Nearby protein, not DNA itself, represses the activity of certain genes
BY JOSEPH BONNER & CATHY YARBROUGH
For a rapidly growing group of scientists, DNA does not tell the whole story of heredity.
Ever since the human genome was deciphered, these scientists, part of the cutting- edge field of epigenetics, have insisted that not only do genes pass information from one generation to the next, but so do spool-like proteins around which DNA is wrapped in the nuclei of cells.
Now, for the first time,Rockefeller’s David Allis and his colleagues have shown how the activity of a normal gene can be abnormally repressed by these histone proteins.
In the September 2 issue of Science Express, a research team led by Allis and Weill Cornell Medical College’s Scott Coonrod describes how an enzyme called peptidylarginine deiminase 4 (PAD4) causes the repression of a gene linked to normal development and cancer.
To understand how the process works, you need to first understand the process of histone methylation. In the nuclei of cells, DNA is wrapped tightly around histones, clusters of proteins that protect the coiled DNA and keep it from unraveling.
When a cell’s molecular machinery wants access to a particular stretch of DNA, it must first trigger a chemical change in the histone that causes it to open up and provide access to that stretch of DNA.The process by which an enzyme attaches a methyl chemical group to a specific amino acid on the histone is called histone methylation. Because histones control access to the DNA, one or more genes can be either turned on or silenced depending on which amino acid on the histone is methylated.
Scientists have discovered several enzymes responsible for attaching methyl groups to the amino acids that make up proteins, but until the research by Allis and Coonrod, no one had identified an enzyme that could remove methyl groups. (Watch an animation on epigenetics.)
An enzyme called PAD4 is involved in a chemical reaction called citrullination, in which an amino acid called citrulline is made from one called arginine.Unlike the 20 amino acids that make up proteins, citrulline is not coded for by our DNA.
Previous research by Coonrod showed that a unique form of PAD is one of the most abundant proteins in mouse eggs. Coonrod hypothesized that histone demethylation may be behind the dramatic changes in chromatin structure and gene activity that are known to occur following fertilization. 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 Allis’ and Coonrod’s labs, performed a series of experiments showing that, in fact, PADs can convert methylated arginine to citrulline in histones.
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 graduate 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 Allis, the Joy and Jack Fishman Professor and head of the Laboratory of Chromatin Biology and Epigenetics at Rockefeller.“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.”
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. Many diseases can’t be readily attributed solely to irregular genes.
“It has been known for some time,” says 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 for some time. But, until now, the way in which this ‘on’ mark is turned off has remained a mystery. Our work shows that PAD4 converts histone methylarginine residues to citrulline and therefore provides a long-sought-after answer to this question.”
In addition to discovering what may prove to be a major advance in our understanding of epigenetic gene regulation, Allis and Coonrod may have shown that PAD activity provides an answer as to how cells can be reprogrammed from an adult state to an embryonic state during cloning. Such reprogramming occurs naturally in stem cells and, though poorly understood, is a critical part of the techniques used to create animal clones. During the nuclear transfer process that produced the cloned sheep Dolly, for example, genetic material from an adult donor cell is injected into the nucleus of an enucleated egg cell.The donor egg cytoplasm then re-sets the adult donor nucleus back to an embryonic state, which gives rise to a cloned animal. Since there is no change in the DNA sequence during the cloning process, this re-setting process is thought to be epigenetic in nature.