NIH-Supported Researchers Map Epigenome of More Than 100 Tissue and Cell Types

NIH-Supported Researchers Map Epigenome of More Than 100 Tissue and Cell Types

Thursday, 19 February 2015

Much like mapping the human genome laid the foundations for understanding the genetic basis of human health, new maps of the human epigenome may further unravel the complex links between DNA and disease. The epigenome is part of the machinery that helps direct how genes are turned off and on in different types of cells.

Researchers supported by the National Institutes of Health Common Fund's Roadmap Epigenomics Program have mapped the epigenomes of more than 100 types of cells and tissues, providing new insight into which parts of the genome are used to make a particular type of cell. The data, available to the biomedical research community, can be found at the National Center for Biotechnology Information website.

Reference epigenomes are available for more than 100 cell and tissue types.
Credit: Image courtesy of Nature and Roadmap Epigenomics Consortium. 

"This represents a major advance in the ongoing effort to understand how the 3 billion letters of an individual's DNA instruction book are able to instruct vastly different molecular activities, depending on the cellular context," said NIH Director Francis Collins, M.D., Ph.D.

"This outpouring of data-rich publications, produced by a remarkable team of creative scientists, provides powerful momentum for the rapidly growing field of epigenomics."

Researchers from the NIH Common Fund's Roadmap Epigenomics Program published a description of the epigenome maps in the journal Nature. More than 20 additional papers, published in Nature and Nature-associated journals, show how these maps can be used to study human biology.

"What the Roadmap Epigenomics Program has delivered is a way to look at the human genome in its living, breathing nature from cell type to cell type," said Manolis Kellis, Ph.D., professor of computer science at the Massachusetts Institute of Technology, Cambridge, and senior author of the paper.

Understanding epigenomics

Almost all human cells have identical genomes that contain instructions on how to make the many different cells and tissues in the body. During the development of different types of cells, regulatory proteins turn genes on and off and, in doing so, establish a layer of chemical signatures that make up the epigenome of each cell. In the Roadmap Epigenomics Program, researchers compared these epigenomic signatures and established their differences across a variety of cell types. The resulting information can help us understand how changes to the genome and epigenome can lead to conditions such as Alzheimer's disease, cancer, asthma, and foetal growth abnormalities.

An epigenomic signature can be made on the

genome in two ways, both of which play a role in

turning genes off or on. The first occurs when

chemical tags called methyl groups are attached

to a DNA molecule directly (DNA methylation).

The second occurs when a variety of chemical

tags attach to the tails of histone proteins that

package DNA (histone modifications). Credit:

Image courtesy of John Stamatoyannopoulos

and Rae Senarighi. 

The value of epigenomic data

Researchers can now take data from different cell types and directly compare them.

"Today, sequencing the human genome can be done rapidly and cheaply, but interpreting the genome remains a challenge," said Bing Ren, Ph.D., professor of cellular and molecular medicine at the University of California, San Diego, and co-author of the Nature paper and several of the associated papers.

"These 111 reference epigenome maps are essentially a vocabulary book that helps us decipher each DNA segment in distinct cell and tissue types. These maps are like snapshots of the human genome in action."

"This is the most comprehensive catalogue of epigenomic data from primary human cells and tissues to date," said Lisa Helbling Chadwick, Ph.D., project team leader and a program director at the National Institute of Environmental Health Sciences (NIEHS), part of NIH.

"This coordinated effort, along with uniform data processing, makes it much easier for researchers to make direct comparisons across the entire data set."

"Researchers from the 88 projects supported by the program, including those from this recent series of papers, have propelled the development of new epigenomic technologies," said John Satterlee, Ph.D., co-coordinator of the Roadmap Epigenomics Program, and program director at the National Institute on Drug Abuse (NIDA), part of NIH. Satterlee added that the work of this program has served as a foundation for continued exploration of the human epigenome through the International Human Epigenome Consortium.

"With this increased understanding of the full epigenome, and the datasets available to the entire scientific community, the NIH Common Fund is striving to catalyse future research, to aid the understanding of how epigenomics plays a role in human diseases, with the expectation that further studies will identify early indications of disease and targets for therapeutics," said James Anderson, M.D., Ph.D., director of NIH Division of Program Coordination, Planning, and Strategic Initiatives that oversees the NIH Common Fund.

NIDA, NIEHS, and the National Institute on Deafness and Other Communication Disorders are co-administrators of the NIH Common Fund's Epigenomics Program.

Source: National Institute of Environmental Health Sciences

Contact: Robin Mackar

Reference:

Integrative analysis of 111 reference human epigenomes

Roadmap Epigenomics Consortium

Nature, (2015); doi: 10.1038/nature14248

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Scientists find that SCNT Derived Cells and iPS Cells are Similar

Scientists find that SCNT Derived Cells and iPS Cells are Similar

Thursday, 06 November 2014

A team led by New York Stem Cell Foundation (NYSCF) Research Institute scientists conducted a study comparing induced pluripotent stem (iPS) cells and embryonic stem cells created using somatic cell nuclear transfer (SCNT). The scientists found that the cells derived from these two methods resulted in cells with highly similar gene expression and DNA methylation patterns. Both methods also resulted in stem cells with similar amounts of DNA mutations, showing that the process of turning an adult cell into a stem cell introduces mutations independent of the specific method used. This suggests that both methods of producing stem cells need to be further investigated before determining their suitability for the development of new therapies for chronic diseases.

The NYSCF Research Institute is one of the only laboratories in the world that currently pursues all forms of stem cell research including SCNT and iPS cell techniques for creating stem cells. The lack of laboratories attempting SCNT research was one of the reasons that the NYSCF Research Institute was established in 2006.

"We do not yet know which technique will allow scientists to create the best cells for new cellular therapies," said Susan L. Solomon, NYSCF CEO and co-founder.

"It is critical to pursue both SCNT and iPS cell techniques in order to accelerate research and bring new treatments to patients."

While both techniques result in pluripotent stem cells, or cells that can become any type of cell in the body, the two processes are different. SCNT consists of replacing the nucleus of a human egg cell or oocyte with the nucleus of an adult cell, resulting in human embryonic stem cells with the genetic material of the adult cell. In contrast, scientists create iPS cells by expressing a few key genes in adult cells, like a skin or blood cell, causing the cells to revert to an embryonic-like state. These differences in methods could, in principle, result in cells with different properties. Advances made earlier this year by NYSCF Research Institute scientists that showed that human embryonic stem cells could be derived using SCNT revived that debate.

"Our work shows that we now have two methods for the generation of a patient's personal stem cells, both with great potential for the development of treatments of chronic diseases. Our work will also be welcome news for the many scientists performing basic research on iPS cells. It shows that they are likely working with cells that are very similar to human embryonic stem cells, at least with regard to gene expression and DNA methylation. How the finding of mutations might affect clinical use of stem cells generated from adult cells is the subject of an ongoing debate," said Dr. Dieter Egli, NYSCF Senior Research Fellow, NYSCF - Robertson Investigator, Assistant Professor in Pediatrics & Molecular Genetics at Columbia University, and senior author on the paper.

The study, published today in Cell Stem Cell, compared cell lines derived from the same sources using the two differing techniques, specifically contrasting the frequency of genetic coding mutations seen and measuring how closely the stem cells matched the embryonic state through the analysis of DNA methylation and of gene expression patterns. The scientists showed that both methods resulted in cell types that were similar with regard to gene expression and DNA methylation patterns. This suggested that both methods were effective in turning a differentiated cell into a stem cell.

The scientists also showed that cells derived using both SCNT and iPS techniques showed similar numbers of genetic coding mutations, implying that neither technique is superior in that regard. A similar number of changes in DNA methylation at imprinted genes (genes that are methylated differentially at the maternal versus the paternal allele) were also found. It is important to note that both types of techniques led to cells that had more of these aberrations than embryonic stem cells derived from an unfertilized human oocyte, or than embryonic stem cells derived from leftover IVF embryos. These findings suggest that a small number of defects are inherent to the generation of stem cells from adult differentiated cells and occur regardless of the method used.

Source: New York Stem Cell Foundation

Contact: David McKeon

Reference:

Comparable Frequencies of Coding Mutations and Loss of Imprinting in Human Pluripotent Cells Derived by Nuclear Transfer and Defined Factors

Bjarki Johannesson, Ido Sagi, Athurva Gore, Daniel Paull, Mitsutoshi Yamada, Tamar Golan-Lev, Zhe Li, Charles LeDuc, Yufeng Shen, Samantha Stern, Nanfang Xu, Hong Ma, Eunju Kang, Shoukhrat Mitalipov, Mark V. Sauer, Kun Zhang, Nissim Benvenisty, Dieter Egli

Cell Stem Cell, Volume 15, Issue 5, p634–642, 6 November 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.002

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Rethinking 'The Code': Histone Modifications

Investigators show that rules governing expression of developmental genes in mouse embryonic stem cells are more nuanced than anticipated

Sunday, 11 August 2013

A decade ago, gene expression seemed so straightforward: genes were either switched on or off. Not both. Then in 2006, a blockbuster finding reported that developmentally regulated genes in mouse embryonic stem cells can have marks associated with both active and repressed genes, and that such genes, which were referred to as "bivalently marked genes", can be committed to one way or another during development and differentiation.

A detailed analysis of the methylation patterns

of histone H3 revealed a nuanced picture of the

epigenetic rules governing expression of

developmental genes in mouse embryonic stem

cells. The image shows a ChIP-seq track file

example of H3K4me3 at mouse Homeobox

(Hox) gene clusters. Credit:  Courtesy of the

study's authors and Nature Structure &

Molecular Biology.

This paradoxical state — akin to figuring out how to navigate a red and green traffic signal — has since undergone scrutiny by labs worldwide. What has been postulated is that the control regions (or promoters) of some genes, particularly those critical for development during the undifferentiated state, stay "poised" for plasticity by communicating with both activating and repressive histones, a state biologists term "bivalency."

A study by researchers at the Stowers Institute for Medical Research now revisits that notion. In this week's advance online edition of the journal Nature Structural and Molecular Biology, a team led by Investigator Ali Shilatifard, Ph.D., identifies the protein complex that implements the activating histone mark specifically at "poised" genes in mouse embryonic stem (ES) cells, but reports that its loss has little effect on developmental gene activation during differentiation. This suggests that there is more to learn about interpreting histone modification patterns in embryonic and even cancer cells.

"There has been a lot of excitement over the idea that promoters of developmentally regulated genes exhibit both the stop and go signals," explains Shilatifard.

"That work supports the idea that histone modifications could constitute a code that regulates gene expression. However, we have argued that the code is not absolute and is context dependent."

Shilatifard has a historic interest in gene regulation governing development and cancer. In 2001, his laboratory was the first to characterize a complex of yeast proteins called COMPASS, which enzymatically methylate histones in a way that favours gene expression. Later, he discovered that mammals have six COMPASS look-alikes — two SET proteins (1A and 1B) and four MLL (Mixed-Lineage Leukaemia) proteins, the latter so named because they are mutant in some leukaemias. The group has since focused on understanding functional differences among the COMPASS methylases. The role of mouse Mll2 in establishing bivalency was the topic of the latest study.

Comprehending the results of the paper requires a brief primer defining three potential methylation states of histone H3. If the 4th amino acid, lysine (K), displays three methyl groups (designated H3K4me3), then this mark is a sign of active transcription from that region of the chromosome. If the 27th residue of histone H3 (also a lysine) is trimethylated (H3K27me3), this mark is associated with the silencing of that region of the chromosome. But if both histone H3 residues are marked by methylation (H3K4me3 and H3K27me3 marks), that gene is deemed poised for activation in the undifferentiated cell state.

The team already knew that an enzyme complex called PRC2 implemented the repressive H3K27me3 mark. To identify which COMPASS family member is involved in this process, the group genetically eliminated all possibilities and came up with Mll2 as the responsible factor. Mll2-deficient cells indeed show H3K4me3 loss, not at all genes, but at the promoters of developmentally regulated genes, such as the Hox genes.

The revelation came when the researchers evaluated behaviours of Mll2-deficient mouse embryonic stem cells. First, the cells continued to display the defining property of a stem cell, the ability to "self-renew," meaning that genes that permit stem cell versatility were undisturbed by Mll2 loss. But remarkably, when cultured with a factor that induces their maturation, Mll2-deficient mouse ES cells showed no apparent abnormalities in gene expression. In fact, expression of the very Hox genes that normally exhibit bivalent histone marks was as timely in Mll2-deficient cells as it was in non-mutant cells.

"This means that Mll2-deficient mouse ES cells that receive a differentiation signal can still activate genes required for maturation, even though they have lost the H3K4me3 mark on bivalent regions" says Deqing Hu, Ph.D., the postdoctoral fellow who led the study.

"This work paves the way for understanding what the real function of bivalency is in pluripotent cells and development."

The study's findings also potentially impact oncogenesis, as tumour-initiating "cancer stem cells" exhibit bivalent histone marks at some genes.

"Cancer stem cells are resistant to chemotherapy, making them difficult to eradicate," says Hu.

"Our work could shed light on how cancer stem cells form a tumour or suggest a way to shut these genes down."

Source: Stowers Institute for Medical Research 

Contact: Gina Kirchweger

Reference:

The Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells

Deqing Hu, Alexander S Garruss, Xin Gao, Marc A Morgan, Malcolm Cook, Edwin R Smith & Ali Shilatifard

Nature Structural & Molecular Biology, 11 August 2013, doi:10.1038/nsmb.2653

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Researchers Chart Epigenomic of Stem Cells that Mimic Early Human Development

Collaborative study will help overcome hurdles to using stem cells to treat diseases and injuries

Thursday, 09 May 2013

Scientists have long known that control mechanisms known collectively as "epigenetics" play a critical role in human development, but they did not know precisely how alterations in this extra layer of biochemical instructions in DNA contribute to development.

Now, in the first comprehensive analysis of epigenetic changes that occur during development, a multi-institutional group of scientists, including several from the Salk Institute for Biological Studies, has discovered how modifications in key epigenetic markers influence human embryonic stem cells as they differentiate into specialized cells in the body. The findings were published May 9 in Cell.

Professor Joseph R. Ecker, Plant Molecular and

Cellular Biology Laboratory, Howard Hughes

Medical Institute and Gordon and Betty Moore

Foundation Investigator, Salk International

Council Chair in Genetics. Credit:  Courtesy of 

the Salk Institute for Biological Studies.

"Our findings help us to understand processes that occur during early human development and the differentiation of a stem cell into specialized cells, which ultimately form tissues in the body," says co-lead author Joseph R. Ecker, a professor and director of Salk's Plant Molecular and Cellular Biology Laboratory and holder of the Salk International Council Chair in Genetics.

Scientists have established that the gene expression program encoded in DNA is carried out by proteins that bind to regulatory genes and modulate gene expression in response to environmental cues. Growing evidence now shows that maintenance of this process depends on epigenetic marks such as DNA methylation and chromatin modifications, biochemical processes that alter gene expression as cells divide and differentiate from embryonic stem cells into specific tissues. Epigenetic modifications - collectively known as the epigenome - control which genes are turned on or off without changing the letters of the DNA alphabet (A-T-C-G), providing cells with an additional tool to fine-tune how genes control the cellular machinery.

In their study, the Salk researchers and their collaborators from several prominent research institutions across the United States examined the beginning state of cells, before and after they developed into specific cell types. Starting with a single cell type-the H1 human embryonic stem cell - the most widely studied stem cell line to date - the team followed the cells' epigenome from development to different cell states, looking at the dynamics in changes to epigenetic marks from one state to another. Were they methylated, an essential process for normal development, or unmethylated?  

What happened to the cells during development? What regulatory processes occurred in the cell lineage?

The scientists found sections of the DNA that activate regulatory genes, which in turn control the activity of other genes, tend to have different amounts of letters of the DNA alphabet, "C" and "G" specifically, depending on when these regulatory genes are turned on during development.  

Additionally, regulatory genes that control early development are often located on stretches of DNA called methylation valleys, or DMVs, that are generally CG rich and devoid of epigenetic chemical modifications known as methylation. Consequently, these genes have to be regulated by another epigenetic mechanism, which the authors found were chemical changes called chromatin modifications. Chromatin is the mass of material-DNA and proteins-in a cell's nucleus that helps to control gene expression.

On the other hand, genes active in more mature cells whose tissue type is already determined tend to be CG poor and regulated by DNA methylation. The results suggest that distinct epigenetic mechanisms regulate early and late states of embryonic stem cell differentiation.

"Epigenomic studies of how stem cells differentiate into distinct cell types are a great way to understand early development of animals," says Ecker, who is also a Howard Hughes Medical Institute and Gordon and Betty Moore Foundation Investigator.

"If we understand how these cells' lineages originate, we can understand if something goes right or wrong during differentiation. It's a very basic study, but there are implications for being able to produce good quality cell types for various therapies."

For example, says Matthew Schultz, a graduate student in Ecker's lab, "understanding how development plays out normally could give us clues about how to reverse the process and turn normal adult cells into stem cells to regenerate tissues."

One area where the findings may help is in the study of tumour development. In normal tissue, DMVs are unmethylated, but in cancer, especially breast, colon and lung cancer, they are hypermethylated, suggesting, says Ecker, that alterations in the DNA methylation machinery might be an important mechanism aiding tumour development. He says further investigation is required to develop a greater understanding of this process.

Source: Salk Institute for Biological Studies

Contact: Andy Hoang

Reference:

Epigenomic Analysis of Multilineage Differentiation of Human Embryonic Stem Cells

Wei Xie, Matthew D. Schultz, Ryan Lister, Zhonggang Hou, Nisha Rajagopal, Pradipta Ray, John W. Whitaker, Shulan Tian, R. David Hawkins, Danny Leung, Hongbo Yang, Tao Wang, Ah Young Lee, Scott A. Swanson, Jiuchun Zhang, Yun Zhu, Audrey Kim, Joseph R. Nery, Mark A. Urich, Samantha Kuan, Chia-an Yen, Sarit Klugman, Pengzhi Yu, Kran Suknuntha, Nicholas E. Propson, Huaming Chen, Lee E. Edsall, Ulrich Wagner, Yan Li, Zhen Ye, Ashwinikumar Kulkarni, Zhenyu Xuan, Wen-Yu Chung, Neil C. Chi, Jessica E. Antosiewicz-Bourget, Igor Slukvin, Ron Stewart, Michael Q. Zhang, Wei Wang, James A. Thomson, Joseph R. Ecker, and Bing Ren
Cell, 09 May 2013, 10.1016/j.cell.2013.04.022

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Study Reveals Dynamic Changes in Gene Regulation in Human Stem Cells

Results suggest researchers implement careful quality control

Friday, 04 May 2012

A team led by scientists at The Scripps Research Institute and the University of California (UC) San Diego has discovered a new type of dynamic change in human stem cells.

Last year, this team reported recurrent changes in the genomes of human pluripotent stem cells as they are expanded in culture. The current report, which appears in the May 4, 2012 issue of the journal Cell Stem Cell, shows that these cells can also change their epigenomes, the patterns of DNA modifications that regulate the activity of specific genes—sometimes radically. These changes may influence the cells' abilities to serve as models of human disease and development.

"Our results show that human pluripotent stem cells change during expansion and differentiation in ways that are not easily detected, but that have important implications in using these cells for basic and clinical research," said team leader Louise Laurent, assistant professor in the UC San Diego School of Medicine.

Human pluripotent stem cells can give rise to virtually every type of cell in the body. Because of this remarkable quality, they hold huge potential for cell replacement therapies and drug development.

Many avenues of stem cell research focus on determining how genes are turned on and off during the course of normal development or at the onset of a disease transformation. It is widely accepted that gene activation and silencing play important roles in transforming all-purpose stem cells into the specific adult cell types that make up the specialized tissues of organs such as the heart and brain.

In the new study, Laurent and her collaborator, Professor Jeanne Loring of Scripps Research, and their colleagues focused on understanding gene silencing via DNA methylation, a process whereby bits of DNA are chemically marked with tags that prevent the genes from being expressed, effectively switching them off. Errors in gene silencing via DNA methylation are known contributors to serious developmental defects and cancer.

Specifically, the team assessed the state of both DNA methylation and gene expression in the most comprehensive set of human stem cell samples to date, comprised of more than 200 human pluripotent stem cell samples from more than 100 cell lines, along with 80 adult cell samples representing 17 distinct tissue types. The researchers used a new global DNA methylation array, developed in collaboration with Illumina, Inc, which detects the methylation state of 450,000 sites in the human genome. The results showed surprising changes in patterns of DNA methylation in the stem cells. Because of the unprecedented breadth of the study, the researchers were able to determine the frequency of different types of changes.

One of the anomalies highlighted by the study centres on X chromosomes. Since female cells contain two X chromosomes and males only one, one of the X chromosomes in females is normally silenced by DNA methylation through a process called X-chromosome inactivation (XCI). The new study demonstrated that a majority of female human pluripotent stem cells cultured in the lab lost their X chromosome inactivation over time, resulting in cells with two active X chromosomes.

This phenomenon could affect stem cell-based models of diseases caused by mutations of the X chromosome, such as Lesch-Nyhan disease, the researchers note. These cell-based models require that only the diseased copy of an X-linked gene be expressed, with the normal copy of the gene in females silenced via XCI. As the originally inactive X chromosome becomes active, the normal copy of the gene is expressed, changing the phenotype of the cells from diseased to normal.

"If an X chromosome that was assumed to be inactive is actually active, scientists may find that their cells perplexingly change from mutant to normal over time in culture," Loring said.

Another epigenomic aberration noted in pluripotent cells was in imprinted genes. Human cells contain two copies of most genes: one inherited from the mother and one from the father. In most cases, both the maternal and paternal copies of a gene are expressed equally. This is not the case, however, for imprinted genes, some of which are only expressed from the paternal chromosomes and others expressed only from the maternal chromosomes. This parent-of-origin specific gene expression involves silencing of one of the copies of the gene. Abnormalities in this selective silencing of genes can lead to serious developmental diseases.

The study found that, while the patterns of DNA methylation required to maintain imprinted gene silencing were stable in all of the somatic tissues, surprisingly, frequent aberrations in the patterns of DNA methylation existed in imprinted genes in the stem cells. Some of these aberrations arose very early in the establishment of the cell lines, while others crept in with the passage of time.

Interestingly, the team was able to link at least some of these aberrations to the conditions under which the stem cells were cultured in the lab. This suggests that researchers who use stem cells to study diseases linked to genomic imprinting will need to use conditions that best maintain imprinted gene silencing.

The researchers found another surprise — this one having to do with the basic process by which stem cells become specialized adult cells. Scientists have assumed that most genes are active at the earliest stages of human development, and that unnecessary ones are switched off as the cells developed specialized functions.

"For example, during the process of differentiation from a stem cell into a neuron, you might expect to observe silencing of all the genes that are important for the kidney, the pancreas, and the liver," said Kristopher Nazor, a Scripps Research Kellogg School of Science and Technology graduate student who is lead author of the study.

"But we found something quite different."

When the team compared stem cells with adult cells taken from tissue samples, rather than seeing mostly active genes in the stem cells and selectively silenced genes in the adult ones, they saw the opposite: in the stem cells, the researchers found that genes linked to the development of specialized tissue cells were silent and methylated, while in the adult cells regions of DNA involved in cell type specification were active and unmethylated. The scientists could reproduce some aspects of the developmental changes in culture: when stem cells were differentiated into neural cells in the culture dish, the patterns of DNA methylation became similar to those seen in human brain tissue.

This implies that, contrary to conventional wisdom, the genes responsible for transforming stem cells into tissue cells were initially silent, and were switched on during the process of differentiation.

Source: Scripps Research Institute 

Contact: Mika Ono

Reference:

Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and Their Differentiated Derivatives

Kristopher L. Nazor, Gulsah Altun, Candace Lynch, Ha Tran, Julie V. Harness, Ileana Slavin, Ibon Garitaonandia, Franz-Josef Müller, Yu-Chieh Wang, Francesca S. Boscolo, Eyitayo Fakunle, Biljana Dumevska, Sunray Lee, Hyun Sook Park, Tsaiwei Olee, Darryl D. D'Lima, Ruslan Semechkin, Mana M. Parast, Vasiliy Galat, Andrew L. Laslett, Uli Schmidt, Hans S. Keirstead, Jeanne F. Loring, Louise C. Laurent

Cell Stem Cell, 2012; 10 (5): 620 DOI: 10.1016/j.stem.2012.02.013

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Dynamic Changes in Methylation Can Determine Cell Fate

Dynamic Changes in Methylation Can Determine Cell Fate Wednesday, 28 September 2011

Scientists at Cold Spring Harbor Laboratory (CSHL) and the University of Southern California (USC) have uncovered intriguing new evidence helping to explain one of the ways in which a stem cell's fate can be determined.

The new data show how the "marking" of DNA sequences by groups of methyl molecules – a process called methylation – can influence the type of cell a stem cell will become. The cellular maturation process, called differentiation, has long been thought to be affected by methylation. Subtle changes in methylation patterns within subsets of a particular cell type have now been observed and closely scrutinized, and they reveal some intriguing mechanisms at work in the process.

A team led by postdoc Dr. Emily Hodges, working in the laboratory of CSHL Professor and HHMI Investigator Gregory Hannon, studied how methylation changes in blood stem cells can affect whether a given stem cell will differentiate into either a myeloid cell or a lymphoid cell. These are the two major lineages of mature blood cells. Sophisticated mathematical analyses of the data were performed under the direction of USC Professor Andrew D. Smith.

The study, which will appear in print October 7 in the journal Molecular Cell, generated some surprising findings that challenge currently held theories about how methylation operates. First, it demonstrated that methylation patterns are more dynamic than they are often thought to be.

"It's not a question of methylation being 'on' or 'off' at a given site in the genome," explains Hodges.

"We find, instead, an interesting fluctuation of the boundaries of regions that are free of methylation marks. This fact, in turn, can have a profound impact upon cell fate."

Areas lacking methylation, called hypomethylated regions, or HMRs, tend to coincide with so-called CpG islands, sites in the genome where adjacent "Cs" and "G's" – cytosine and guanine nucleotides – are seen in strings of repeats. These unmethylated regions tend to be ones associated with nearby genes that are capable of being expressed. In contrast, sites in the genome that are methylated are typically not expressed.

The new study, which looks at these areas at high resolution in cells of the different blood cell lineages and in blood stem cells, finds that in many cases, a core portion of the unmethylated region is shared in common, but that adjacent areas, sometimes called "CpG shores" – the outlying areas around CpG islands – differ markedly in breadth. The CSHL-USC team refines the notion of islands and shores, preferring to describe the narrowing and widening of the "shoreline" as a tidal phenomenon.

"We observed that the boundaries of these unmethylated regions goes in and out, like the tides," says Hodges.

"The key question is what drives these changes. We found that the width of these regions depends on the gene that is associated with the region. We showed in blood cells that the variation is lineage-specific."

The team deduced this after making close study of the methylation patterns in genomic regions containing genes known from other research to be expressed specifically in lymphoid cells, but not in myeloid cells, or vice versa. In these cases, all blood cells share a narrow "core" region of hypomethylation; but only in one lineage did the unmethylated region widen – a widening that opens the promoter of the "underlying" gene to the cellular machinery initiating gene expression. In other words, the lack of methylation over a wider area enables the underlying gene to be activated – only in the specified cell-type, but not in any of the others.

Another striking observation made from this data is the directional preference of this expansion. For example, in the widening of the unmethylated region seen in the case of the lymphoid cell, the direction of the widening was toward the area occupied by the underlying gene, which in this case was a gene encoding a B cell surface marker called CD22.

It has generally been thought that methylation is a stable epigenetic mark and that change in methylation are unidirectional; and further, that cells become increasingly methylated as they move through the differentiation process toward their mature identity. And in fact, the only known direction of active change is from an unmethylated state to a methylated state.

The new data suggests, however, that dynamic changes in methylation status may be possible. The relevant evidence comes from blood stem cells, which were observed to have methylation patterns that the team describes as "intermediately methylated," seemingly in dynamic equilibrium of the two extreme states of "methylated" and "unmethylated."

According to Hodges, this raises the possibility that methylation might in fact be bidirectional, and that there might be an as yet undiscovered, active mechanism that performs de-methylation. No known enzyme has this ability to remove methyl groups from DNA; DNA methyltransferase is the well-known enzyme that catalyzes the addition of methyl groups.

Yet another of the team's unexpected findings concerns the position of HMRs relative to know genic regions. While unmethylated regions tend to be associated with nearby genes that are capable of being expressed, the team found, according to Hodges, "a lot of HMRs located far away from any annotated gene locus."

One notable thing about these regions, she says, "is that they were highly enriched for binding sites of specific regulatory molecules that are involved in chromatin organization."

Chromatin consists of DNA and the protein complexes called histones around which genomic DNA is packed. In a given cell, chromatin organization, like methylation, helps to determine whether specific genes can be expressed or not.

About CSHL

Founded in 1890, Cold Spring Harbor Laboratory (CSHL) has shaped contemporary biomedical research and education with programs in cancer, neuroscience, plant biology and quantitative biology. CSHL is ranked number one in the world by Thomson Reuters for impact of its research in molecular biology and genetics. The Laboratory has been home to eight Nobel Prize winners. Today, CSHL's multidisciplinary scientific community is more than 400 scientists strong and its Meetings & Courses program hosts more than 8,000 scientists from around the world each year. Tens of thousands more benefit from the research, reviews, and ideas published in journals and books distributed internationally by CSHL Press. The Laboratory's education arm also includes a graduate school and programs for undergraduates as well as middle and high school students and teachers. CSHL is a private, not-for-profit institution on the north shore of Long Island.

Source: Cold Spring Harbor Laboratory

Contact: Peter Tarr

Reference:

Directional DNA Methylation Changes and Complex Intermediate States Accompany Lineage Specificity in the Adult Hematopoietic Compartment
Emily Hodges, Antoine Molaro, Camila O. Dos Santos, Pramod Thekkat, Qiang Song, Philip J. Uren, Jin Park, Jason Butler, Shahin Rafii, W. Richard McCombie, Andrew D. Smith and Gregory J. Hannon
Molecular Cell October 7, 2011, doi:10.1016/j.molcel.2011.08.026
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