Highlights

The fast moving world of epigenetics

Boston Children's researchers are leading the way in understanding histone modification

Nucleosome

Epigentics reveals the structures and processes that organize and control access to the DNA code. It coordinates different combinations of gene activity that allows cells with identical genomes to turn into ~ 200 specialized tissues and organs in the human body.

Epigenetic regulation is imposed by chemical modifications to specific sites within the genome, either to the underlying DNA or to the proteins that package the DNA (histones), which in turn modifies the expression of those genes. There are different types of modification, including acetylation or methylation of specific amino acid sites within the histone proteins and methylation and hydroxymethylation of DNA. A host of enzymes perform the chemical modifications on specific targets at specific sites in the genome. Not surprisingly, abnormal activity of any one of these enzymes leads to substantial changes in gene expression for many genes across the genome, and can have gross changes for the entire organism, leading to developmental abnormalities, diseases or cancers.

Children’s researchers have been studying epigenetic modifications with the hope of finding new treatments for deadly diseases such as melanoma, lung cancer and mental retardation. Below, we tell specific stories about Boston Children's research and new approaches that could lead to novel treatments for patients. These stories represent strong opportunities to establishing collaborative research and development partnerships with industry.

Carla Kim, PhD

The research of Carla Kim, PhD, principal faculty in the Stem Cell Program, explores the use of novel EZH2 inhibitors to modulate chemotherapy response in non-small cell lung cancer. In lung cancer, it is thought that chemotherapy resistant cells may rely heavily on gene expression controlled by histone modifying complexes. Polycomb Repressive complexes (PRCs) are one class of histone modifying proteins expressed in tumors and are thought to silence many tumor suppressor genes. Expression of EZH2, a PCR2 histone methyltransferase, in lung tumors is correlated with a poor overall survival of lung cancer patients. Because EZH2 is an enzyme, it is an attractive target for development of a small molecule inhibitor. Based on this idea, Dr. Kim’s lab tested the effects of EHZ2 inhibition on chemotherapy response of non-small cell lung cancer cell lines. They found that knocking down EZH2 in an lung adenocarcinoma line increased the line’s sensitivity to the chemotherapeutic drug etoposide, showing that inhibition of EZH2 in combination with chemotherapy may have benefits for cancer patients. To build upon this, they used a small molecule inhibitor of EZH2 (DZNep) in non-small-cell lung cancer cell lines. Half of the cell lines were sensitized to etoposide by the compound.

Dr. Kim plans to continue this work by exploring the phenotypes that identify cell lines or tumors that are sensitized to chemotherapy by EZH2 inhibition. On June 16, 2011, Carla Kim won Cell Stem Cell's 2010 best paper contest. Last year, her lab identified human lung cancer stem cells and found that, from one lung tumor to another, those stem cells are not the same.

Yang Shi, PhD

Yang Shi

Dr. Yang Shi, PhD, Merton Bernfield Professor of Neonatology, Division of Newborn Medicine, firmly established the notion that histone methylation is dynamically regulated by both histone methylases and demethylases. Dr, Shi is a co-founder of Constellation Pharmaceuticals and a pioneer in the field of epigenetics who has a history developing technologies that can be commercialized.

In his recent studies, Dr. Shi found that a subtle mutation affecting the epigenome may lead to an inherited form of mental retardation in boys. It appears to depend upon an enzyme in the pathway that could be a drug target. The Enzyme is a histone demethylase and works with a genetic partner to keep neuronal cells alive during development of the embryonic brain. The findings may help scientists understand the underlying reasons why x-linked disorders cause cognitive impairment and help develop new therapies to treat or prevent them.

Several years ago, Shi and his colleagues identified the first enzyme that detaches a molecule known as a methyl group, previously thought to be a permanent fixture, from a histones tails. Then his team and a number of other research groups independently discovered members of a second known family of these enzymes, known collectively as histone demethylases.

In his recent studies, Dr. Shi found that a subtle mutation affecting the epigenome may lead to an inherited form of mental retardation in boys. It appears to depend upon an enzyme in the pathway that could be a drug target. The Enzyme is a histone demethylase and works with a genetic partner to keep neuronal cells alive during development of the embryonic brain. The findings may help scientists understand the underlying reasons why x-linked disorders cause cognitive impairment and help develop new therapies to treat or prevent them.

Several years ago, Shi and his colleagues identified the first enzyme that detaches a molecule known as a methyl group, previously thought to be a permanent fixture, from a histones tails. Then his team and a number of other research groups independently discovered members of a second known family of these enzymes, known collectively as histone demethylases.

His latest study began with a gene mutated in several male patients with X-linked mental retardation and craniofacial abnormalities. He found that gene codes for a specific enzyme looked a lot like a member of the second family of histone demethylases. The mutations in these patients removed the working part of the enzyme that plucks the methyl group from the histone tail.

The researchers found that the enzyme, PHF8, indeed works as a histone demethylase. And it is the first known demethylase discovered for a strategic methylation point on the tail of histone H4 (the twentieth amino acid, a lysine “H4K20”), which other evidence suggests plays a critical role in gene expression and regulation and in the DNA damage response.) In this case, by removing the methyl group, the enzyme appears to maintain active gene transcription.

Despite its widespread presence, the enzyme seems to have a narrowly targeted biological effect on a master genetic regulator of craniofacial development, the transcription factor MSX1. Dr. Shi’s group and his collaborators, Madathia Sarkissian and Thomas Roberts at Dana-Farber Cancer Institute, tested the normal enzyme function in zebrafish.

They found that the dramatic impact on craniofacial development was evident. Fish without the enzyme developed no jawbone, a condition that could be partially prevented by providing the functioning enzyme, showing its importance in development. As importantly, providing more of the fish version of the MSX1 gene (whose activity the demethylase enzyme encourages) also prevented cell death in the brain caused by the missing enzyme.

Most recently, Dr. Shi’s laboratory has been particularly interested in the relationships between epigenetic gene errors on the X chromosome and neurodevelopmental, cognitive and craniofacial disorders. Dr. Shi, along with collaborators from Tsinghua University and Memorial Sloan Kettering Cancer Center, recently announced in Nature Structural and Molecular Biology that one section of the ATRX gene, called the ADD domain, is actually a hitherto unknown epigenetic reader. Read more here.

Leonard Zon, MD

Len Zon

Dr. Leonard Zon, MD, director of the Stem Cell Research Program, and his lab recently identified a novel epigenetic target that may inhibit the growth of the deadly skin cancer melanoma. Dr. Zon’s lab established SETDB1 as a new oncogene in melanoma, the first to be identified using a zebrafish model of the disease.

Mutations in the BRAF gene are present in about 50 to 60 percent of human melanomas. But BRAF mutations are also found in benign moles and are not themselves sufficient to cause cancer. In the study, the histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. The researchers set out to pinpoint other candidates in a region of chromosome 1 called 1q21, which contains 54 genes that is amplified in about 30 percent of melanoma patients. Of the 54, the gene SETDB1 was the only one in this region to work with BRAF to feed tumor development.

SETDB1 encodes an enzyme that helps turn other genes on or off, and is overactive in numerous other tumors (e.g., ovarian, breast, and liver cancers). The gene could be a valuable target for prognostic testing or for designing new melanoma treatments because the level of a tumor's malignancy rose with the level of SETDB1 activity in the model.

Currently, there limited therapies for melanoma and other aggressively metastatic cancers. This technology also provides an attractive in vivo screening model for melanoma therapies targeting SETDB1 histone methyltransferase.

Scott Armstrong, MD, PhD

Scott Armstrong

While 80 percent of childhood leukemia patients are cured, children with a subtype of acute lymphoblastic leukemia (ALL) known as MLL-AF4 have a lower chance of survival. This particular pathogenic fusion gene accounts for only 5 percent of all ALLs, and 70 percent of ALLs striking infants. Children with this form of leukemia often suffer rapid relapses and have a cure rate of about 50 percent with chemotherapy. Scott Armstrong, MD, PhD, Associate in Medicine, and his colleagues found that a small but potent epigenetic change that induces leukemia could potentially reverse ALL, preventing cancer-promoting genes from being turned on.

Dr. Armstrong’s group first developed a mouse model of MLL-AF4 and then showed MLL-AF4, a abnormal "fusion" protein that characterizes the disease, targets the cell's DNA and causes abnormal modification of one of the histones, "scaffolding" proteins that give chromosomes their shape and help control gene activation. As a result of this epigenetic change, chromosome structure is altered. This activates activity of genes known to be critical in initiating leukemia.

"The fusion protein modifies histones and turns on genes that are not supposed to be turned on, and that initiate the development of the cancer," says Armstrong. "If you could inhibit that abnormal histone modification, you might be able to reverse the tumorigenic properties of the fusion protein."

Dr. Armstrong’s group then showed that MLL-AF4 recruits an enzyme called DOT1L, which modifies the histone H3 by attaching a methyl group to a particular amino acid. While MLL-AF4 itself is not a good target chemically, enzymes are easier to target with small-molecule drugs. By inhibiting DOT1L, a few of the genes that contribute to the malignancy could be inhibited.

When the researchers suppressed DOT1L indirectly through RNA interference techniques, the abnormally activated genes were turned off.

Based on this research, Dr. Armstrong created an in vivo and in vitro model system to generate, isolate and characterize leukemia stem cells (LSC) utilizing the MLL-AF9 oncogene. He identified the gene expression signature unique to LSC, providing the means not only to distinguish these cells from normal progenitor cells, but to identify and test specific gene targets that prevent leukemia stem cell proliferation and survival, rescuing the recipient from death.

Anjana Rao, PhD

Anjana Rao

Epigenetics researchers have identified two alternate forms of cytosine whose biology differs enough from that of their parent base that may count as fifth and sixth DNA bases. These additional cytosines, called 5-methylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc), each have a group of atoms called a methyl group added onto their central ring, a feature normal cytosine lacks.

To help clarify the specific roles of these two new bases and shed light on the nature of what they do, a team of researchers at Children’s Hospital Boston has developed a pair of methods for mapping the locations of two methylated cytosines within a cell’s genome with almost GPS-like accuracy. The methods – published in Nature by Anjana Rao of the Immune Disease Institute and the Program for Cellular and Molecular Medicine at Children’s lab (and now also at the La Jolla Institute of Immunology and Allergy in California), use markedly different techniques to achieve the same goal: pinpointing where in the genome 5hmc can be found.

The new work adds to the growing body of knowledge from the Rao lab on the two new cytosines and genetic regulation. Two years ago, Rao’s team found that a family of enzymes called TET turns 5mc into 5hmc. Since then, her lab has also shown that the TET enzymes help control the fate of embryonic stem cells and that TET mutations in leukemia cells impair the enzymes’ catalytic capabilities.

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