Intramural Papers

Intramural By Nicholas Alagna, Mimi Huang, Sanya Mehta, Prashant Rai, and Saniya Rattan

Assessing the potential carcinogenicity and toxicity of botanical dietary supplements, such as Ginkgo biloba extract (GbE), in rodent systems is challenging due to the variability in commercially available products. To address this issue, scientists at the NIEHS Divisions of the National Toxicology Program (NTP) and Intramural Research used several analytical chemistry and chemometric techniques to compare the composition of GbE used in recent NTP research with other GbE samples on the market, as well as with the National Institute of Standards and Technology (NIST) GbE standard reference materials. Their results suggested that a simple analysis for the presence, absence, or amount of compounds unique to GbE would be adequate to determine the authenticity of a GbE sample.

The scientists conducted chemometric analysis of chromatographic and spectrophotometric data to determine that GbE used in rodent experiments was most similar to the NIST reference materials and two unfinished products, as well as finished commercial products containing an authentic GbE extract, based on their constituent profiles. Because the team found its particular GbE was representative of other high quality GbE products, the results of the NTP carcinogenicity and toxicity study could be more broadly applied to other similar GbE products on the market. (NA)

Citation:Collins BJ, Kerns SP, Aillon K, Mueller G, Rider CV, DeRose EF, London RE, Harnly JM, Waidyanatha S. 2020. Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches. Anal Bioanal Chem 412(25):67896809.

Researchers from NIEHS and its Division of NTP found that subtle changes to mitochondrial function led to long-lasting epigenomic effects in the mouse. Scientists identified that mitochondrial inhibition during early development persistently altered DNA methylation and liver gene expression throughout the life of the mouse. The long-term effects observed in this study shed light on potential mechanisms involved with health outcomes under the developmental origins of health and disease paradigm.

To assess the effects of subtle alteration of mitochondrial function on epigenetics, researchers used the viable yellow agouti mouse, Avy, which reveals DNA methylation changes through changes in the coat color of the animals. Mice born from mothers fed with a mitochondrial inhibitor, rotenone, solely during gestation and lactation, showed an altered coat color distribution. The mice also showed altered DNA methylation and gene expression patterns in the liver, effects that were observed up to 18 months after rotenone exposure had ceased. Finally, the animals showed that prenatal rotenone exposure led to altered mitochondrial and antioxidant function later in life, demonstrating that early exposure influenced long-term health outcomes.

This study places mitochondria as players in controlling gene expression through epigenetic mechanisms and highlights how environmental toxicants could influence long-term health outcomes. (SR)

Citation:Lozoya OA, Xu F, Grenet D, Wang T, Grimm SA, Godfrey V, Waidyanatha S, Woychik RP, Santos JH. 2020. Single nucleotide resolution analysis reveals pervasive, long-lasting DNA methylation changes by developmental exposure to a mitochondrial toxicant. Cell Rep 32(11):108131.

To uncover novel deletion patterns in mitochondrial DNA (mtDNA), NIEHS researchers and their collaborators developed LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. The team used the technique to reveal links between mitochondrial DNA replication, aging, and mitochondrial disease.

A mutation in POLG, a nuclear gene responsible for maintaining the mitochondrial genome, is known to be the most common cause of mitochondrial disease, a condition where the mitochondria fail to produce enough energy for the body to function properly.

The scientists analyzed mtDNA from skeletal muscle biopsies of 41 patients with mitochondrial disease with wild-type (WT) and mutated POLG. They used LostArc to detect loss of mtDNA segments by mapping split-reads in the samples to a normal mtDNA reference. Thirty-five million deletion segments were detected in the biopsies. They spanned more than 470,000 unique segments, 99% of which were novel. The scientists also found that ablation, or loss of mtDNA segments to deletion, significantly increased with age. Mutated POLG samples had higher ablation levels than WT. These changes in mtDNA ablation help explain some of the muscular symptoms of aging and disease. The additional potential applications of LostArc include genetic screening, diagnostic applications, and medical forensics. (SM)

Citation:Lujan SA, Longley MJ, Humble MH, Lavender CA, Burkholder A, Blakely EL, Alston CL, Gorman GS, Turnbull DM, McFarland R, Taylor RW, Kunkel TA, Copeland WC. 2020. Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging. Genome Biol 21(1):248.

The cellular signaling molecule InsP7 can modify the abundance of cytosolic, membrane-less condensates called P-bodies through a new epitranscriptomic process identified by NIEHS researchers and their collaborators. P-bodies contain structurally and metabolically stabilized mRNAs, which can subsequently be released to drive protein expression in response to environmental stress. The mRNA release can sometimes promote protein expression in a specific location within the cell and can also direct stem cell differentiation. The possible role of P-bodies in aging and neurodegenerative disease suggests potential therapeutic implications of these findings.

The relationship between InsP7 and P-bodies is centered on the enzyme NUDT3, a phosphate-cleaving enzyme that not only metabolizes InsP7 but also removes the structurally stabilizing cap from the mRNA. By competing with NUDT3-catalyzed mRNA cap removal, InsP7 increases mRNA transcript levels and P-body abundance. It appears that de-capping enzymes like NUDT3 each have specific mRNA targets, so this regulatory ability of InsP7 is likely specific to NUDT3. Thus, the authors study is a significant step forward in the understanding of how the cell can selectively regulate the fate and function of specific mRNA transcripts. (MH)

Citation:Sahu S, Wang Z, Jiao X, Gu C, Jork N, Wittwer C, Li X, Hostachy S, Fiedler D, Wang H, Jessen HJ, Kiledjian M, Shears SB. 2020. InsP7 is a small-molecule regulator of NUDT3-mediated nRNA decapping and processing-body dynamics. Proc Natl Acad Sci U S A 117(32):1924519253.

NIEHS scientists and their collaborators reported that spatial protein-DNA interaction between transcription factors and their specific binding sites in the genome is a key step to initiating the transcription of genetic code. The study was conducted on a transcription factor called GATA3, which can bind directly to the condensed structural units of the human genome, called nucleosomes, and drive transcription of genes necessary for cellular reprogramming and differentiation.

The study used high-resolution mapping to reveal that GATA3 binding near the central axis of the nucleosome coil was associated with remodeling of local DNA structure to facilitate a productive reading of genetic code. This binding was found to be independent of the genetic sequence of nucleosomes. The study used an in vitro model of a nucleosome and cryoelectron microscopy and found that nonproductive GATA3 binding, without any structural changes to the nucleosome, occurred efficiently at the periphery of the nucleosome coil.

Binding of a transcription factor, such as GATA3, in a specific spatial arrangement on the nucleosome surface affects the local genomic scaffolds and could determine the overall outcome of such binding in gene regulation. (PR)

Citation:Tanaka H, Takizawa Y, Takaku M, Kato D, Kumagawa Y, Grimm SA, Wade PA, Kurumizaka H. 2020. Interaction of the pioneer transcription factor GATA3 with nucleosomes. Nat Commun 11(1):4136. (Story)

(Nicholas Alagna is an Intramural Research Training Award [IRTA] fellow in the NIEHS Mechanisms of Mutation Group. Mimi Huang, Ph.D., is an IRTA fellow in the Division of the National Toxicology Program Systems Toxicology Group. Sanya Mehta is an IRTA postbaccalaureate fellow in the NIEHS Matrix Biology Group. Prashant Rai, Ph.D., is a visiting fellow in the NIEHS Clinical Investigation of Host Defense Group. Saniya Rattan, Ph.D., is an IRTA fellow in the NIEHS Reproductive Developmental Biology Group.)

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November 2020: Intramural Papers of the Month - Environmental Factor Newsletter