaces of the 1:1 complex to form the transcriptional machinery. In contrast with the localized surface implicated in the 2:1 complex, NMR evidence shows that the residues involved are diffusely distributed with many buried in the PU.1 monomer, leading to a conformationally destabilized dimer. The CSPs observed in the DNA-free dimer, namely, H1 and the wing (S3/S4), were also recently observed for the interaction of PU.1 with a disordered peptide from the SRR domain of Ets-1 (29). These regions may, therefore, represent interaction hotspots for protein/protein partnerships for PU.1 in the absence of DNA. Beyond the minimal ETS domain, the short C-terminal IDR acts in concert with the PEST domain to reinforce the dimerizing properties of the ETS domain. Structurally, this suggests that the two IDRs likely interact physically, either antagonistically in the monomeric state or cooperatively in the dimeric state. In the cytoplasm, the ETS domain mediates nuclear import of PU.1 (49), so dimerization may also help regulate subcellular trafficking. Further studies to solve the structures of dimeric PU.1 and map their distributions in subcellular compartments will define their dynamics in vivo and contributions to target gene expression.

DNA encoding fragments of human PU.1 encompassing the ETS domain with and without various segments of its N- and C-terminal IDRs were synthesized by Integrated DNA Technologies (IDT) (Midland, IA) and subcloned into the Nco I/Hind III sites of pET28b (Novagen). For truncated constructs harboring the PEST domain (i.e., N117, sN117, D2N117, and D4N117), Cys118 was mutated to Ser to facilitate purification and biophysical experiments. Full-length PU.1 used in cell-based experiments was fully wild type. Various PU.1-sensitive enhancer sequences as described in the text were also purchased from IDT and inserted between the Age I/Bgl II sites of pD2EGFP (Clontech, CA). All constructs were verified by Sanger sequencing.

THP-1 and HEK293 cells were purchased from the American Type Culture Collection and were routinely cultured in RPMI 1640 and Dulbeccos modified Eagles medium, respectively, supplemented with 10% heat-inactivated fetal bovine serum. Where indicated, cells were induced with a single dose of PMA at 16 nM for 72 hours (final dimethyl sulfoxide concentration: 0.1%, v/v). All cell lines were maintained at 37C under 5% CO2.

Cellular PU.1 transactivation was measured using a PU.1-dependent EGFP reporter construct under the control of a minimal enhancer harboring only cognate binding sites for PU.1. In PU.1-negative HEK293 cells, the reporter was transactivated in the presence of an expression plasmid encoding wild-type full-length PU.1 and a cotranslating iRFP marker (18). Cells (7 104) were seeded in 24-well plates and cotransfected with a cocktail consisting of the EGFP reporter plasmid (250 ng) and up to 25 ng of expression plasmids for full-length PU.1, using jetPRIME reagent (Polyplus, Illkirch, France) according to the manufacturers instructions. The total amount of plasmid was made up to 500 ng with empty pcDNA3.1(+) vector. Twenty-four hours after transfection, cells were trypsinized and analyzed by flow cytometry using an FCS Fortessa instrument (BD Biosciences). Live cells were gated for iRFP and EGFP fluorescence using reporter and full-length PU.1 only controls, respectively, in FlowJo (BD Biosciences) before computing the total fluorescence of the dually fluorescent population.

Following extraction of total RNA using a spin column kit (Omega) and RT (Thermo Fisher Scientific), RT-PCR reactions were performed on a QuantStudio 3 instrument (Applied Biosystems) with SYBR Green PCR Master Mix (Thermo Fisher Scientific). Expression levels of genes were normalized to gapdh. The primer sequences used for pu.1, csf1ra, e2f1, and gapdh are given in table S2.

Heterologous overexpression in BL21(DE3)pLysS Escherichia coli was performed as previously described (20). In brief, expression cultures in LB or M9 media (the latter containing 15NH4HCl or U-13C6-glucose as required) were induced at an optical density (OD600) of 0.6 with 0.5 mM isopropyl -d-1-thiogalactopyranoside for 4 hours at 25C. Uniformly 15N- and 15N/13C-labeled constructs were expressed in appropriate M9-based media. Harvested cells were lysed in 10 mM NaH2PO4/Na2HPO4 (pH 7.4) containing 0.5 M NaCl by sonication. After centrifugation, cleared lysate was loaded directly onto a HiTrap Sepharose SP column (GE) in 10 mM NaH2PO4/Na2HPO4 (pH 7.4) containing 0.5 M NaCl. After extensive washing in this buffer, the protein was eluted in a gradient at ~1 M NaCl in phosphate buffer. Purified protein was dialyzed extensively into various buffers, as described in the text, and diluted as needed with dialysate. Protein concentrations were determined by UV absorption at 280 nm.

DNA binding by protein was measured by steady-state fluorescence polarization of a Cy3-labeled DNA probe encoding the optimal PU.1 binding sequence 5-AGCGGAAGTG-3. In brief, 0.5 nM of DNA probe was titrated with protein in a 10 mM tris-HCl buffer (pH 7.4) containing 0.1% (w/v) BSA and NaCl at concentrations as stated in the text. Steady-state anisotropies r were measured at 595 nm in 384-well black plates (Corning) in a Molecular Dynamics Paradigm plate reader with 530-nm excitation. The signal represented the fractional bound DNA probe (Fb), scaled by the limiting anisotropies of the ith bound ri and unbound states r0, as followsr=Fb(i=1nrir0)+r0=Fbi=1nri+r0(1)where Fb as a function of total protein concentration was fitted to various models as follows. In all cases, the independent variable was the total titrant concentration as taken.

For DNA binding, the two stepwise dissociation constants describing the formation of the 1:1 and 2:1 PU.1/DNA complexes areKD1=[P][D][PD]KD2=[PD][P][P2D]=KD1(2)where P and D denote PU.1 and DNA. In this analysis, the binding affinities were not further constrained by interactions of the unbound states. The ratio of KD2/KD1 = defines the nature of the cooperativity of the 2:1 complex in the paradigm of McGhee and von Hippel. Values of below, equal to, or above unity denote positively cooperative, noncooperative, and negatively cooperative formation of the 2:1 complex with respect to the 1:1 complex, respectively.

In direct titrations of the DNA probe by PU.1, the observed anisotropy change represented the summed contributions of the two complexes as expressed by Eq. 1. The most efficient approach is to determine binding in terms of the unbound protein, P. The solution, which is cubic in [P], is0=0+1[P]+2[P]2+3[P]3{0=KD1KD2[P]t1=KD1KD2KD2[P]t+KD2[D]t2=KD2[P]t+2[D]t3=1(3)where the subscript t represents the total concentration of the referred species. [P] was solved numerically from Eq. 3, rather than analytically via the cubic formula, to avoid failure due to loss of significance. With [P] in hand, [D], [PD], and [P2D] were computed from Eq. 2 and the corresponding equations of state. In the limit of no formation of the 2:1 complex (i.e., KD2 ), Eq. 3 simplifies to a quadratic, corresponding to formation of only the 1:1 complex0=KD1[P]t+(KD1[P]t+[D]t)[P]+[D]2(4)

Analyses were performed on a Waters Q-TOF (quadrupole orthogonal accelerationtime-of-flight) micro mass spectrometer equipped with an ESI source in positive ion mode. Samples were dialyzed extensively against 0.01 M NH4HCO3 (pH 8) and introduced into the ion source by direct infusion at a flow rate of 5 l/min. The instrument operation parameters were optimized as follows: capillary voltage of 2800 V, sample cone voltage of 25 V, extraction cone voltage of 2.0 V, desolvation temperature of 90C, source temperature of 120C, and collision energy of 3.0 V. Nitrogen was used as nebulizing and drying gas on a pressure of 50 and 600 psi, respectively. MassLynx 4.1 software was used for data acquisition and deconvolution. A multiply charged spectra were acquired through a full scan analysis at mass range from 300 to 3000 Da and then deconvoluted by a maximum entropy procedure (27) to the zero-charge spectra presented. Samples were diluted with dialysate to different concentrations for acquisition and data processing under the same conditions.

Spectra were acquired in 10 mM NaH2PO4/Na2HPO4 (pH 7.4) plus NaCl as a function of concentration or temperature as indicated in the text in a Jasco J-810 instrument. Thermal denaturation experiments were performed at 45C/hour with a response time of 32 s. The path length for near-UV scans was typically 1 or 0.1 mm for far-UV scans. Spectral analysis following blank subtraction and normalization with respect to path length and concentration was performed by singular value decomposition as follows.

For each experiment, a matrix A with column vectors represented by CD intensities at each protein concentration was factorized into the standard decompositionA=UVT(5)where the left-singular unitary matrix U contained the orthonormal basis spectra ui scaled by the singular values i from the diagonal matrix . The row vectors in the right singular unitary matrix VT gave the concentration- or temperature-dependent contribution of each basis spectrum to the observed data and is termed as transition vectors vT in the text. For ease and clarity of presentation, the scaling due to is captured into the transition vector, i.e., a = u(vT) (matrix multiplication is associative), which has no effect on the fitted parameters. The transition vectors were fitted to titration models describing a two-state transition with dissociation constant K as followsX=F1n(XnX1)+X1=F1nX+X1(6)where X = vT and the subscripts 1 and n refer to monomer and oligomer (n = 2 for dimerization), respectively. F1n is given byKn=nptn1(1F1n)nF1n(7)where F1n is the fractional two-state 1-to-n oligomer at equilibrium and pt is the total protein concentration. As detailed elsewhere (26), a fundamental feature of Eq. 7 is that dimerization is uniquely nonsigmoidal on linear scales, which is diagnostic for two-state dimers. Any higher-order oligomer processes are invariably sigmoidal on linear scales.

NMR experiments were conducted at 25C using Bruker BioSpin 500, 600, or 800 MHz spectrometers. For DOSY experiments, unlabeled protein and DNA were co-dialyzed in separate compartments against the required buffer, lyophilized, and reconstituted to 250 M in 100% D2O before data acquisition at 500 MHz with a 5-mm total body irradiation probe. For two-dimensional (2D)/3D experiments, uniformly labeled N165 and N117 ( unlabeled DNA) were dialyzed against the required buffer at 11/10 excess concentration and adjusted 10% D2O at 400 to 700 M protein. The dependence of the DOSY-derived self-diffusion coefficients on total protein concentration was fitted using Eq. 6, with X corresponding to the diffusion coefficients of the oligomer and monomer.

1H-15N correlated measurements were made using a phase-sensitive, double inept transfer with a garp decoupling sequence and solvent suppression (hsqcf3gpph19). Spectra were acquired with 1k 144 data points and zero-filled to 4k 4k. Steady-state heteronuclear {1H}15N-NOE was acquired at 600 MHz from the difference between spectra acquired with and without 1H saturation and a total recycle delay of 3 s. The data were processed with TopSpin 3.2 to extract peak intensities and fitted as single exponential decays.

Spectra were assigned with purified 13C/15N-labeled constructs in a standard suite of 3D experiments: HNCA, HNCACB, HN(CO)CACB, HNCO, and HN(CA)CO at 800 MHz using a 5-mm TCI cryoprobe for bound protein to DNA and at 600 MHz using a 5-mm QXI resonance probe for unbound protein. Spectra were processed using NMNRFx software, referenced to 4,4-dimethyl-4-silapentane-1-sulfonate (DSS), and peak picked/analyzed with NMRFAM-Sparky. Automated Assignments were made using the NMRFAM Pine server and verified manually.

The intrinsic fluorescence from three tryptophan residues in the PU.1 ETS domain was excited at 280 nm and detected at 340 nm with a slit of 15 nm for excitation and 20 nm for emission. Intensity data recorded in the vertical and horizontal polarizer positions were corrected for the grating factor and by blank subtraction. Concentration-dependent anisotropies were fitted to Eqs. 6 and 7, where X = r. For denaturation studies, PU.1 at 100 M and 1 M in 10 mM tris-HCl buffer (pH 7.4) containing either 0.15 or 0.05 M NaCl was titrated with urea. Blank-subtracted intensity data were directly fitted with the linear extrapolation method.

Protein samples were exhaustively dialyzed against 10 mM NaH2PO4/Na2HPO4 (pH 7.4) and 0.05 M NaCl over 48 hours with at least three buffer changes. The final dialysate was reserved and used to rinse and fill the reference cell as well as diluent for the samples. Thermal scans were carried out at 45C/hour from 10 to 80C using a MicroCal VP-DSC instrument (Malvern). All scans were carried out only when the baseline was reproducibly flat. Thermograms were fitted to two-state transition models. Nonpolar and polar solvent-accessible surface area (SASA) for monomeric N165 was estimated from the solution NMR structure 5W3G (29) based on a 1.4- probe. SASA for the unfolded state ensemble was provided by the ProtSA algorithm. The change in SASA in angstrom was converted to heat capacity change in kilojoule mol1 Kelvin1 using coefficients as followsCp0=(0.320.04)Anonpolar(0.140.04)Apolar(8)

Solution densities were measured in 10 mM tris-HCl (pH 7.4) at 25C, containing 150 mM NaCl using an Anton Paar model DMA-5000 vibrating tube densimeter with a precision of 1.5 106 g/ml. The partial molar volume of the solute V was determined from the following relationship=0+(MV0)c(9)where 0 is the density of the buffer, c is the molar solute concentration, and M is the molecular weight of the solute. For a two-state dimeric species, the observed density was analyzed as followsobs=F1n2+(1F1n)1(10)where F1n is as defined by Eq. 7, with n = 2. Because the observed density varies with the concentration of any species, 1 and 2 are each treated as linear functions as described by Eq. 9.

Acknowledgments: We thank D. Beckett and W. D. Wilson for insightful discussions and L. McIntosh for providing a structure of the unbound PU.1 ETS monomer (5W3G) before publication. We also acknowledge with appreciation the many excellent suggestions from the reviewers. NMR data presented here were collected, in part, at the City University of New York Advanced Science Research Center (CUNY ASRC) Biomolecular NMR Facility. Funding: This investigation was supported by NSF grant MCB 15451600 and NIH grant R21 HL129063 to G.M.K.P. S.X., H.M.K., and S.E. were partially supported by GSU Molecular Basis of Diseases Fellowships. V.T.L.H. was supported by the GSU University Assistantship Program. Author contributions: S.L. and H.M.K. carried out cell-based studies. G.M.K.P. cloned the molecular constructs. S.X. and S.E. expressed and purified the recombinant protein constructs. S.X., S.E., J.M.A., and M.W.G. performed and analyzed the NMR experiments. S.W. performed and analyzed the data from the ESI-MS studies. V.L.T.H. performed the densimetric experiments and analyzed the volumetric data. S.X., M.K., G.L.F., and A.V.A. performed the binding, thermodynamic, and other spectroscopic experiments. S.X., M.W.G., and G.M.K.P. jointly designed the studies, analyzed data, composed the figures, and wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Intrinsic disorder controls two functionally distinct dimers of the master transcription factor PU.1 - Science Advances