Findings Get a Handle on Stemness

‘Bivalent Domains’ May Enable Pluripotency of Stem Cells

During development, cells of the embryo diverge into different forms with increasing specificity. Each cell carries the same genetic information, yet each puts its own spin on it. Research into epigenetics has shown how cell-specific information is carried in the structure of chromatin, the packed mass of DNA and proteins in the nucleus. Different chromatin structures give rise to patterns of gene expression unique to each type of cell, with certain regions silenced and others active.

Photo by Graham Ramsay

Bradley Bernstein (left) and Eric Lander led a team that uncovered a novel pattern in the histones associated with DNA in embryonic stem cells that they believe may be a structural correlate for “stemness.”

But what is the epigenetic signature of embryonic stem cells? These cells are not yet differentiated into the specific patterns found in the body’s tissues, but retain the potential to become any kind of cell. A new study, led by Bradley Bernstein and published in the April 21 Cell, shows that the chromatin of stem cells contains regions with characteristics of both active and repressed gene expression. These “bivalent domains” are found at key regulatory sites in the genome. The researchers believe these domains may be a structural correlate to stemness, a mechanism by which stem cells keep their plasticity.

Deciphering Histones
Epigenetics is “a way to remember which genes are on and which genes are off in a given tissue or cell type,” said Bernstein, HMS assistant professor of pathology at Massachusetts General Hospital and the Broad Institute. For instance, when a muscle cell divides, the resulting daughter cell is not a tabula rasa—it inherits its parent’s unique interpretation of the genome that makes it a muscle cell. But how this memory first emerges is unclear.

In this study, the researchers focused on a particular type of epigenetic signature—methyl groups attached to histone proteins associated with DNA (also see this issue's molecular biology story). A cell’s chromatin is divided into bundles called nucleosomes, and each nucleosome contains four pairs of histone proteins that act as spools around which DNA is wound. Each histone spool has a tail, which can be chemically modified in many possible ways. Some chemical modifications cause the nucleosomes to wind more tightly, keeping genes silenced, while others promote a looser structure more accessible to gene transcription. It has been thought that different modifications might even form a “histone code” that determines a pattern of gene expression.

Bernstein began studying histone modifications while a postdoc in the lab of Stuart Schreiber, the Morris Loeb professor of chemistry and chemical biology at Harvard. Together with Emily Humphrey and Chih Long Liu, graduate students in the lab, he developed ways to use a chromatin immunoprecipitation (ChIP) assay to study epigenetic signatures on a genomewide level. Using this assay, the researchers expose chromatin to antibodies that bind to a particular chemical modification on histones. They separate out the histones with DNA attached, and these pieces of DNA can then be analyzed with DNA microarrays to yield the sequences of the genome that are linked to a particular epigenetic signature.

Unsteady state. The chromatin of embryonic stem cells has “bivalent” domains with marks of both gene activation and repression. In these domains, the tail of histone protein H3 has a methyl group attached to lysine 4 (K4) that is activating and a methyl group at lysine 27 (K27) that is repressive (above). This contradictory state may keep the genes silenced but poised to activate if needed. When the cell differentiates (right), only one tag or the other remains, depending on whether the gene is expressed or not.

The most recent study, a collaboration with members of Eric Lander’s group at the Broad, examined chromatin in embryonic stem cells. (Lander is an HMS professor of systems biology.) It focused on two different histone modifications; both occur when a methyl group is attached to the tail of histone H3, but on one position, the methylation promotes gene activation, and on the other, it represses it. In differentiated cells, the team found, the regions marked by the active tag are almost always separate from regions marked by the repressive one. In undifferentiated cells, however, the pattern is different. “In embryonic stem cells, most of the repressive regions have active regions within them,” Bernstein said.

The genes within these bivalent domains are silenced, but the researchers speculate that the presence of an active region within a repressive one may be a mechanism for quickly reactivating these genes if and when the cell differentiates. “These bivalent marks may leave the cell poised to be differentiated in several different ways,” said Schreiber, a co-author on the paper.

Epigenetic Memory
The pattern of bivalent domains suggests they may have an important role in stem cell biology. A majority of the domains overlap with genes that regulate development, such as Hox genes that help lay out the body plan. The domains also overlap with genetic sequences that do not code for genes, but are highly conserved across vertebrates and often appear alongside important developmental regulators. This fact is intriguing, Bernstein said, because “you could almost predict where bivalent domains would be by DNA sequence,” suggesting that DNA sequence determines epigenetic patterns in stem cells. For Bernstein, the key question is, “Where did epigenetic memory come from?” The link between genomic sequence and histone codes might help explain how these epigenetic signatures first form. He added that an epigenetic signature could also be used to identify and characterize stem cells, which is now a tricky task.

Schreiber, who leads the Chemical Biology Program at the Broad Institute, studies fundamental processes in the cell by developing small molecules to manipulate them. He is interested in pursuing the work from a different angle. “Brad’s work has had a significant impact on the direction of our research,” he said. “We are focusing our research efforts today on small-molecule screens that might identify small-molecule regulators of chromatin function.” This study suggests that methyl groups on histones hold a key to controlling the state of stem cells—an ability that could have far-reaching impact. One of the major challenges of stem cell biology is gaining control of stem cell development—maintaining stemness in stem cell lines or inhibiting it in cancer stem cells that nourish tumors. “You could imagine inhibiting specific demethylases that might prevent the removal of methyl groups and therefore impact on embryonic, tissue-specific, and cancer stem cell biology,” Schreiber said.

—Courtney Humphries

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