Leukemia: Cancer Of The Blood – Modern Ghana

DEFINITIONLeukemia is a Cancer of blood forming tissues including bone marrow and the lymphatic system.This type of cancer hinders the body's ability to fight infection, leukemia involves the white blood cells.

CAUSES OF LEUKEMIALeukemia can develop due to a problem with blood cell production.

Several factors have been identified which increase the risk of having the cancer:

A family history of leukemiaSmokingGenetic disorder such as Down syndromeExposure to chemicals such as benzeneExposure to high levels of radiationTYPES OF LEUKEMIALeukemia can be Acute or Chronic. In Acute leukemia, Cancer cell multiply quickly while in Chronic leukemia, the disease progresses slowly and early symptoms may be very mild.

Leukemia can also be classified according to types of cells which are myelogenous leukemia and lymphotic leukemia involving myeloid cells and lymphocytes respectively.

SYMPTOMS OF LEUKEMIAExcessive sweating most especially in the nightFatigue and weaknessUnintentional weight lossFever or chillsFrequent infectionsBleeding easily and bruising easilyEnlargement of the liver or spleenLEUKEMIA IN PREGNANCYLeukemia affects approximately 1 in 10000 pregnancies.Women with leukemia have non-specific symptoms and some of them could also be attributed to pregnancy.

The damage in the fetus is correlated with the time of exposition and the fetus is most vulnerable during organnogenesis phase, Although chemotherapy has effect in the fetus, there are reports of cases with successful pregnancy.

It is necessary to study the relation among chemotherapy, the leukemia and the fetus in long term studies where fetus has been exposed to chemotherapy agents and is reported with normal characteristics at birth.

ENZYMES DEFICIENT IN LEUKEMIABlast cells from 100 cases of Acute leukemia were evaluated for the presence of methylthioadenosine phosphorylase (MTAase), an enzyme important in polyamine metabolism.

Ten cases (10%) had undetectable levels of MTAase activity. A relatively high frequency (38%) of MTAase deficiency was seen in all of T-cell origin.

MTAase deficiency occurs in a wide variety of acute leukemia, that the lack of enzyme activity is specific in malignant cells. The absence of MTAase in some leukemia may be therapeutically exploitable.

TREATMENT OF LEUKEMIALeukemia is usually treated by a hematologist-oncologist. These are doctors who specialize in blood disorders and cancer.

The treatment depends on the type and stage of the cancer, the treatment includes the following:

Chemotherapy uses drugs to kill leukemia cells.Radiation therapy uses high energy radiation to damage leukemia cells and inhibit their growth.

Stem cell transplantation replaces diseased bone marrow with healthy bone marrow.

Biological or immune therapy uses treatments that help your immune system recognize and attack cancer cells.

Targeted therapy uses medication advantages of vulnerabilities in cancer cells.

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Locking in and preserving your healthy stem cells has never been easier and more accessible as Acorn Biolabs partners with Coverdale Clinics. – Canada…

Appointments for stem cell & DNA collection now available.

TORONTO, Feb. 20, 2020 /CNW/ - With the rapid emergence of regenerative medicine therapies and genetic analysis testing reaching mainstream medicine, consumers are demanding increased opportunities to prepare for their future healthcare needs, including banking a viable source of cells to preserve their current health.

To meet this growing demand, Acorn Biolabs, the leading non-invasive stem cell collection, cryopreservation and analysis company, today announced its partnership with Coverdale Clinics Inc., a premium network of specialty care clinics in Canada. Together, Acorn and Coverdale will help make stem cell collection more accessible and affordable in the West Greater Toronto Area.

Through their partnership, Coverdale Clinics' Oakville location be offering Acorn's non-invasive stem cell collection services. The simple process involves plucking a few hair follicles from a client's head, which are then analyzed and cryopreserved for future use.

Acorn's innovative solution for live cell collection enables the collection of stem cells without the need for surgery or other painful and invasive procedures, making stem cell collection for life-long storage significantly more affordable and accessible for everyone.

"Our partnership with Acorn Biolabs opens up a great opportunity for us to expand Coverdale Clinics service offering into the emerging regenerative medicine and geneticsmarket. We're pleased to be able to leverage our specialty clinic in Oakville to offer a service that improves access to exciting and novel health care technologies," said Chris Dalseg, BioScript Solutions' Vice President of Strategic Growth and Marketing. "We have always been at the forefront of providing innovative health care services to Canadians, and adding stem cell collection services from Acorn exemplifies our ongoing commitment."

Once stem cells are collected, Acorn uses its proprietary capabilities to keep cells alive during transportation before going into long term cryogenic storage. The process turns collected hair follicles into a highly valuable and accessible resource for further regenerative medicine and genetics. Not only are these stem cells securely stored for future use, but the company's scientists will also be able to extract critical genetic information through DNA tests, for clients that want it, that will unlock valuable data about a person's health over their lifetime.

"We are excited to bring Acorn's preventative, personalized healthcare services to more people through this partnership with Coverdale Clinics. The cells collected are a life-long resource for these clients, not only in regenerative medicine, but also for advanced analytics, helping to identify diseases even before the first symptom," said Dr. Drew Taylor, Co-founder and CEO at Acorn Biolabs Inc. "The partnership is an important extension for Acorn to serve health-minded individuals in more geographies, freezing the clock on their stem cells so they can use them later in life, when they will need them most."

Clients can book their non-invasive stem cell collection appointment in Oakville, Ontario through Acorn's website today at http://www.acorn.me

About Acorn Biolabs, Inc.

Acorn helps you live a longer, healthier tomorrow by freezing the clock on your cells today. Founded in 2017 by Steven ten Holder, Patrick Pumputis and Dr. Drew Taylor and borne out of years of research, Acorn is a healthcare technology company based at Johnson & Johnson INNOVATION JLABS in Toronto. Acorn is focused on giving everyone the best chance to experience more healthy years with its easy, affordable and non-invasive stem cell collection, analysis and cryopreservation service. For more information, visit acorn.me.

About Coverdale Clinics

Coverdale Clinics is a premium network of specialty care clinics, providing patients with a safe, comfortable environment to receive specialty medications by infusion or injection. With more than 100 clinics nationwide, our nurses take a personalized approach to patient care that includes education and counselling to better support medication adherence.

About BioScript Solutions

BioScript Solutions is committed to helping patients with chronic illnesses achieve the best possible health outcomes. With our total care approach, we simplify access to complex, specialty drug therapies and deliver full-service specialty care solutions at every stage of the patient's treatment journey. Through our specialty pharmaceutical distribution, pharmacies, patient support programs and clinical services, BioScript has the capability to manage the needs of manufacturers, payors, prescribers and health care practitioners across Canada today, and tomorrow. To learn more, please visit bioscript.ca.

SOURCE Acorn Biolabs

For further information: Press and media inquiries please contact: Sonya Verheyden, E: [emailprotected] C: 226-747-4600

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Locking in and preserving your healthy stem cells has never been easier and more accessible as Acorn Biolabs partners with Coverdale Clinics. - Canada...

Chinese Scientist Unite Together to Tackle With COVID-19 – Benzinga

BEIJING, Feb. 18, 2020 /PRNewswire/ -- Reported by Science & Technology Daily:

Astothe battle against the COVID-19, Chinese Ministry of Science and Technology (MOST) has been launched the program of "Dealing With the Outbreak of COVID-19 Using Science and Technology", a program including four batches of 20 emergency projects until now. Moreover, MOST re-examining two major scientific and technological projects, including the "Creation of Important Innovative New Drugs" and the "Prevention and Treatment of Severe Contagions", and a series of national essential research and development plans. Recently, some important achievements from these projects have already been implemented in the front line of epidemic prevention and control.

When it comes to drug screening, front-line staff from different research teams collaborated on the basis of existing researches, making great efforts to systematically and massively screen the drugs that have been on the market already or already go into clinical trials. As a consequence, they discovered potentially effective anti- coronavirus drugs, such as Chloroquine Phosphate, Remdesivir, and Favipiravir. Furthermore, in recent days, researchers have urgently launched some clinical trials and the results show that the curative effect of drugs on patients is increasingly obvious.

Based on the R&D level and preliminary accumulation in the field of cell therapy, traditional Chinese medicine and plasma therapy in China, the clinical trials of the three therapies organized by the "Joint Prevention and Control Group" have been gradually carried out, and they are playing significant role in curing critically ill patients.

For the purpose of providing more cutting-edge and effective solutions to the epidemic using stem cell therapy, the leaders of the MOST went to the Innovation Institute of Stem Cell and Regenerative Medicine of the Chinese Academy of Science to confirm their progressing. A new cellular drug called CAStem supposed to cure COVID-19 was created, and it has already made major progress in the experiment about the treatment of the severe acute respiratory distress syndrome (ARDS) previously. The research team has applied for emergency approval from China National Medical Products Administration, and it is cooperating with relevant medical institutions. So far, the CAStem has already been approved by the Ethics Committee, and is ongoing clinical observation and evaluation.

An emergency project titled "Clinical Study on the Prevention and Treatment of COVID-19 by Integrated Chinese and Western Medicine" was officially launched on February 3 with the support from the MOST. "The positive curative effect has been shown in treating COVID-19," said Zhang Boli, the project leader and the headmaster of Tianjin University of Traditional Chinese Medicine. It was reported that a total of 23patients had been cured and discharged from Hubei Provincial Hospital of Integrated Traditional and Western Medicine and Wuhan Hospital of Traditional Chinese Medicine.

With regard to the field of plasma therapy,China National Biotech Group has completed the collection of plasma from some convalescent patients, as well as the preparation of special immune plasma products and special immune globulin of novel coronavirus on February 13. Through strict blood biosafety testing, virus inactivation, and antiviral activity testing, etc., special immune plasma has been successfully prepared for clinical treatment and has already been put into clinical treatment of severely ill patients.

In terms of field of vaccine research and development, the Chinese Center for Disease Control and Prevention (CDC) had successfully isolated the first COVID-19 virus strain on January 24, which was recognized by the World Health Organization and other international agencies. Several new type of vaccines for COVID-19 have started the animal testing phase, which bring hope to all.

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Chinese Scientist Unite Together to Tackle With COVID-19 - Benzinga

Army spouse dances her way through chemotherapy – We Are The Mighty

It is not uncommon to stumble upon live videos while scrolling through Facebook. And for the hundreds of people who follow Army wife Sofia de Falco who is an adjunct professor of Italian language and literature it is not uncommon to come across her videos where she is smiling and dancing, uplifting them with a joyful and serene expression on her face. As the hundreds of comments on her posts highlight, Sofia is a source of inspiration and a true beacon of light to many.

But in those videos, Sofia is in a hospital room, wearing a shirt that lightly uncovers the right side of her chest, revealing the central venous catheter that feeds her chemotherapy medicine directly into her bloodstream.

In February 2019, Sofia was diagnosed with lymphoma. "I found a lump in my groin," Sofia said. "But I didn't give it much thought because it wasn't the first time. I always had them removed and nothing suspicious ever came of it."

During her Christmas vacation in Naples, Italy where she is originally from Sofia developed a dry and irritating cough. "I decided to go to a local doctor and see if there was anything he could do." After the doctor dismissed her because he couldn't find anything wrong, Sofia made a follow-up appointment with her PCM in Virginia, where she and her family are stationed.

"As I was leaving my PCM's office," Sofia said, "I turned around and told him about the lump in my groin, which had grown in size by then." The doctor had Sofia lie down, checked the lump and told her to see a hematologist and a surgeon. Although he didn't explicitly verbalize it at the time, the doctor suspected Sofia had lymphoma.

He was right. "Since February 2019, I have been going through countless tests and surgical procedures," Sofia revealed. After being told the first round of chemotherapy which she faced in "warrior mode," she said had worked and she was clear, in November 2019 Sofia's positive attitude and bright outlook on life was put to the test again. "The cancer came back," she said. "And this time, I have to fight even harder." Sofia will have to undergo a stem cell transplant and several rounds of high-dose chemotherapy.

Yet, she dances. As if those tubes were not attached to her body. As if the machine next to her was not feeding her chemo medicine. As if she didn't suffer from nausea and migraines. She dances as if she were by the beach in downtown Naples, with a bright sun glittering over the Mediterranean Sea in the background, its warm rays caressing her exposed skin.

"I dance on it," she said. "Dancing makes me happy, so I know it's what I'm supposed to do. My body feels so much better after I get up and start dancing, just like one, two, three, four," she said snapping her fingers as if following the rhythm of an imaginary song.

"Dancing is a way for me to keep away the pain, the sorrow and the negative thoughts," she admitted. "I believe that it is possible to defeat this beast because I believe in the power of hope."

And as her hundreds of followers are inspired by her inner strength that shines through her smile, and as the stunned nurses watch her from outside her hospital room while she dances through chemo, she laughs out loud confessing, "You know, I'm actually really bad at dancing!"

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The Winnipeg Foundation Innovation Fund supports cutting-edge research – UM Today

February 21, 2020

Thanks to a grant from The Winnipeg Foundation Innovation Fund, an interdisciplinary team in the Rady Faculty of Health Sciences will create heart tissue using a 3D bioprinter to study the development of diabetes.

Dr. Adrian West, an assistant professor of physiology and pathophysiology, Max Rady College of Medicine, said it is truly an honour for his team to receive the first-ever Winnipeg Foundation Innovation Fund grant.

The fact that The Winnipeg Foundation has the vision to invest in something like this is fantastic for our program because it really lets us develop these unusual ideas and push them forward to the national level, he said. High risk, high reward is always going to be difficult to fund and I think thats the project were doing.

The Winnipeg Foundation committed $1 million last year to give health sciences researchers at the UM the opportunity to work on innovative interdisciplinary research and develop projects to the point of qualifying for additional external funding.

West is collaborating on the project with Dr. Vernon Dolinsky, associate professor of pharmacology and therapeutics, Max Rady College of Medicine, and Dr. Joseph Gordon, associate professor in the College of Nursing and of human anatomy and cell science, Max Rady College of Medicine. All three are research scientists at the Childrens Hospital Research Institute of Manitoba.

The project, which was awarded close to $100,000, is looking at the origins of diabetic cardiomyopathy, a cardiovascular disease that makes it harder for the heart to pump blood throughout the body and can lead to heart failure. West said this research is important because 80 per cent of people with diabetes ultimately die of heart disease.

The team will build heart tissue using a 3D bioprinter, which creates the tissue by combining cells, biomaterials and growth factors that imitate natural tissue. West said that 3D bioprinting allows them to create a more realistic environment for cells than in a petri dish.

Cells arent flat, West said. Cells arent squished. Cells are three dimensional in nature and by creating these 3D bioprinted culture models it makes it much like it is in the body.

Theyre building the heart tissue with samples that are exposed to diabetes-like conditions during growth and development to see what underpins diabetic cardiomyopathy. Once theyve developed a model that replicates the disease, they can test a variety of different treatments and scientists around the world can follow their model.

West said that this research will lead to building tissues with human stem cells. The idea is that they will take a cell from a patient, grow it in a dish, build a piece of their own heart and test a treatment that is personalized to them, he said. It will give them the ability to potentially test drugs and treatments on a patients own cells rather than just relying on the results they derive from standard cell cultures and animal models.

Dr. Peter Nickerson, vice-dean (research), Rady Faculty of Health Sciences, said that The Winnipeg Foundation Innovation Fund is unique because it can allow transformative research like Wests to take place.

It is a bit higher risk, but if it pays off, it really will be not just a couple steps forward, but a leap forward from where we are today, Nickerson said. What were trying to achieve with The Winnipeg Foundation Innovation Fund is bringing teams together with really innovative ideas. Well then give them the seed money to bring that idea to life and see if they can get enough of a launch to then apply for national funding to move it to the next level.

The Winnipeg Foundation Innovation Fund grant has already helped Wests team with future funding.

Knowing that somebody had the confidence to fund this project, its already strongly advanced our other grant applications, West said. Just by The Winnipeg Foundation Innovation Fund grant being available has helped us apply for that grant a year, two years earlier.

Matthew Kruchak

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The Winnipeg Foundation Innovation Fund supports cutting-edge research - UM Today

Lung-MAP: A five-year recap on the first master protocol trial in cancer research – The Cancer Letter

publication date: Feb. 21, 2020

Roy S. Herbst, MD, PhD

Lung-MAP Study Chair,

Chief of Medical Oncology,

Yale Cancer Center and Smilow Cancer Hospital;

Ensign Professor of Medicine and Professor of Pharmacology,

Yale School of Medicine

Lyudmila Bazhenova, MD

Lung-MAP Sub-Study Chair,

Professor of Medicine,Medical Oncology,

University of California San Diego School of Medicine,

Moores Cancer Center at UC San Diego Health

Joel Neal, MD, PhD

Lung-MAP Medical Oncology Chair,

Assistant Professor of Medicine,Medical Oncology,

Stanford Cancer Institute,

Stanford University School of Medicine

Saiama N. Waqar, MBBS, MSCI

Lung-MAP Medical Oncology Chair,

Lung-MAP Sub-Study Chair,

Associate Professor of Medicine,

Director, Hematology and Oncology Fellowship Program,

Washington University School of Medicine in St. Louis

This is a formidable challenge. Cancer trials were, and remain, notoriously time-consuming to launch, expensive to run, and difficult to enroll patients to. A deeper understanding of cancer biology and the genomics revolution in medicine have changed how we approach clinical research.When the Lung-MAP trial was launched in June 2014, the goal was simple: Make drug development faster and more collaborativeand do it for lung cancer, the leading cause of cancer death in the United States.

We now have the ability to sequence a patients tumor tissue, determine which proteins or DNA sequences are associated with their cancer, then select a drug designed to attack that type of abnormality in order to create precision, or personalized, treatments. Great news for patients, but tough for researchers.

Once clinical trials incorporate genomic biomarkers, the patient pool splinters. For example, EGFR mutations are observed in 10% to 15% of non-small cell lung cancer patients in the U.S. The ALK gene rearrangement occurs in fewer than 7% of patients, and ROS1 rearrangements are found in only 1% to 2%. In the era of personalized medicine, we are required to sort through a much bigger haystack in order to find much smaller needles.

To address these challenges, the scientific leadership of Lung-MAP adopted an emerging trial design called a master protocolmaking us one of the first to adopt this model in cancer research and the first to do so with drug registration intent.

Master protocols, while complex, are based on the illusion of simplicitymany drugs or patient populations, but only one protocol. No need to open and close the whole trial every time you want to test a new agent or bring on a new group of patients. You simply use the master platform and plug and play independent sub-studies to incorporate promising new treatments.

After an expansion last year, Lung-MAP is open to the vast majority of patients with NSCLC. Its a biomarker-driven protocol that tests several drugs at the same time, a so-called umbrella design. The trial is streamlined, with uniform genomic screening, highly harmonized eligibility requirements, coordinated treatment plans, and consistent endpoints. The sub-studies are also designed to get answers to safety and effectiveness questions quickly and, particularly, to pave the way to regulatory approval.

Finally, because biomarker trials require screening large patient populations, we tapped into the NCIs National Clinical Trials Network. By working within the NCTN, we get access to tens of thousands of lung cancer patients who get their care in more than 2,200 affiliated cancer centers, academic medical centers, and community hospitals and clinics worldwide.

So, here we are, five years after the Lung-MAP launch. What have we accomplished?

Quite a bit. The first NCI-backed precision medicine trial has a track record of accelerating drug discovery and development and fostering innovation in cancer research. We have strong scientific partnerships and a robust drug pipeline. Currently, Lung-MAP has five sub-studies open. In 2020, one more is targeted for activationwith seven others under discussion with pharmaceutical partners. Its our largest pool of potential trial drugs yet.

Lung-MAPs primary mission is to create efficiency and operate collaborativelyall to benefit patients. Weve succeeded on those counts.

Phase II/III trials testing a drug against a biomarker at one or just a few clinical sites historically face slow accrual, especially if the prevalence of the biomarker is low. Similarly, patients with rare molecular biomarkers may have difficulties accessing precision trials due to a lack of comprehensive genomic screening and geographic distance from enrolling sites.

Lung-MAP has opened and completed eight drug-centered sub-studies testing 12 novel therapies since activating in June 2014. Thats 12 new therapies tested in five years. While no comparative studies have been conducted to show how much time master protocols and other innovative trial designs save, we can credibly claim a much faster track record than a typical single drug, single biomarker cancer trial.

Weve also tested a model that was, and remains, a unique public-private partnership. Lung-MAP is a result of years of work by the NCI, NIH, FDA, Friends of Cancer Research, the Foundation for the NIH, SWOG Cancer Research Network and many other research institutions, patient advocacy groups, and industry partners.

This ongoing partnership has been instrumental to fostering innovation and creating a strong value proposition for industry participation. Many Lung-MAP innovations are built into the trial design.

The Lung-MAP leadership team at a Feb. 3 planning meeting held in the Washington, D.C., offices of Friends of Cancer Research

Under Lung-MAP, clinicians have the ability to pre-screen patients while they are on a prior therapy, a feature that shortens the time needed to move a patient into the trial once they progress on front-line therapy. Another innovation: patients who dont qualify for biomarker-driven sub-studies are offered a non-match option. In non-match sub-studies, Lung-MAP patients receive an immunotherapy or another investigational drug expected to work on a range of molecular sub-types.

Additionally, as novel immunotherapies were approved by FDA and quickly became the standard of care in NSCLC, we changed the design in 2015 to allow evaluation of molecularly-targeted compounds to continue in immunotherapy-nave patients, and also provided these cutting-edge drugs as our non-match sub-study for those who were eligible. In 2019, we further revised the trial to allow the addition of sub-studies that evaluate treatment combinations in immunotherapy-resistant patientsan unmet medical need that is increasing as more patients are exposed to immunotherapies.

Drugs and combinations tested on the Lung-MAP trial

Company Names

Drugs Tested

AstraZeneca

AZD4547

AbbVie Inc.

telisotuzumab vedotin

Amgen Inc.

rilotumumab + erlotinib

Bristol-Myers Squibb

nivolumab

nivolumab + ipilimumab

Clovis Oncology, Inc.

rucaparib

Genentech, Inc.

taselisib

Loxo Oncology at Lilly

selpercatinib

MedImmune/AstraZeneca

durvalumab

durvalumab + tremelimumab

Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc./ Eli Lilly and Company

pembrolizumab + ramucirumab

Pfizer Inc.

palbociclib

talazoparib

talazoparib + avelumab

Pioneering the use of next-generation sequencing and the Foundation Medicine platform in a large-scale cancer clinical trial.

Lung-MAP was one of the first trials to partner with Foundation Medicine to use its innovative FoundationOne DNA next generation sequencing platform, now an industry standard (F1CDx) for comprehensive genomic screening of all patients.

F1CDx detects gene substitutions, insertions and deletion alterations, and copy number alterations in 324 genes and select gene rearrangements and can evaluate complex molecular targets (e.g., DNA repair deficiency, microsatellite instability, tumor mutational burden, and loss of heterozygosity.

This broad range of molecular signatures defined from F1CDx are used as the molecular biomarkers for Lung-MAP sub-studies, allowing numerous therapies to be evaluated in a biomarker-driven manner and providing historical and real-time information on molecular target prevalence in study populations useful in future Lung-MAP studiesas well as other drug discovery and development efforts. The short turnaround time established for generating screening results from tissue submission to results reporting16 days or less, with an average of 12 daysis critical to the success of Lung-MAP.

Beyond Lung-MAP, F1CDx-derived molecular signatures are now serving as companion diagnostics for 19 drugs, and F1CDx and other NGS platforms are widely used in establishing genomic profiles and diagnostic signatures in clinical research and practice settings. Even with all this success, Foundation Medicine remains a valuable partner and continuously works with us to adapt their platform for new biomarker-driven sub-studies and provide critical insights on developments in the field.

Increasing the amount of genomic screening data available on lung cancer patients, advancing medicine and science.

As of Feb. 6, 2020, Lung-MAP had screened 2,813 patients and paired 799 patients to some type of trial therapy. Data from these patients are being shared to stimulate research that will further our understanding of common NSCLC mutations and to discover future treatment targets.

Influencing the design of other master protocols within and outside of cancer research.

The Lung-MAP design was a pioneer that paved the way for a dozen master protocol trials to launch in recent years, including the landmark NCI-MATCH and Pediatric MATCH cancer trials.

It has also influenced master protocol designs for other diseases, including infectious diseases and Alzheimers disease. Benefits of the Lung-MAP design were highlighted in an October 2018 FDA draft guidance, Master Protocols: Efficient Clinical Trial Design Strategies to Expedite Development of Oncology Drugs and Biologics.

Establishing a site coordinators committee to provide fast, critical feedback and insights on trial conduct.

Site staff are linchpins to patients when it comes to trial enrollment and management, and we hold their perspectives in high regard when it comes to Lung-MAP conduct.

We selected 12 coordinators based on geography and site-type diversity (e.g. cancer center, community clinic, etc.) to advise us on all aspects of the master protocol to ensure that both patients and staff needs are considered. This committee helps us streamline processes to minimize complexity of the trial.

Providing access to biomarker-targeted drugs and immunotherapies to hundreds of patients, with some success of extending and improving lives.

This broad access is a product of the study design.

As we described above, Lung-MAP patients whose tumor doesnt include a biomarker that matches a trial drug, or who dont otherwise qualify for a biomarker sub-study, still have an option to enroll in a non-match sub-study. These test a novel immunotherapy or other drug expected to have activity across multiple molecular subtypes. Although the squamous NSCLC sub-studies did not lead to the approval of new therapies, it is clear that some patients have benefited from Lung-MAP participation.

For example, about 450 Lung-MAP patients have gotten access to immunotherapies to date. One is Carol Annie Burke, an artist from Savannah, GA, who enrolled on Lung-MAP when she was diagnosed with stage IV squamous cell lung cancer at age 56. After chemotherapy failed to stop her cancer from spreading, she was eligible for a non-match immunotherapy sub-study. The cancer in her lungs, and brain, was gone. The Lung-MAP treatment saved my life, she said.

In addition, trial partner SWOG Cancer Research Network has a unique outreach program with U.S. Department of Veterans Affairs medical centers, which is aimed at getting more military veterans enrolled onto cancer clinical trials. The partnership has given Lung-MAP access to hundreds of veterans, who are at higher risk for lung cancer.

Exploring new, less invasive methods for pairing patients with the best treatments.

With the latest revision of Lung-MAP, the team is collecting blood from patients that consent both during patient screening and while they are being treated on study to allow for retrospective circulating tumor DNA.

These results can eventually be compared to the NGS results and build the appropriate evidence for the eventual use of the less invasive ctDNA testing for clinical placement of patient who are not able to provide a suitable biopsy for participation in the trial, as well as potentially lead to the discovery of blood-based biomarkers of response.

If Lung-MAP has taught us anything these last five years, its that even with a master protocol, you cant stand still. The science moves too fast, always changing the drug development and treatment landscape. This is particularly true of the immunotherapies and targeted drugs we test.

Last year, we expanded Lung-MAP eligibility from squamous cell lung cancer patients to patients with all types of NSCLCmaking investigational drugs available to thousands more eligible people and expanding our patient pool exponentially. That decision to expand has, in part, revived the trial, which is surpassing accrual goals and boasting a pipeline of exciting new drugs, including cutting-edge, promising targeted therapies and the already noted treatments for immunotherapy-refractory patients.

Another reason behind the renewed energy is a change in management structure. There are now eight Lung-MAP study chairstwo each from SWOG, the Alliance for Clinical Trials in Oncology, ECOG-ACRIN Cancer Research Group, and NRG Oncology. This new format of equal representation of all four adult cancer clinical trial groups in the NCI-supported NCTN allowed us to bring in new leaders with broader knowledge of emerging NSCLC therapies, expand access to companies whose drugs are ideal candidates for the trial, and invigorate a broader NCTN commitment to enrolling patients onto the trial.

Continued here:
Lung-MAP: A five-year recap on the first master protocol trial in cancer research - The Cancer Letter

Transplant for Szary Syndrome is Patient’s First Step in Returning to the Dance Floor – Dana-Farber Cancer Institute

The first time Bill Cronin Googled his own cancer diagnosis in 2016, his heart sank. He had Szary syndrome, a rare and aggressive form of cutaneous T-cell lymphoma and staring back at him were countless articles predicting a negative prognosis.

However, after receiving a stem-cell transplant at Dana-Farber/Brigham and Womens Cancer Center, Cronin is returning to the life he enjoyed before cancer.

Im at a place I never thought Id get to, Cronin says.

In 2015, Cronin, then 60, started feeling incredibly itchy and developed an accompanying rash. He went to his dermatologist, who diagnosed him with eczema and told him to return in five months. The rash continued to grow, however, and at the five month mark, Cronins dermatologist encouraged him to undergo further testing at Dana-Farber.

A blood test revealed that Cronins T-cells a type ofwhite blood cells that make up part of the immune system had becomecancerous. In the case of Szary syndrome, lymphoma cells will circulatethrough the blood stream and deposit in different areas of the skin. This willgenerally lead to a full-body rash and intense itchiness.

Cronin would need a stem cell transplant to combat the disease, but before he could receive one, his care team had to get him into remission. Patients who do not achieve remission prior to transplant have a high chance of relapsing.

When they first told me everything, I was really scared, says Cronin. But I knew I was in one of the best places in the world to figure out and treat this rare disease.

Cronins pre-transplant care was spearheaded by oncologists David Fisher, MD, and Nicole LeBoeuf, MD, MPH, clinical director of Cutaneous Oncology at Dana-Farber, with his transplant conducted by Corey Cutler, MD, MPH, medical director of the Adult Stem Cell Transplantation Program at Dana-Farber. Initially, Cronins disease was incredibly resistant; for nearly three years, mainstay drugs including steroids, monoclonal antibodies, and enzyme blockers all failed to put his disease into remission.

Ultimately, it would take a new drug, mogamulizumab (a type of immunotherapy that directly kills T-cells involved with Sezary Syndrome) to get Cronins disease into remission.

In May 2019, Cronin was cleared to undergo an allogeneic transplant, a type of transplant that uses a donors stem cells, in this case, Cronins brother. Since his transplant Cronin has remained in remission.

We had to use all of our big guns to get him totransplant, but Im pleased with where we are now, says Cutler.

I know the situation can always change, but it was great tobe able to share some good news with my family and friends, adds Cronin.

Patients like Cronin serve as a reminder of how stem cell transplants have improved and continue to impact patient outcomes, Dana-Farber experts note. Initially offered to only an incredibly small patient population when first performed at Dana-Farber in the 1970s, research advancements have, and continue to, broaden who is eligible for a transplant. In 2019, Dana-Farber/Brigham and Womens Cancer Center (DF/BWCC) surpassed 10,000 total adult transplants.

This milestone indicates our success as a program and our volume has allowed us to do the research to help move the field forward rather impressively, says Joseph Antin, MD, chief emeritus of Adult Stem Cell Transplantation at DF/BWCC.

In 1996, Dana-Farber Cancer Institute and Brigham and Womens Hospital merged their then separate transplant centers. By pooling together physical and intellectual resources, the new combined program was able to more than double the number of transplants each hospital could perform individually.

We always felt collaboration was better than competition, explains Robert Soiffer, MD, vice chair of Medical Oncology for Hematological Malignancies and chief of the Division of Hematologic Malignancies, who oversaw the merger with Antin. Each side could learn from the other, and that helped to catapult us into the leadership position we have today.

The Stem Cell Transplantation Program is also bolstered by the Connell and OReilly Families Cell Manipulation Core Facility (CMCF), which was established in 1996. The state-of-the-art center, led by Jerome Ritz, MD, not only processes the stem cells for transplant; it also assists researchers in developing new cell-based therapies for patients.

Another key component to the programs success has been the creation of the Ted and Eileen Pasquarello Tissue Bank. The Pasquarello Tissue Bank receives, processes, banks, and distributes research samplesof blood, bone marrow, and other tissues. Through a database overseen by Vincent Ho, MD, the Institute is able to log, assess, and later review every patients disease, including all complications and mutations. This technology allows researchers to explore the genetic makeup of past donors and better understand why a transplant was or was not successful.

Were still learning from biological specimens we collected 20 years ago, and it will continue to impact care 20 years from now, Soiffer says.

Today, there is a continuous push to develop new and more precise therapies to complement and improve stem cell transplants. The hope is to bring new treatment options to patients like Cronin who are facing rare and difficult diseases.

Before his diagnosis, Bill, and Barbara Finney, his partner ofnearly 30 years, were avid English Country dancers. English Country dancingevolved from the court dances of Europe in the early 17th century, and Croninand Barbara have friends from all over the country who share their passion forit.

While Cronin isnt dancing just yet, as hes stillrecovering from his transplant, he says he couldnt have gotten through thiswithout his partner on the dance floor and in life.

Barbara has been amazing and has helped take care ofeverything I couldnt do, he adds. Ive been fortunate and privileged to notonly have her, but to have been able to come to Dana-Farber.

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Transplant for Szary Syndrome is Patient's First Step in Returning to the Dance Floor - Dana-Farber Cancer Institute

Eternal youth and good health from stem cells soon within reach? – Innovation Origins

Could one of humankinds wildest dreams eternal youth and eternal good health perhaps come true after all? Well, were probably not quite there yet. But a new discovery in stem cell research may well constitute a step in this direction. Researchers led by biologist Vlad Cojocaru from the Dutch Hubrecht Institute, together with colleagues from the Max Planck Institute in Mnster, Germany, have discovered how normal human cells can be transformed into stem cells.

Differentiation between cell types is based on whether the DNA is read or not read at a specific point in time. Here, proteins known as transcription factors send the signal to read DNA in the cell or to halt the process of reading. Identity transformations, whereby cells move from an undesignated cell type to a designated cell type, occur naturally during development. However, these transformations can also be reversed. Japanese researchers were awarded the Nobel Prize in 2012 after they succeeded for the first time in reverse engineering a normal skin cell back to a stem cell.

However, it is not yet fully understood how this transformation of a skin cell into a stem cell takes place on a molecular level. A thorough understanding of the processes involving atomic details is essential if we are to produce such cells reliably and efficiently for individual patients in the future, says research leader Vlad Cojocaru. It is assumed that these types of artificially produced cells could in future be part of a remedy for diseases such as Alzheimers and Parkinsons. But the production process would have to become more efficient and predictive.

A key role in stem cell formation is played by a protein known as Oct4. This triggers the activity of the proteins that reset the adult cell as a stem cell. These genes are located in the same structure that stores the DNA in the nucleus the chromatin and are no longer active in the adult cells. Known as a pioneer transcription factor, Oct4 contributes to the opening-up of the chromatin and hence facilitates gene expression.

The study data reveals how the binding of Oct4 to DNA on the nucleosomes works. We have modelled Oct4 in various configurations, explains Cojocaru. The molecule consists of two domains, only one of which is capable of binding to a specific DNA sequence on the nucleosome at this stage of the process. With our simulations, we have been able to find out which of these configurations are stable. And how the dynamics of the nucleosomes influences the binding of Oct4. The models have been validated by experiments carried out by our colleagues Caitlin MacCarthy and Hans Schler in Mnster.

More IO articles on stem cell research here.

It was the first time that computer simulations were able to show how a pioneer transcription factor binds to nucleosomes that opens up the chromatin and regulates gene expression. Cojocaru explains that this computational approach for acquiring Oct4 models could also be used to screen other transcription factors and find out how they bind to nucleosomes.

In the following step, Cojocaru plans to fine-tune the current Oct4 models in order to find a definitive structure for the Oct4 nucleosome complex. We have known for almost 15 years that Oct4, together with three other pioneering factors, transforms adult cells into stem cells. But we still dont know how they work. The scientist emphasizes that the experimental structure definition for such a system is extremely expensive and time-consuming. Thats why he and his colleagues hope to use computer simulations in combination with a range of laboratory experiments to get a definitive model for the binding of Oct4 to the nucleosome. We hope that our definitive model will enable us to pioneer the development of transcription factors for the efficient and reliable production of stem cells and other cells required in regenerative medicine.

Results of the study were published in the Biophysical Journal.

Title photo: The pioneer transcription factor Oct4 (blue) binds to the nucleosome {a complex of proteins (green) and the DNA wrapped around these proteins (orange)}. Jan Huertas and Vlad Cojocaru, MPI Mnster, Hubrecht Institute.

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Eternal youth and good health from stem cells soon within reach? - Innovation Origins

How do body parts grow to their right sizes? – The Week Magazine

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Living things just seem to know how big to grow and how big to grow their sundry parts. A human liver maintains itself at just the right volume to do its job. A fruit fly's wings, on opposite sides of its body, somehow wind up the same size as each other, correctly scaled to sustain flight.

In everyday life, we expect body parts to be in proportion, because they usually are. "You notice if somebody comes up in front of you and one leg is way bigger than the other," says Ben Stanger, a gastroenterologist and researcher at the University of Pennsylvania's Perelman School of Medicine, who has studied organ growth.

But as much as we take this basic aspect of life on Earth for granted, scientists don't fully understand it. How do body parts know when to start and stop growing?

In some cases, cells seem to follow an intrinsic program carried out by the activity of their genes. At other times, cells appear to react to a cacophony of messages they receive from other cells and their environment, turning growth on and off as needed.

A lot of times, they seem to do a little of both. And when they're cancer cells, the whole business has gone awry.

"We don't get it," says Stanger, author of a 2015 article in the Annual Review of Physiology that described mechanisms that control liver growth.

Starting with salamanders

Scientists have been trying to "get it" for a long time. In the 1930s, Yale zoologists Victor Twitty and Joseph Schwind conducted experiments in salamanders, cross-transplanting limb buds from a smaller species, Ambystoma punctatum, with those of a larger but closely related species, Ambystoma tigrinium. In some experiments, the researchers found that taking a limb bud from the small salamander and grafting it onto the larger salamander resulted in an animal with three large limbs and one small one (and vice versa). This suggests that "the information for size was embedded in that group of cells very early on and didn't care what was happening in the animal," Stanger says.

But Twitty and Schwind found in other experiments that nutrition an external regulator also affected limb size. "It's nature and it's nurture," Stanger says. "In biology, it's never either/or."

Developmental biologists soon discovered a variety of ways that organs and structures achieve their ultimate sizes. In one famous 1960s experiment, researcher Donald Metcalf implanted 6 or 12 fetal mouse spleens into individual adult mice whose own spleens had been removed. He found that each implanted spleen grew to a proportional fraction of the size of a normal adult spleen leaving the animal with a normal total amount of spleen material. This suggests that spleen tissue has a way of understanding how much of it there is in relation to the body, says Jamie Davies, an experimental anatomist at the University of Edinburgh in Scotland. But "really annoyingly," Davies says Metcalf also found that multiple thymus grafts implanted in an adult mouse behave completely differently: Each grows to its full adult size.

Decades later, Stanger found similar growth differences in the mouse liver and pancreas: Cells that give rise to the liver use environmental cues to determine how much the developing organ should grow, while those that form the pancreas follow an "autonomous trajectory" they always achieve a preprogrammed size, no matter what is going on around them.

Pumping the brakes on growth

Scientists have sussed out a reasonable amount of detail about some of the feedback-based programs that direct growth. A protein called myostatin, for instance, helps to suppress muscle growth. When the tissues get large enough to pump out a threshold amount, muscle cells stop growing. The molecular processes that dynamically regulate liver size seem to involve tissues in the gut: When levels of bile acid fall (a sign of reduced liver function), those gut tissues produce factors that disengage a brake on liver growth known as the Hippo pathway. As a result, growth kicks into gear allowing the liver to grow to its proper size. When bile acid levels rise to normal, Hippo comes back on, and liver growth turns off again. And so on.

The Hippo pathway is a super-popular subject of study today, both because of its job in regulating organ size and because of its potential role in controlling cancers. Many questions about it remain unanswered.

Mysteries also remain for cases where instructions for size are baked in, as seen in those early experiments with salamander limbs, says Laura Johnston, a geneticist and developmental biologist at Columbia University Medical Center. Labs are delving into a number of inputs that may play a role in directing cells to grow, from information about cell fates and cell organization that are hardwired in the DNA, to mechanical forces on tissues.

Johnston's own research, some of which she and her coauthors described in a 2009 article in the Annual Review of Cell and Developmental Biology, focuses on a phenomenon known as cell competition interactions that lead to the deaths of unfit or unneeded cells. It seems to play a role in stabilizing organ size. When researchers in her lab blocked cell-death mechanisms in the cells that give rise to fruit fly wings, they found that the bell-curve-shaped distribution of wing sizes normally seen in fly populations broke down. A larger-than-usual number of flies developed overly large wings, or overly small ones. It's as if, she says, "the precision of size regulation is lost if the cells can't do these competing interactions."

There's still much to learn out about the deceptively simple, fundamental questions of how an arm matches its corresponding limb or how a liver ends up just the needed size. But the questions have practical ramifications too. Many growth studies today, including myriad explorations of the Hippo pathway, are conducted in the service of understanding and treating cancers. "Researchers are saying, 'Look cancer is development gone wrong, and it's obviously growth-connected, so we really need to understand growth on its own,'" Davies says.

Also interested in organ growth are researchers who want to engineer tissues using stem cells. "There's always the worry that if you build something that will grow up inside the body," Davies says, "will it know when to stop?"

This article originally appeared in Knowable Magazine, an independent journalistic endeavor from Annual Reviews. Sign up for the newsletter.

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How do body parts grow to their right sizes? - The Week Magazine

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