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For the first time, scientists have shown how the activity of a gene associated with normal human development, as well as the occurrence of cancer and several other diseases, is repressed epigenetically by modifying not the DNA code of a gene, but instead the spool-like histone proteins around which DNA tightly wraps itself in the nucleus of cells in the body.

By studying how and when these histone changes occur, many scientists hope to explain human diseases that can't be readily attributed only to irregular genes.

In the September 2 issue of Science Express (and September 3 issue of Science), researchers at Weill Cornell Medical College and Rockefeller University report what may prove to be a major advance in understanding epigenetic gene regulation.

In exquisite detail, Rockefeller's C. David Allis, Ph.D., and Weill Cornell's Scott Coonrod, Ph.D., and colleagues describe a novel activity that is carried out by a poorly characterized enzyme that leads to the repression of specific genes.

Since the human DNA code, or human genome, was deciphered, Dr. Allis, Dr. Coonrod, and other researchers have begun to investigate how cells determine when one or more of the 30,000 or so genes within the human genome should be turned on and off at just the right time points, allowing for normal development and functioning. Understanding the regulation of gene expression is important because if our genes are not expressed in the right time, place and amount, then disease may occur.

Until recently, scientists thought that regulation of gene expression was entirely determined by information contained within the DNA sequence. Now, thanks to the research of Dr. Allis and other leaders in the new field of epigenetics, scientists know that this gene regulatory information is also stored on the histones around which DNA wraps itself. Together DNA and histones form a package called chromatin, which makes up chromosomes.

Chromatin stretches open to allow the cell's molecular machines to have access to DNA and thereby to switch on genes. However, when chromatin is closed, the same gene-activating machinery cannot penetrate the protective barrier, and gene expression cannot occur. Dr. Allis and other researchers have shown that chemical changes to tail-like structures of histones can open or close chromatin, thus regulating gene expression.

(Please go to http://www.rockefeller.edu/interactive/allis/silence.html to view an animation that explains how chemical modification of histones can silence genes.)

In previous studies of gene expression, scientists found that one particular chemical reaction, called histone methylation, has been shown to be a crucial mark for gene expression. Methylation describes the process by which an enzyme attaches a methyl chemical group to specific amino acids on the histone tails. Genes can either be turned on or silenced depending on which amino acid is methylated. For example, methylation of the amino acid lysine can be a gene-silencing mark, while scientists believe that methylation of arginine is generally a gene-activating mark.

Scientists have discovered several enzymes responsible for attaching methyl groups to amino acids, but until the research by Drs. Allis and Coonrod, no one had identified an enzyme that could remove methyl groups.

In Science, Drs. Allis and Coonrod, and colleagues report that specific genes are repressed by the novel activity of histone demethylation, which is carried out by the poorly characterized enzyme known as peptidylarginine deiminase 4 (PAD4).

We've shown for the first time that an enzyme can remove a 'methyl mark' from a histone and repress gene expression, says Dr. Allis, the Joy and Jack Fishman Professor and Head of the Laboratory of Chromatin Biology and Epigenetics at Rockefeller. These epigenetic marks are extremely important in normal human development and in cancer and other diseases.

It has been known for some time, adds Dr. Coonrod, Assistant Professor of Genetic Medicine at Weill Cornell, that the 'on' and 'off' dynamics of histone modifications are important for the regulation of gene expression and that misregulation of these modifications can lead to disease. Also, the identity of the enzymes responsible for putting the gene activating 'methyl mark' on histones has been known. But, until now, the way in which this 'on' mark is turned 'off' has remained a mystery. Our work shows that PAD4 converts histone methyl-arginine residues to citrulline and therefore provides a long-sought-after answer to this question.

In addition, the Rockefeller and Weill Cornell scientists speculate that this research may provide a better understanding of the reprogramming of cells that allows animals to be cloned from adult cells and holds promise for the therapeutic potential of stem cells.

Drs. Allis and Coonrod focused on the enzyme called PAD (peptidylarginine deiminase), which is involved in a chemical reaction called citrullination, in which arginine loses a chemical group called an imino and becomes the amino acid citrulline. Unlike the 20 amino acids that make up proteins, citrulline is not coded for by our DNA.

Previous research by Dr. Coonrod showed that a unique form of PAD is one of the most abundant proteins in mouse eggs. The fact that PADs were so abundant in eggs and were recently shown by others to target histones led him to hypothesize that a histone demethylase activity of egg PADs may be behind the dramatic changes in chromatin structure and gene activity that are known to occur following fertilization. This prediction was supported by preliminary work with mouse eggs by Dr. Coonrod's graduate student Olga Sarmento. It showed a dramatic loss of methylated arginine residues on histones at certain stages of the cell cycle.

Dr. Coonrod then teamed up with Dr. Allis to determine whether PADs were indeed the enzymes that could remove the methyl mark from histones. Using enzymatic assays and antibodies that recognize methylated arginine and citrulline on histones, postdoctoral researchers Yanming Wang and Joanna Wysocka, both of whom share appointments in Drs. Allis's and Coonrod's labs, performed a series of experiments showing that, in fact, PADs can convert methylated arginine to citrulline in histones.

Drs. Allis and Coonrod then recruited Yali Dou, a postdoc in Robert Roeder's lab at Rockefeller, and Young-Ho Lee, a postdoc in Michael Stallcup's lab at the University of Southern California, to conduct further experiments, which showed that the histone demethylation activity of PAD4 results in repression of gene expression. UCLA's Steven Clarke and grad student Joyce Sayegh determined PADs' enzymology.

The discovery that PAD4 removes a methyl group from arginine and represses gene activity adds to our understanding of epigenetic gene regulation, says Dr. Allis. We already know that two different enzymes can methylate arginine and activate genes, and now we have a mechanism that essentially reverses this process and represses gene activation.

Dr. Allis notes that much research has been conducted on enzymes called HATs and HDACs, which attach and remove, respectively, acetyl chemical groups from histones. HATS and HDACs have been shown to be involved in the development of certain human cancers.

HATs and HDACs are among the most exciting targets for cancer therapies, says Dr. Allis. We think that, similar to what we are learning about these enzymes,more research may reveal clues about PAD4's role in human disease.

Drs. Allis and Coonrod also believe that PAD activity could also provide an answer as to how cells can be reprogrammed from an adult state to an embryonic state during cloning. Such reprogramming is evident in the regenerative properties of stem cells and in animal cloning techniques, such as the nuclear transfer process that produced the cloned sheep, Dolly. In nuclear transfer, genetic material from an adult donor cell is injected into the nucleus of an enucleated egg cell. Poorly characterized factors within the donor egg cytoplasm then reset the adult donor nucleus back to an embryonic state, which can then give rise to a cloned animal. Since there is no change in the DNA sequence during the cloning process, this resetting process is thought to be epigenetic in nature.

As Dr. Coonrod explains, an egg cell is terminally differentiated. That is, it cannot develop any further on its own. The egg only becomes totipotent able to produce any and all kinds of cells in the embryo and adult after it is fertilized by a sperm cell.

Sperm and egg chromatin is very condensed and only allows for a restricted subset of genes to be expressed. Around the time of fertilization, however, unknown factors within the egg somehow cause the chromatin to become more open, thereby allowing embryonic genes to be expressed, explains Dr. Coonrod.

This is extremely speculative, but if arginine methylation is the 'on switch' for genes in the oocyte, Dr. Coonrod continues, PAD, through the 'demethylation' of arginine, may erase certain epigenetic marks and shut down the maternal program, allowing the embryonic program then to become activated.

The National Institutes of Health supported this research.