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Scientists Gain Crucial Insights into Traffic Cop Function of Gene Expression Protein

UNC scientists led by Brian Strahl, PhD, and Raghuvar Dronamraju, PhD, uncovered a key regulatory step in gene expression that may be a significant factor in cancers and other major diseases.

Media contact: Mark Derewicz, 984-974-1915, mark.derewicz@unchealth.unc.eud

December 18, 2018

CHAPEL HILL, NC – Scientists at the UNC School of Medicine have discovered a crucial quality-control mechanism inside cells that, when it fails, might contribute to major diseases including cancers.

The discovery, published in Cell Reports, concerns the process of gene transcription or gene expression – the copying of a gene’s DNA into RNA – the essential first step in turning genetic information into functional molecules such as proteins. The UNC-Chapel Hill scientists found that an enzyme called casein kinase II adds small chemical modifications or “molecular tags” onto Spt6, a key protein in the transcription process that enables Spt6 to wind back up the DNA after transcription. This rewinding prevents inappropriate transcription from occurring in the wrong direction along the gene. Such a “wrong way” situation can lead to serious health conditions.

“We made this finding in yeast cells, but all the players are found in human cells, too, and many of them are linked to human disease,” said senior author Brian Strahl, PhD, professor, vice chair, and Oliver Smithies Investigator in the department of biochemistry and biophysics and member of the UNC Lineberger Comprehensive Cancer Center.

How gene transcription works so efficiently and accurately most of the time is one of the great mysteries of biology, in large part because of DNA’s almost unbelievably tight packaging. The DNA in a single human cell would be about six feet long if unraveled, but somehow it manages to fit within the cell nucleus.

“One of the big questions in our field,” Strahl said, “is how cells manage to wind up so much DNA but keep it carefully organized and indexed so DNA can be accessed at the right place and time?”

Scientists know part of the answer already. The DNA in our cells is wound onto millions of spool-like structures made of proteins called histones. Each histone spool and its wrapped DNA is referred to as a nucleosome. When a cell needs to transcribe a stretch of DNA, the histones and their helper proteins called “histone chaperones” loosen up the nucleosome so that the molecular machinery of transcription can access the hidden DNA. When the process of transcription is complete, the histone chaperones then usually put nucleosomes back together and keep DNA firmly wrapped. How this crucial and complex job is performed so well across the genome is not fully known.

In their new study, Strahl and his team found an important piece of this puzzle: a new function of an enzyme that makes a histone chaperone function properly, which in turn helps prevent the serious errors in DNA-wrapping that lead to disrupted gene transcription. Both the enzyme casein kinase II and the histone chaperone Spt6 were already known to be involved in gene transcription, though their precise roles were unclear.

The UNC scientists found that casein kinase II activates Spt6 in a way that allows the chaperone protein to work with histones to properly batten down DNA. The enzyme performs this activation by chemically attaching phosphorylation groups (i.e., molecular tags) onto Spt6. “Phosphorylation” is a common method by which cells switch a protein’s function from “on” to “off” or the other way around.

To study the importance of the phosphorylation on Spt6, the UNC scientists reengineered Spt6 slightly so that it could no longer be modified by the kinase, and found that Spt6 wasn’t performing its job well in putting nucleosomes back together. Curiously, this effect was more pronounced on the nucleosomes at the starting point of gene transcription, which led to a peculiar form of abnormal transcription.

“We observed an increase in transcripts that run from the start of the gene backwards through the gene’s regulatory or so called “promoter” region – where transcription begins – not downstream as normal,” said study first author Raghuvar Dronamraju, PhD, a research assistant professor in the Strahl laboratory.

The scientists concluded that casein kinase II’s crucial phosphorylation of Spt6 allows it to place histones back at the beginning of genes in order to help it enforce normal gene transcription in the right direction.

“It’s like a traffic cop at an intersection, allowing cars to travel in one direction but not another,” Strahl said.

This basic science discovery could lead to medically important insights because loss of the ability to control transcription can be harmful to cells, for example by allowing stretches of DNA that don’t code for genes to be expressed. This can interfere with normal transcription. It can also lead to neighboring genes being improperly activated if nucleosomes are not re-inserted at their promoters to block new transcription after a gene is supposed to be shut down.

Such transcriptional mis-regulation can result in serious diseases such as cancer (for example, if a tumor suppressor gene is affected). In fact, any derangement of the normal dynamics of DNA packaging is potentially a cause of major illness. Strahl said many of the proteins involved in packaging and unpackaging our DNA have been found at abnormal levels or are mutated in many cancers, and some of these proteins are being eyed as potential cancer drug targets.

In follow-up experiments, Strahl, Dronamraju, and colleagues hope to determine how exactly Spt6 exerts its DNA-organizing effect particularly at the upstream ends of genes and how it interacts with other histone chaperone proteins.

The National Institutes of Health (GM126900, CA166447, CA198482), the Corn-Hammond Fund for Pediatric Oncology, and the National Science Foundation (MCB 1515748) funded this research.

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Scientists discover new gene expression mechanism with possible role in human disease

UNC School of Medicine researchers, led by Brian Strahl, PhD, found surprising role for a protein called Spt6, which is crucial to the maintenance of proper messenger RNA levels in cells, a discovery that opens new research avenues and suggests a target for basic understanding, diagnosis, and treatment of human diseases.

June 21, 2018 CHAPEL HILL, NC –When cells grow and divide to ensure a biological function – such as a properly working organ – DNA must be unwound from its typical tightly packed form and copied into RNA to create proteins. When this process goes awry – if too little or too much RNA is produced – then the result could be diseases such as cancers. UNC School of Medicine researchers have discovered that a protein called Spt6, previously known to have a key role in making RNA and repackaging DNA after RNA copying, also facilitates RNA degradation so that cells have just the right amount of RNA for the creation of proteins. 

The discovery, published in Molecular Cell, represents a revolutionary new understanding of gene expression control and suggests a potential target for treating cancers and other diseases.

“By revealing and understanding this mechanism, we can start to think about targeting parts of it therapeutically in diseases in which Spt6 isn’t working properly,” said study senior author Brian D. Strahl, PhD, the Oliver Smithies Investigator and Professor and Vice-Chair in the Department of Biochemistry & Biophysics at UNC-Chapel Hill.

Every human cell carries a large amount of DNA – called the genome – composed of roughly 3.5 billion letters that assemble into the genetic code. Researchers have been studying how large genomes fit into the tiny confines of cells. We know that proteins called histones carefully organize and package DNA in cells. Much like wrapping yarn around its spool, the DNA wraps around the histones to be condensed into a smaller space. Although histones help to keep DNA packaged, this packaging creates a barrier to “reading” the genetic information housed within DNA. The DNA needs to be “opened” much like a book needs to be opened for the pages to be read – except that “opening DNA” is a little complicated.

Accessing DNA information is a highly controlled process that involves temporarily removing the histones so the genetic code can be copied into RNA and the RNA can then be used to create proteins. Normally, cells destroy the copied RNA “messages” once they are no longer needed. Diseases such as cancer may arise when the ability of cells to either produce or destroy the messages goes awry.

When a gene is copied into a strand of RNA, the DNA in and around the gene must be loosened from its normal tightly wound configuration. Scientists have known that Spt6 has the crucial job of helping DNA become tightly re-wound when the copying process is completed. But that’s not its only function. “Spt6 seems to be a bit like a Swiss Army Knife,” said Strahl, a member of the UNC Lineberger Comprehensive Cancer Center. “Spt6 has many functions, from helping the cell create messenger RNAs, to putting histones back onto the DNA after they were removed. Our study now shows that Spt6 also helps control how much of the messenger RNA remains after it’s copied from DNA.” The first thing Strahl’s lab investigated was how Spt6 binds to RNA Polymerase II, which is the enzyme machine that copies DNA into RNA. The function of this Spt6-Polymerase II interaction has been unclear. So the Strahl lab wanted to determine whether a non-binding version of Spt6 still performed its DNA-histone rewrapping function. 

“To our surprise, we found that Spt6 was still able to get to genes, although at sub-optimal levels,” Strahl said. “But Spt6 still did its job of adding back histones.” 

Although Spt6 still functioned, the researchers witnessed a big problem: the RNA amounts were extremely high, and these high RNA amounts did not occur because there was more copying with the defective form of Spt6. “It dawned on us that there is more to Spt6 function than just re-wrapping the DNA around histones and facilitating RNA Polymerase copying of DNA,” said first author Raghuvar Dronamraju, PhD, research assistant professor in Strahl’s lab.

The researchers measured the amounts of all the RNAs in cells that had the mutant form of Spt6 and found abnormal amounts of many RNAs. This suggested there was a loss of the usual control mechanism that maintained just the right amount of each RNA.

It wasn’t clear at first how the disruption of Spt6’s binding to the polymerase caused RNA misregulation, but further experiments revealed a completely unexpected mechanism. 

Normally, RNAs in the process of being made are exposed to enzymes that protect or degrade them so that the cumulative actions of these enzymes create a precise amount of RNA that a cell needs for protein synthesis. The UNC scientists found that the form of Spt6 that could not bind to RNA Polymerase II disrupted this balance between RNA protection and RNA degradation, specifically the degradation side. They found that many RNAs survived in cells longer than they normally would have, allowing the RNA levels to rise to abnormal levels. 

Strahl’s team went further and connected the dots to show that Spt6 interacted with one of the cell’s major RNA degradation machineries – a protein complex called Ccr4-Not. Strahl’s team showed that Spt6 used its interaction with RNA Polymerase II to recruit Ccr4-Not during gene expression to ensure the proper balance of enzymes that protect and degrade RNA.

Moreover, the researchers discovered that mutant Spt6 did not affect the levels of all RNAs. A large number of affected RNAs encode proteins that control cell division. Ordinarily, RNAs that contribute to cell division are rapidly degraded as cells pass from one part of the cell division cycle to another. But the abnormal failure to remove these RNAs in the mutant Spt6 cells caused the cells to develop profound growth and cell division defects. 

The study by the Strahl lab thus revealed a previously unknown, fundamental mechanism of RNA degradation, and the results suggest that defects in the RNA degradation function of Spt6 may underlie some diseases, particularly cancers, which feature uncontrolled cell division. 

“Given Spt6 in humans is sometimes found mutated or misregulated in cancers, it will be important to examine this RNA control mechanism further to determine whether its failure contributes to cancer,” Strahl said. His team will turn to researching this with the hope that future studies could identify new therapeutic targets to treat human disease.

The researchers still have many questions about Spt6’s involvement in regulating RNAs. But already it’s apparent that Spt6’s influence on RNA stability represents “a new twist in transcription,” as Strahl calls it. 

This research was performed with baker’s yeast, a classic basic science organism that researchers use to investigate the intricate details of how cells perform and control many biological functions. Importantly, the yeast studies can be extended to human cells because the same proteins occur in yeast and humans. 

Other co-authors are Austin Hepperla, Yoichiro Shibata, PhD, Alexander Adams, Terry Magnuson, PhD, and Ian Davis, PhD.

The National Institutes of Health and the Corn-Hammond Fund for Pediatric Oncology funded this research

Media contact: Mark Derewicz, 984-974-1915, mark.derewicz@unchealth.unc.edu

The protein Spt6 wears many hats, including one for RNA degradation, which the lab of Brian Strahl, PhD, discovered. (By Christ-claude Mowandza-ndinga, UNC Health Care)

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Brian D. Strahl Awarded NIGMS Outstanding Investigator Grant

Brian D. Strahl, Professor of Biochemistry & Biophysics and Oliver Smithies Investigator, has been awarded a 5-Year Outstanding Investigator R35 Maximizing Investigators’ Research Award (MIRA) for his research on gene expression and chromatin regulation. The goal of the MIRA award is to increase the efficiency of NIGMS funding by providing investigators with greater stability and flexibility to enhance scientific productivity and the chances for important breakthroughs.  

Brian D. Strahl, PhD, receives R35 MIRA award

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UNC Researchers show how a cancer gene protects genome organization

UNC study uncovers crucial function of a yeast enzyme Set2 whose well-conserved human counterpart is often mutated in cancers, especially kidney cancer.

(Illustration by Christ-claude Mowandza-ndinga)

June 13, 2017

CHAPEL HILL, NC – UNC School of Medicine researchers have cracked a long-standing mystery about an important enzyme found in virtually all organisms other than bacteria. The basic science finding may have implications for understanding cancer development and how to halt it.

Researchers have known that the enzyme Set2 is important for transcribing genes – the process of making strands of RNA from the DNA. Transcription is critical for making proteins and other functional molecules. But Set2’s precise role in transcription hasn’t been clear. Now, UNC scientists discovered that the enzyme is particularly important for keeping transcription working properly when cells are under stress. Without Set2, cells that become stressed through the lack of nutrients begin mis-transcribing genes in a way that prevents cells from adapting properly to the stress.

“We think this solves a mystery about the purpose of Set2, and we now understand much better how gene transcription is prevented from happening at the wrong place and time,” said study senior author Brian Strahl, PhD, professor of biochemistry and biophysics and member of the UNC Lineberger Comprehensive Cancer Center.

Set2 enzymes in yeast and other lower organisms have close relatives in all animal species and plants. Its human cousin SETD2 is often found mutated in cancerous cells.

“These fundamental findings may help explain how SETD2 mutations could lead to inappropriate transcription within genes, which might then promote cancer initiation or progression,” Strahl said. His team’s research on SETD2 is ongoing.

The research, published in Cell Reports, involved collaboration between Strahl’s laboratory and that of Ian J. Davis, MD, PhD, associate professor of pediatrics and genetics at the UNC School of Medicine and member of the UNC Lineberger Comprehensive Cancer Center.

The discovery comes 15 years after the first studies of Set2 by Strahl and others, who found that the enzyme works by attaching molecules known as methyl groups to a support protein – or histone – around which DNA is spooled.

This methyl-attaching process is called methylation. Research has shown in recent years that the particular histone methylation performed by Set2 serves as a quality control check on gene transcription.

Transcription of a gene should start at a precise spot at the beginning of a gene and then continue until the end in order to fully transcribe the RNA. But in the absence of histone methylation laid down by Set2, transcription begins at the wrong places in the middle of a gene instead of at the beginning. If that is allowed to happen, the production of “cryptic” RNA transcripts can then interfere with the normal expression of a gene. The mis-expression of our genetic material can result in diseases such as cancer.

Strahl’s team thought Set2 might have something to do with these cryptic transcripts arising during stress. Previously, it was shown that Set2’s histone-methylating activity has the effect of attracting another enzyme to clear away chemical tags in the middle of a gene that, otherwise, can lead to inappropriate new transcription from within that gene.

“But under typical laboratory conditions, the deletion of Set2 and the subsequent increase in cryptic transcripts didn’t seem to harm cells very much,” Strahl said.

Strahl’s team then thought about cells under stress, which is what cells are like in disease states. His team conducted experiments to observe what happens in cells that don’t have Set2 when vital nutrients are removed. In this stressed state, cells normally activate a complex set of gene expression programs to help cope with the reduced nutrient resources.

“Nutrient depletion more accurately mimics what yeast cells experience in the wild,” Strahl said.

The scientists examined yeast cells that were deprived of nutrients, or were exposed to chemicals that reliably trigger the low-nutrient response. In these cells, not having Set2 proved to have major consequences.

“We found that this inappropriate transcription at the wrong place in genes exploded to high levels in stressed cells, and often interfered with the normal genes,” Strahl said. “As a result, the normal changes in genes that help cells survive under low-nutrient conditions did not happen correctly, and the cells became extremely sick.”

To Strahl and colleagues, the finding suggests that Set2 evolved to guard against harmful abnormal transcription in times of stress, when cells seem particularly vulnerable to this type of error. Why would cells be so vulnerable to cryptic transcription during the nutrient stress response? Strahl isn’t sure. But his team suspects that when there’s a sudden and widespread rearrangement of the molecular machinery of gene transcription, genes across the genome are left relatively open to inappropriate transcription.

“We found that a lot of the genes that show this crazy jump in cryptic transcription were not even related to the nutrient stress response,” Strahl said. “It’s as if there are genes throughout the genome that are just predisposed to this error, especially at this time when transcription is shifting dramatically.”

Strahl and Davis and their colleagues plan further research to determine why cryptic transcription rises so dramatically during nutrient stress. They also intend to find out whether Set2 is important for safeguarding transcription during other types of cellular stress.

In addition, the scientists are now studying Set2’s human counterpart, SETD2, which for unknown reasons is often mutated in tumor cells, especially in kidney cancers.

“It’s possible that SETD2 normally works as a major tumor suppressor by preventing inappropriate transcription,” Strahl said.

The lead author of the study was Stephen L. McDaniel, PhD, a former graduate student in the Strahl Laboratory. Other co-authors were Austin Hepperla, Jie Huang, Raghuvar Dronamraju, PhD, Alexander T. Adams, and Vidyadhar G. Kulkarni, PhD.

The National Institutes of Health funded this work.

Media Contact: Mark Derewicz, 919-923-0959, mark.derewicz@unchealth.unc.edu

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Strahl lab 2017!

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New Strahl lab photo for 2015

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New antibody portal bolsters biomedical research reliability

UNC’s Brian Strahl, PhD, and Van Andel Research Institute’s Scott Rothbart, PhD, create a robust online interactive database to address science’s ‘antibody crisis’.

Media contacts: Mark Derewicz, UNC School of Medicine, 919-923-0959, mark.derewicz@unch.unc.edu; Beth Hinshaw Hall, Van Andel Research Institute, 616-234-5519, beth.hinshawhall@vai.org

July 23, 2015

CHAPEL HILL, NC – For years, a crisis has been brewing in molecular biology. The problem is that antibodies – research tools used to identify key proteins at work in a cell – aren’t always what they seem. Unreliable antibodies have led to numerous instances of false findings, failed experiments, and wasted money and samples.

Enter the Histone Antibody Specificity Database (www.histoneantibodies.com), a newly launched online portal that lets scientists find the right antibodies for their research with a much higher degree of confidence than ever before. Rather than relying on the claims of antibody manufacturers, the database is populated with validated test results, allowing researchers to access and compare real-world data and pick the most reliable antibody for each experiment.

A paper published today in the journal Molecular Cell describes the database and the science behind it.

“I can’t think of any field of biomedical research that doesn’t rely on antibodies; they’re typically the linchpin in many studies,” said Brian D. Strahl, PhD, professor of biochemistry and biophysics in the University of North Carolina School of Medicine and the study’s co-senior author. “But how reliable are these tools? There’s been a real awakening in the biological community that we need better ways to know if the antibodies are recognizing what they’re intended to recognize. That’s what we aim to accomplish with this portal.”

The web portal focuses on histone antibodies, a subset of antibodies that are central to the fast-growing field of epigenetics. Epigenetics researchers examine how layers of information outside the genome control the ‘on’ and ‘off’ state of genes, much like a light switch, that in turn affect all aspects of human health. Scientists use histone antibodies to track the roles of various proteins in this vital process of gene regulation. 

“Epigenetic regulation of our genome is at the core of all aspects of biology, and basic research in this area is uncovering key mechanisms that contribute to human health and disease,” said Scott B. Rothbart, PhD, assistant professor in the Center for Epigenetics at Van Andel Research Institute (VARI) and co-corresponding author of the study. “Drugs targeting the epigenetic machinery are being tested in oncology clinics now, and a better understanding of what the antibodies are recognizing will help researchers define more precisely how epigenetics works and ultimately lead to better, stronger conclusions that can make a difference for drug discovery and future therapeutic interventions.”

 Initially developed in the 1970s, laboratory-produced antibodies have been commercially available to scientists for decades. Since then, many researchers have simply ordered antibodies from among the hundreds of thousands on the market, taking their labels at face value. But a growing body of evidence has revealed that an alarming number of antibodies do not perform as advertised.

Each histone antibody is supposed to bind to a specific protein target, allowing scientists to track where and how that protein is being regulated in healthy and diseased cells. However, some antibodies have been observed binding to other proteins in addition to their primary target while other antibodies have been shown to bind to just one target, but not the target indicated on the product label. In some cases, an antibody’s accuracy and specificity vary from one lot to the next, suggesting quality-control issues in the manufacturing process.

All of these errors muddy – and in some cases, completely undermine – research results.

While some vendors have stepped up their testing and quality control efforts, others have not, and few researchers have the resources or time to do the battery of tests recommended to ensure their antibodies are performing as indicated.

The new database offers an easy way to access detailed information about the accuracy and specificity of more than 100 commonly used and commercially available histone antibodies.

The testing method used to populate the database is based on custom-designed peptide microarrays that Strahl and his colleagues developed. The arrays present a library of all of the chemical modifications or “tags” known to occur on histone proteins. Analyzing antibodies that are developed to target these different tags on the arrays quickly illuminates the types of tags to which each antibody is capable of binding. This matters a lot. Each tag is thought to have a different function. For example, turning a gene ‘on’ or ‘off’.

Once word got out about the team’s peptide array platform, Strahl and his colleagues started fielding calls from other scientists asking for advice on antibody selection. Such interest from the scientific community sparked the team’s idea to buy all of the most common histone antibodies, test them on the array, and publish the results in an online portal accessible to everyone.

“We wanted it to be much more than a simple search-and-go database,” Strahl said. “Peptide arrays like the ones we developed are now available commercially, so any researcher, antibody manufacturer, or pharmaceutical company can run these tests. We built the portal so that people can easily upload their own results and the database can continue to grow.”

The portal allows users to search for antibodies using multiple parameters. A quality-control plan is in place to ensure the validity of user-submitted data.

Strahl said he hoped the database would enable more accurate and reproducible epigenetic findings.

“You don’t expect every antibody to be 100% perfect, but we think it’s important for researchers to be able to know what the flaws are,” Strahl said. “That way, at least people can interpret their results with the right amount of caution based on what the antibodies see – or don’t see.” 

This work was supported by grants from the NIH and the W.M. Keck Foundation

Additional UNC coauthors include Terry R. Magnuson, PhD, the Sarah Graham Kenan Professor and chair of the department of genetics in the UNC School of Medicine; Erin Shanle, PhD, a postdoctoral research associate in Strahl’s lab; Jesse R. Raab, PhD, a postdoctoral fellow in Magnuson’s lab; Krzysztof Krajewski, PhD, research assistant professor; and Angela H. Guo, an undergraduate researcher in Strahl’s lab. Other coauthors include Bradley M. Dickson, PhD, a computational biophysicist in Rothbart’s lab at VARI, graduate student Adrian T. Grzbowski and Alexander J. Ruthenburg, PhD, of the University of Chicago; Steven Z. Josefowicz, PhD, and David Allis, PhD, of The Rockefeller University; and Stephen M. Fuchs, PhD, of Tufts University.

Van Andel Institute (VAI) is an independent biomedical research and science education organization committed to improving the health and enhancing the lives of current and future generations. Established by Jay and Betty Van Andel in 1996 in Grand Rapids, Michigan, VAI has grown into a premier research and educational institution that supports the work of more than 270 scientists, educators and staff. Van Andel Research Institute (VARI), VAI’s research division, is dedicated to determining the epigenetic, genetic, molecular, and cellular origins of cancer, Parkinson’s, and other diseases, and translating those findings into effective therapies. The Institute’s scientists work in on-site laboratories and participate in collaborative partnerships that span the globe. Learn more about Van Andel Institute or donate by visiting www.vai.org. 100% To Research, Discovery & Hope®

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UNC Program in Chromatin and Epigenetics Launches

The new UNC Program in Chromatin and Epigenetics aims to address the fundamental gaps inknowledge surrounding epigenetic regulation, with a long-term goal of developing novel therapeutic approaches towards treating human disease. The program is centered around a highly collaborative and team science environment that has a dedicated goal of solving fundamental and challenging problems in chromatin biology – with an emphasis on developing novel approaches towards treating human disease. The large number of research faculty at UNC specializing in different areas of epigenetic research are optimally positioned to meet this challenge.

 Faculty at UNC are focused on the basic mechanisms of epigenetic regulation using a variety of model organisms, high-throughput drug discovery efforts to target epigenetic machinery and clinical research that emphasizes epigenetic translational science employing the knowledge gained by basic research and drug discovery efforts. These researchers employ a range of cutting edge technologies, such as genomic and proteomic techniques, to address a vast number of epigenetic problems – and this research is supported by top-notch core facilities. The Chromatin and Epigenetics Program currently encompasses 34 faculty members, and participation of several laboratories from the National Institute of Environmental Health.

 Along with the many resources and events associated with this new program, a highlight is the Chromatin and Epigenetics Certificate Program. Doctoral students interested in being formally part of the Program in Chromatin and Epigenetics can apply for entrance into a certificate program. The goal of this program is to provide students with more emphasis on epigenetic mechanisms and further exposure to approaches and techniques used in epigenetic research.

 The leadership behind the program are Terry Magnuson, PhD, and Brian Strahl, PhD. Magnuson is a professor and chair of Genetics at UNC and the Executive Associate Dean for Research for the School of Medicine. Work in the Magnuson lab focuses on the role of mammalian genes in unique epigenetic phenomena such as genomic imprinting, X-chromosome inactivation, stem cell pluripotency and the tumor suppressor role of chromatin remodeling complexes. Strahl is a professor and director of graduate studies of Biochemistry and Biophysics at UNC. The Strahl lab addresses how histone post-translational modifications, and the combinatorial codes they create, contribute to the structure and function of chromatin.

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Scientists from UNC-Chapel Hill have created a new way to investigate epigenetic mechanisms important in diseases ranging from Alzheimer’s to cancers.

Robert Duronio, PhD; Daniel McKay, PhD; Greg Matera, PhD; Brian Strahl, PhD. 

CHAPEL HILL, NC –University of North Carolina scientists have created a new research tool, based on the fruit fly, to help crack the histone code.  This research tool can be used to better understand the function of histone proteins, which play critical roles in the regulation of gene expression in animals and plants.

This work, published in the journal Developmental Cell, opens the door to experiments that are expected to uncover new biology important for a host of conditions, such as neurological diseases, diabetes, obesity, and especially cancer, which has become a hotbed of epigenetic research.

“People think cancer is a disease of uncontrolled proliferation, but that’s just one aspect of it,” said Robert Duronio, PhD, professor of biology and genetics and co-senior author. “Cancer is actually a disease of development in which the cells don’t maintain their proper functions; they don’t do what they’re supposed to be doing.” Somehow, the gene regulation responsible for proper cell development goes awry. 

One aspect of gene regulation involves enzymes placing chemical tags or modifications on histone proteins – which control a cell’s access to the DNA sequences that make up a gene. Properly regulated access allows cells to develop, function, and proliferate normally. The chemical modification of histones is thought to be a form of epigenetic information – information separate from our DNA – that controls gene regulation. This idea is based on the study of the enzymes that chemically modify histones. However, there is a flaw in this argument.

“In complex organisms, such as fruit flies, mice, and humans, scientists have only been able to infer how these enzymes mechanistically accomplish their tasks,” said Daniel McKay, PhD, assistant professor of genetics and biology and first author of the paper. “It’s been technically impossible to directly study the role of histone modifications. Now, through our collaboration between UNC biologists, we’ve been able to develop a tool in fruit flies to directly test the function of histones independently of the enzymes that modify them.”

This is crucial because therapies, such as cancer drugs, can target histones. With this new research tool, scientists will be able to better study thousands of enzyme-histone interactions important for human health.

 “If you think of the genome as a recipe book, then you could say we’ve made it possible to know that there are hidden ingredients that help explain how specific recipes turn out correctly or not,” said Greg Matera, PhD, professor of biology and genetics and co-senior author of the paper.  “That’s the first step in scientific discovery – knowing that there are things we need to look for and then searching for them.”

 Beyond Yeast

Before now, a lot of this epigenetic research had been done in yeast – single cell organisms that also use enzymes to lay chemical tags on histone proteins. This work has yielded many interesting findings and has led to the development of therapeutics. But some of this work has led to an oversimplification of human biology, leaving many questions about human health unanswered.

For instance, in complex organisms, enzymes in cells typically do more than one thing. One likely reason for this is that animals undergo cellular differentiation; human life begins as a single cell that differentiates into the various cell types needed for different organs, body parts, blood, the immune system, etc. This differentiation has to be maintained throughout life.

“Because of this, animals likely have a greater requirement for epigenetic regulation than yeast do,” Matera said. “Animal cells have to ‘remember’ that they must express genes in specific ways.” When cancer cells start dividing rapidly to form tumors, these cells are actually reverting to an earlier time in their development when they were supposed to divide rapidly. The gene regulation that was supposed to rein them in has gone haywire.

Whereas in yeast, a histone-modifying enzyme might have a single regulatory task, the human version of that same enzyme might have other regulatory tasks that involve additional proteins.

“In fact, maybe the really critical target of that one modifying enzyme is some other protein that we don’t know about yet,” Matera said. “And we need to know about it.”

The best way to figure that out would be to make it impossible for the enzyme to modify a histone by changing – or mutating – the histone protein. If a histone protein could be disabled in this way and cells still behaved normally, then that would mean there was some other protein that the enzyme acted on. To do this, however, would require replacing a histone gene with a genetically engineered one that could not be modified by an enzyme.

The problem is that in animals, such as mice and humans, there are many histone genes and they are scattered throughout the genome. This makes replacing them with ‘designer’ histone genes difficult.  In addition, other genes are located in between the histone genes. Therefore, deleting the portion of the chromosome with histone genes in order to replace them with a modified one would wind up deleting other genes vital for survival. This would make such an approach in, say, a mouse, useless.

“It has been technically impossible to do this kind of research in complex organisms,” Duronio said. “But fruit flies have all their histone genes in one place on the chromosome; this makes it feasible to delete the normal genes and replace them with designer genes.”

Designer genes

Matera, Duronio, and McKay led an effort to delete the histone genes in fruit flies and replace them with specific designer histone genes they created. These new genes were created so they could not be the repositories of epigenetic tags or modifications. That is, the modifying enzyme would not be able to do its job on that particular protein.

As shown in the Developmental Cell paper, the researchers put their new tool to the test. They “broke” one histone protein that had been identified to interact in a specific way with a modifying enzyme, and they got the outcome in fruit flies they expected. But for another enzyme-histone interaction, the researchers got an unexpected result.

 Previously, in mammalian cells, other researchers had discovered that when you mutate a specific modifying enzyme, the result is death because the cells can’t replicate.

With their new fruit fly research model, the UNC researchers altered the histone gene so that this particular enzyme could not modify its histone protein target. The result was not death. In fact, the flies lived and flew as normal flies do. This meant that the enzyme, which was previously proven to be vital to life, must do something else very important.

 “There must be another target for that modifying enzyme,” Matera said. “There must be another hidden carrier of epigenetic information that we don’t know about.”

McKay added, “This is a demonstration of the potential of our epigenetic platform. Going forward, we’re going to do a lot more experiments to identify more discrepancies and hopefully other targets of these enzymes. We’re on the ground floor of a long-term project.”

 This research shows that the epigenetic recipe book for yeast is thin. The recipe book for humans, which is genetically akin to the one for fruit flies, is much thicker, more complex, and full of hidden ingredients scientists have yet to discover. Now, scientists have a tool to test the recipes.

Brian Strahl, PhD, professor of biochemistry and Biophysics in the School of Medicine, is also a principal investigator of this study.

Duronio, Matera, and McKay are professors in the UNC School of Medicine and the College of Arts and Sciences.

Strahl, Duronio, and Matera are members of the UNC Lineberger Comprehensive Cancer center. Matera, Duronio, and McKay are members of the Integrative Program for Biological and Genome Sciences. Duronio is the director of the program.

National Institutes of Health and the University of North Carolina funded this research.

Media Contact: Mark Derewicz, 919-923-0959, mark.derewicz@unchealth.unc.edu

February 9, 2015

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All the Cell’s a Stage

Brian Strahl, PhD, and his band of biochemists unravel the complicated mysteries of the epigenetic code to find a culprit in cancer development.

By Mark Derewicz

  Every single human cell contains every single human gene.  But depending on the cell, only some of these genes need to be expressed or “turned on.” For instance, a heart cell has all the genes needed for, say, proper kidney function. But that heart cell won’t express those genes. In a heart cell, those genes are “turned off.” When one of these “wrong” genes is turned on by mistake, the result can be rampant cell growth – cancer.

How this happens used to be the stuff of science fiction. Now, scientists know that there are tiny proteins –epigenetic proteins - that sit atop the genetic code inside cells. These proteins are responsible for turning on or off the genes.

Now, UNC researchers discovered that one gene-regulating protein called Bre1 must be maintained in the proper amount for other epigenetic players to do their jobs properly. It’s a key coordinator in the sort of cellular scenes that can turn a healthy cell into a cancer cell.

Setting the scene

Within each cell of the body is an ongoing and intricate performance with genes playing some of the leading roles. As with all performances, the actors do not act alone, but instead, rely on support from behind the scenes. This supporting staff provides the script and cues for what the genes are supposed to say and do – how genes are accessed and used.  Important members of the support staff are histones – the proteins that package genes inside cells and allow them to be used for various cellular functions that keep us healthy; they allow the plot to unfold perfectly.

Unfortunately, sometimes cues are missed or lines are forgotten and the show doesn’t go as planned.  This causes the actors to speak when they should be quiet or stay quiet when they should speak.  And if one of these actor genes happens to be essential for, say, cell growth, then the result can be disastrous. The actors take the story in an unintended direction.

All this supporting staff is part of epigenetics – epi meaning on or above – a field that focuses on the environment and the players that allow our genes to act.

“I think epigenetics is a new frontier of cancer research,” says Brian Strahl, Ph.D., a professor of biochemistry and biophysics in the UNC School of Medicine. “We can now sequence the entire genome of a cancer cell, and what we’re finding is that many cancers have mutations in the epigenetic machinery. We’re not just finding this in cancer cell lines in the lab but in cancer patients.”

The director’s cut

Strahl, who’s a member of the UNC Lineberger Comprehensive Cancer Center, said major questions surround how histones wrap up the DNA into chromatin – a structure that allows or denies access to the genetic information inside our cells.

This is what Strahl studies. His goal is to figure out precisely how histones contribute to basic biological functions and, in turn, contribute to cancers and other diseases.  Adding a twist to this idea, however, is the fact that not every histone is the same.

“We’ve already learned that the histone proteins found at the sites of genes can be chemically modified with a variety of small chemical “tags” that either promote or further prevent access to our genetic information – our DNA. And this access or denial ultimately affects genes so they are either activated or not.”

These chemical tags come from a variety of sources – mainly the food we eat, the chemicals in the environment that gets inside us through our skin and lungs, for example, and the various biological chemicals that simply make us tick. Proper nutrients, for instance, allow for the formation of chemical tags to direct the histones to activate genes in the proper ways. Nasty environmental stuff, such as cigarette smoke, can mess up the epigenetic machinery.

Yet, these chemical tags are not ultimately in charge of the genes.  Another layer of proteins above the histones are responsible for putting on the chemical tags.

“Something has to ensure that these chemical tags on histones are regulated properly, to ensure that the tags are only present on the right genes at the right time,” Strahl said.

Strahl and graduate student Glenn Wozniak focused on one of the proteins that add these chemical tags – a protein called Bre1, which keeps one tag –  ubiquitin – in check.

In a sense, Bre1 hires ubiquitin; it allows ubiquitin to do its job.

Ubiquitin is known to help a histone open up the cell’s chromatin to expose genes for activation. When ubiquitin is finished, it is removed from the histones, and the genes become inactivated.

If this process goes awry – if the genes are allowed to remain active indefinitely – then normal cells can turn into cancer cells. And the entire cellular performance collapses.

The Goldilocks effect

Until now, how this happened was unclear. Through a series of experiments, Strahl and Wozniak found that, like the chemical tags themselves, a precise amount of Bre1 must be maintained to ensure that just the right amount of ubiquitin is added to histones.

“We found that if there’s too little Bre1, then the gene doesn’t turn on,” Strahl said. “If there’s too much, the gene doesn’t shut off. We call it the Goldilocks effect.”

Wozniak added, “We also found that when Bre1 is not needed or when it doesn’t perform its function, it’s removed as a control mechanism. There won’t be as much ubiquitin on histones because Bre1 is not there.”

Strahl and Wozniak’s finding illuminates what had been an epigenetic mystery. Scientific literature on Bre1 had been mixed.

“Some studies indicated that Bre1 had a role as a tumor suppressor,” Strahl said. “Other studies showed that it’s a cancer promoter. So there’s been conflicting evidence about all of this. Now we know. If there’s too little Bre1, the gene won’t turn on.” This could turn off the genes that protect the cell from cancer. “If there’s too much,” Strahl said. “Then the genes might not turn off.” This could also trigger cancer development.

 “When you think about it, Bre1 could be a really good target for a cancer drug,” Strahl said. “Cancer cells divide rapidly. A lot of chemotherapies involve creating DNA damage within all rapidly dividing cells. But if you just target the Bre1 protein and maybe shut it off, you could have very bad outcomes specifically for rapidly dividing cancer cells. They wouldn’t be able to transcribe genes anymore.”

Strahl and Wozniak’s study appeared in the journal Genes and Development. The National Science Foundation funded this work

      Illustration by Max Englund/UNC Health Care

New story posted here

                                              Brian Strahl, PhD

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Brian Strahl receives NIH and NSF grants to study gene expression

Congratulations to Dr. Brian Strahl, Professor of Biochemistry and Biophysics for receiving grants from the NIH and NSF

The National Institutes of Health and the National Science Foundation have given Dr. Brian Strahl grants to study the basic functions of gene regulation in the context of chromatin (DNA that is compacted by the action of histone proteins).  One grant is centered on the role of histone chaperones, which are proteins that insert or remove histones in the chromatin environment, and the other is on how enzymes that modify histones with chemical “tags” or post-translational modifications regulate the opening and closing of chromatin to make the DNA assessable for gene transcription.  These grants will significantly advance our understanding of gene expression.

Brian Strahl obtained his PhD in 1998 from North Carolina State University before performing his postdoctoral studies with Dr. C. David Allis at the University of Virginia. In 2001, Brian joined the faculty at the University of North Carolina at Chapel Hill, where his group has been addressing how the chemical “tags” on histones influence the structure and function of chromatin.  Dr. Strahl’s scientific contributions have been recognized with a number of awards, including a Presidential Early Career Award for Scientists and Engineers (PECASE) and the ASBMB Schering-Plough Research Institute Award for outstanding research contributions to biochemistry and molecular biology.

Link to Strahl Lab.

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Brian Strahl’s lab pinpoints new role for enzyme in DNA repair

The discovery, from the lab of Brian Strahl, PhD, offers insights for the creation of better, more targeted therapies for various forms of cancer.

Twelve years ago, UNC School of Medicine researcher Brian Strahl, PhD, found that a protein called Set2 plays a role in how yeast genes are expressed – specifically how DNA gets transcribed into messenger RNA. Now his lab has found that Set2 is also a major player in DNA repair, a complicated and crucial process that can lead to the development of cancer cells if the repair goes wrong. “We found that if Set2 is mutated, DNA repair does not properly occur” said Strahl, a professor of biochemistry and biophysics. “One consequence could be that if you have broken DNA, then loss of this enzyme could lead to downstream mutations from inefficient repair. We believe this finding helps explain why the human version of Set2 – which is called SETD2 – is frequently mutated in cancer.”

The finding, published online June 9 in the journal Nature Communications, is the first to show Set2’s role in DNA repair and paves the way for further inquiry and targeted approaches to treating cancer patients.  In previous studies, including recent genome sequencing of cancer patients, human SETD2 has been implicated in several cancer types, especially in renal cell carcinoma – the most common kind of kidney cancer. SETD2 plays such a critical role in DNA transcription and repair that Strahl is now teaming up with fellow UNC Lineberger Comprehensive Cancer Center members Stephen Frye, PhD, director of the UNC Center for Integrative Chemical Biology and Drug Discovery (CICBDD), Jian Jin, PhD, also with the CICBDD, and Kim Rathmell, MD, PhD, an associate professor in the department of genetics. Their hope is to find compounds that can selectively kill cells that lack SETD2. Such personalized medicine is a goal of cancer research at UNC and elsewhere.

In recent years, scientists have discovered the importance of how DNA is packaged inside nuclei. It is now thought that the “mis-regulation” of this packaging process can trigger carcinogenesis. This realm of research is called epigenetics, and at the heart of it is chromatin – the nucleic acids and proteins that package DNA to fit inside cells.

Proper packaging allows for proper DNA replication, prevents DNA damage, and controls how genes are expressed. Typically, various proteins tightly regulate how these complex processes happen, including how specific enzyme modifications occur during these processes. Some proteins are involved in turning “on” or turning “off” these modifications. For instance, protein and DNA modifications involved in gene expression in kidneys must at some point be turned off.

In 2002, Brian Strahl found that Set2 in yeast played a role as an off switch in gene expression – particularly when DNA is copied to make RNA. Now, Brian Strahl’s team found that Set2 also regulates how the broken strands of DNA – the most severe form of DNA damage in cells – are repaired. If DNA isn’t repaired correctly, then that can result in disastrous consequences for cells, one of them being increased mutation that can lead to cancer.

Through a series of biochemical and genetic experiments, Deepak Jha, a graduate student in Strahl’s lab, was able to see what happens when cells experience a break in the double-strand of DNA.  “We found that Set2 is required when cells decide how to repair the break in DNA,” said Jha, the first author of the Nature Communications paper. He said that the loss of Set2 keeps the chromatin in a more open state – not as compact as normal. This, Strahl said, leaves the DNA at greater risk of mutation. “This sort of genetic instability is a hallmark of cancer biology,” Jha said.

Strahl and Jha said they still don’t know the exact mechanism by which Set2 becomes mutated or why its mutation affects its function. But that’s the subject of their next inquiry. They are now collaborating with Rathmell and Ian Davis, also members of UNC Lineberger Comprehensive Cancer Center, to study how the human protein SETD2 is regulated and how its mutation contributes to cancer.  Strahl said, “We think this work will lead to a greater understanding of cancer biology, and open the door to future therapeutic approaches for patients in need of better treatment options.”

This research was funded through a grant from the National Institutes of Health.
Link to Strahl Lab.

Original story published on news.unchealthcare.org

 (Mark Derewicz, writer and Max Englund, graphic designer)

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Brian Strahl Promoted to Full Professor

Brian Strahl’s laboratory has been at the forefront of understanding how histones and their covalent modifications regulate chromatin structure and function, with a particular emphasis on how chromatin impacts gene regulation. His career began at the University of North Carolina (UNC) at Greensboro, where he majored in Biology and Chemistry. He then obtained his doctorate degree in Biochemistry from North Carolina State University in 1998, where he provided new insights into the transcriptional regulation of the Follicle Stimulating Hormone-ß (FSHß) gene. His curiosity in transcriptional regulation led him to pursue his postdoctoral studies in the laboratory of Dr. C. David Allis at the University of Virginia.  In David’s lab, he made a number of seminal discoveries in the area of histone methylation and histone function.  In particular, Dr. Strahl identified new sites of histone lysine methylation and linked this chromatin modification to gene regulation using the model organism Tetrahymena. His work also helped to identify the first lysine-specific histone methyltransferase in humans and several others in the budding yeast S. cerevisiae.  Dr. Strahl, with David Allis, also coined the idea of the histone code – a highly influential review that has been cited well over 5000 times. In December of 2001, Dr. Strahl initiated his lab at UNC-Chapel Hill, where he has now been promoted to the rank of Full Professor in the Department of Biochemistry & Biophysics. Dr. Strahl is also the Director of Graduate Studies and is the Faculty Director of the UNC High-Throughput Peptide Synthesis and Arraying Core Faculty. With his colleagues, his group has been at the forefront of determining how small chemical additions or molecular “tags” on histone proteins regulate the accessibility of DNA and the genetic information it contains.  Histones are central to the organization of our DNA in cells.  These proteins come in a variety of types or isoforms – defined as histone H3, H4, H2A and H2B, and they associate with themselves as a means to package our DNA within the small nuclei of cells.  Two copies each of each histone type come together to form what is called an octamer, which wraps approximately 147 base pairs of DNA around it.  This structure (histones + DNA) makes up the fundamental building block of chromatin – the nucleosome. Strings of nucleosomes make up the chromatin fiber, and they organize into higher-order structures that are poorly defined but allow large genomes (e.g., ~3 billion base pairs making up the human genome) to fit in the confines of a 2-10 micron nucleus.  With all this compaction, a fundamental question Dr. Strahl’s group has been addressing is how our genome is made accessible at the right place and time for all of the fundamental processes that occurs with DNA (e.g., gene expression, DNA repair and replicating the genome). Dr. Strahl’s UNC group has made a number of key contributions into the role of these chemical tags or modifications on histones (e.g., methylation and ubiquitylation), and more recently, DNA methylation.  Using budding yeast as a model system, his lab has helped to show how histone-modifying enzymes “hitch a ride” with RNA polymerase II (RNAPII) during gene transcription, and how the modifications they put on histones contributes to the transcription process. More recently, the Strahl group has focused on how patterns of histone modifications (i.e., the ‘histone code’) regulate the structure and function of chromatin. To understand how patterns of histone modifications function, They developed a high-throughput peptide microarray platform, where hundreds of synthetic histone peptides that are combinatorially modified with distinct chemical modifications are arrayed on glass slides.  With this technology, the lab has been interrogating chromatin-associated proteins that are critical for cell growth and development, and/or are dysregulated in human cancer.  One such protein his lab has recently been focused on is UHRF1, an E3 ubiquitin ligase essential for DNA methylation. Dr. Strahl’s lab showed that this protein binds to a particular pattern of histone modification to regulate the maintenance of DNA methylation in human cells.  They are continuing these lines of studies to address how the chromatin-machinery engages histones and DNA, and how these factors influence fundamental processes in the cell such as gene transcription. Work in Dr. Strahl’s lab is funded by the National Institutes of Health (NIH), the Keck Foundation and the National Science Foundation (NSF).

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Brian Strahl, Higher Education: The authoritative source of Brian Strahl's personal information, links, and social activity.

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Brian Strahl, PhD featured in history of epigenetics story

Dr. Brian Strahl, associate professor of biochemistry & biophysics at UNC-Chapel Hill, is featured in a video and story about the history of epigenetics in the Jan 15, 2014 (Vol. 34, No. 2) issue of GEN (Genetic Engineering & Biotechnology News). The video and related featured story can be found here.

Drs. David Allis and Brian Strahl formally proposed the ‘histone code’ about 14 years ago. At that time, Dr. Strahl was a postdoctoral fellow in David Allis' lab. This hypothesis provided an explanation for how distinct histone modifications, such as acetylation and methylation, could regulate epigenetic inheritance, gene expression and the control of cell growth and differentiation. However, limited experimental support exists for this hypothesis, and to date, it is unclear whether the binding of DNA-associated proteins to combinatorially-modified histones is a universal phenomenon of these regulators or is restricted to a subset of histone-binding proteins.

To address this long-standing question, Dr. Strahl’s lab is investigating how DNA-associated proteins bind to one or more histone modifications to regulate cellular function.  With his colleagues, the Strahl lab has been utilizing high-density histone peptide microarrays to determine how proteins with specialized histone interaction domains associate with multiple histone modifications to regulate chromatin structure and function.  Recent work has uncovered how the E3 ubiquitin ligase UHRF1 binds to histone H3 in a combinatorial manner – a binding event that governs the epigenetic inheritance of DNA methylation. To learn more, visit Brian Strahl’s page.

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brian-d-strahl:

Providing researchers with high quality services for synthesis, purification, and characterization of synthetic peptides and preparation of custom designed peptide arrays. The core director is Dr. Krzysztof Krajewski, PhD; Faculty Director of the facility is Dr. Brian D Strahl, Associate Professor of Biochemistry & BIophysics at UNC Chapel HIll.

For more information, please email us at: peptides@unc.edu

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Brian Strahl and colleagues identify another piece of the ‘histone code’ puzzle

UNC-CHAPEL HILL – DNA is often called the blueprint of life, but the four letter combinations that make up the genetic code are just part of the story.  Built upon the DNA lies additional ‘epigenetic’ information in the form of a complex ensemble of chemical tags attached to the DNA itself and on proteins that package our DNA – called histones – which ultimately control how our genetic code is accessed and used.  Interestingly, histones are decorated with many types of chemical tags, and their particular combinations have been referred to as the ‘histone code’.  But understanding how this ‘code’ is interpreted by the cell has proven challenging due its sheer complexity and a lack of tools to study the ‘code’ inside the cell.

Now research from the University of North Carolina at Chapel Hill School of Medicine has shown how a protein called UHRF1 “reads” the ‘histone code’ in a specific way to perform an important cellular function. “Because the protein has been found to be defective in cancer, the finding not only lends new insight into functions downstream of the ‘histone code’ but could also point the way toward novel strategies for cancer treatment and prevention,” said senior study author Brian Strahl, PhD, associate professor of biochemistry and biophysics and member of the UNC Lineberger Comprehensive Cancer Center.

The research, which appears June 1st, 2013, in the journal Genes and Development, is the latest of many studies to investigate the ‘histone code’ hypothesized more than ten years ago by Strahl and his former postdoctoral advisor C. David Allis. The hypothesis suggests that distinct combinations of histone modifications work together to form a ‘code,’ akin to the classic genetic code, in which three-letter combinations of nucleotides make an amino acid. These histone modifications – chemical changes like phosphorylation, acetylation and methylation -- generate an epigenetic language that is interpreted through the ability to recruit proteins to DNA and histones that in turn modulate cellular functions.

“This study provides important support for the ‘histone code’ hypothesis, and also reiterates how difficult it will be to crack this ‘code.’ It is not enough to understand how one tag works in isolation, we now have to look at all different combinations of tags on both histones and DNA to piece together the puzzle encrypting this second layer of information,” said Strahl.

Over the last decade, researchers have pinpointed a number of different “domains” that proteins use to interact with, or read, the ‘histone code’. Scott Rothbart, PhD, a postdoctoral research fellow in Strahl’s laboratory, previously showed that one such domain on the protein UHFR1 – called the tandem Tudor -- helps it bind to a histone in the cell that is methylated at a specific place. Adjacent to the Tudor was another domain called a PHD finger that helped the protein also bind the unmodified end of a histone. Rothbart and Strahl wondered if these neighboring domains might function together to help UHRF1 to read the ‘histone code’ and, subsequently, influence its ability to function in the cell. 

To investigate this question, the researchers used a highly sophisticated peptide microarray technology developed in the Strahl lab. Just as DNA microarrays contain sections of DNA sequence spotted on glass slides, these peptide arrays contained sections of modified histone proteins. When the researchers applied the UHRF1 protein to the array, they found it bound the histone differently when it contained the linked Tudor and PHD domains than when it contained the domains in isolation. They then used biochemical techniques to show that the two domains of UHRF1 functioned together in cells – whereby each domain is making a key contribution to promote binding to the histone protein in a specific way.

One of the main functions of UHRF1 is the maintenance of a critical modification known as DNA methylation. The researchers showed that when these domains of UHRF1 were not functioning together to read the ‘histone code’, DNA methylation patterns in the cell were eventually lost.

“Abnormalities in the patterning of DNA methylation are a hallmark of many cancers. In addition, UHRF1 has been found to be defective in a number of cancers including prostate, breast, kidney, and lung cancer,” said Scott Rothbart, PhD, who is lead author of the study.

“UHRF1’s function in maintaining DNA methylation seems to be reversible – if you take it out of the cell you lose DNA methylation, but if you add it back you restore DNA methylation. We therefore think that by using small molecules to disrupt the recognition of the ‘histone code’ by UHRF1, we may be able to reprogram DNA methylation patterns in cancer cells.”

The research was supported in part by the National Institutes of Health, the Carolina Partnership and the University Cancer Research Fund, the Natural Sciences and Engineering Research Council of Canada, and the American Cancer Society.

Study co-authors from UNC were Bradley M. Dickson, PhD, postdoctoral research associate; Krzysztof Krajewski, PhD, research assistant professor; and Dmitri B. Kireev, PhD, research professor.  Other collaborators on the story were Cheryl Arrowsmith, PhD, professor; Michelle Ong, postdoctoral research associate; and Scott Houliston from the University of Toronto.

Media contacts:  Tom Hughes, (919) 966-6047, tahughes@unch.unc.edu