http://www.eurekalert.org/pub_releases/2001-08/cshl-cj080601.php ~~~~~~~~~~~~~~~~~~~~~~~~~~~ Researchers Focus on Histone Code Studies offer support for an emerging hypothesis By Brendan A. Maher Histones, the proteins around which DNA coils to form chromatin, are moving toward the forefront of epigenetic research (see also, "The Meaning of Epigenetics"). A recently floated hypothesis states that the highly modifiable amino termini, or tails, of these proteins could carry their own combinatorial codes or signatures to help control phenotype, and that parts of this code may be heritable. Histones are perhaps more intimately linked with DNA than any other protein. Transcriptional regulation, recombination, repair, and replication--basically anything that happens to the DNA--must happen within the context of its packaging. That alone lends importance to the field. Leukemia therapeutics based on inhibition of histone modifiers have already made it to the clinic.1 And in light of this new "histone code" hypothesis, researchers are quick to tout further possibilities in human development, fertility, and other types of cancer. Shelley L. Berger, associate professor of molecular genetics at the Wistar Institute, University of Pennsylvania, says, "I think it's going to be really critical in human biology. It's not just some dry academic pursuit." Though first voiced in 1993 by Bryan Turner, professor of experimental genetics, University of Birmingham Medical School, Birmingham, U.K.,2 the histone code hypothesis was formally named last year by C. David Allis, Byrd professor of biochemistry and molecular genetics, University of Virginia Medical School, and his postdoc, Brian Strahl.3 Before the new hypothesis, the reigning theory had stated that modifications like acetylation and phosphorylation alter the electrostatic charge of histone tails, thereby loosening their grip on the DNA. While not excluding old assumptions, the new hypothesis states that specific modifications, like acetylation, phosphorylation, or methylation, to specific amino acid residues on histone tails facilitate processes by forming combinatorial codes that other proteins read. According to Allis, modification patterns might form a sort of landing pad upon which specialized regulatory proteins dock. Birth of this notion came from observations that the same modification could bring about different regulatory outcomes depending on where it appears on the histone tails. A case in point, says Allis, are the recent indications that the distinct methyl marks occurring only a few amino acids away from each other are associated with transcriptionally active euchromatin or unexpressed heterochromatin. "It has such a wonderfully split personality that it really makes you think about a code," Allis says. Though intentionally vague, the hypothesis finds support in recent Science articles.4-5 Berger and her lab found that in budding yeast, a certain phosphorylation event on the histone H3 tail is required for acetylation at a different amino acid on the same tail. Further, the kinase that phosphorylates at one promoter sequence does not necessarily phosphorylate at others.4 Berger says, "The very clear establishment of that pattern leads to support for the idea that there is a code, a histone code, and that the modifications vary at different places in the genome and are doing very specific things to regulate transcription." For the past five years--basically since the first histone acetyltransferase and histone deacetylases were found,1 acetylation has been a hot research topic. Now, say Allis and others, methylation is coming into its own. Because methyl groups are relatively stable and there is yet no known histone demethylase to remove the marks, histone methylation, which can activate or silence a gene, is believed to pass its signature on to daughter cells. In a nontransient form of histone modification, it's possible, says Turner, "to maintain a particular gene expression pattern--either on or off--over a long period." Berger explains, "The methyl transferase enzymes actually have domains... that recognize the methylated histone. Why would you need that? Well, it would allow you to provide a heritable mark." Allis points out that methylation at one amino acid coincides quite thoroughly with X inactivation in sex determination and the phenomenon of gene imprinting. Shiv Grewal, assistant professor of molecular biology and genetics at Cold Spring Harbor Laboratory, showed that differential sites of histone H3 methylation mark active and inactive regions of the chromosomes in fission yeast to establish a specific histone code.5 In inactive heterochromatic regions, histone H3 tails were specifically methylated at lysine 9, and in the adjacent active region, the tails were methylated at lysine 4 only a few amino acids away. Specific DNA sequences stand in between different chromatin neighborhoods. When Grewal's lab mutated the boundaries, the methlyation pattern associated with heterochromatin spread past them, silencing formerly expressed genes. "You have implications," Grewal says, "not only at the molecular level, but at a cellular level ... and all the way up to population biology." Yet with four different histone proteins, each with between 10 and 30 amino acid residues on their N-termini, along with numerous other types of histone modifications like ubiquitination and ADP-ribosylation, researchers are just beginning to unravel this mystery. For now, the limitations lie in the technique. Chromatin Immunoprecipitation (ChIP) is really the only tool researchers have for examining these ideas, says Turner. "Advances in this area are going to depend on advances in the chromatin immunoprecipitation approach," he says. And, while leaders in the field will continue to be those who are best equipped to produce the antibodies, new technologies could aid in moving the field forward. Grewal notes that some are moving toward amalgamating the advances in ChIP with advances in microarray technology, "So we have what we call, 'ChIP on a chip." He believes that in 10 years, scientists will have a clearer picture of the kind of epigenetic map the histone code could make up. But don't wait for a Rosetta stone, Turner cautions. This will not be static like the genetic code, he says, but something that is context-dependent--meaning different things in different situations. "I think the potential is enormous," Turner says. The genome, relatively small in size, is just the starting point. "What matters is how you use it." References 1. E. Russo, "Hot Papers in acetylation," and "Hot papers in deacetylation," The Scientist, 13:13, March 1, 1999. 2. B.M. Turner, "Decoding the nucleosome," Cell, 75:5-8, 1993. 3. B.D. Strahl and C.D. Allis, "The language of covalent histone modifications," Nature, 403:41-5, Jan. 6, 2000. 4. W. Lo et al., "Snf1--A histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription," Science, 293:1142-6, Aug. 10, 2001. 5. K. Noma et al., "Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries," Science, 293:1150-55, Aug. 10, 2001. ~~~~~~~~~~~~~~~~~~~~~~~~~ Translating the Histone Code: A Tale of Tails at Stetten Talk By Alison Davis News stories appearing over the last year would have everyone believe that scientists have ? once and for all ? cracked the human genetic code. Indeed, two teams of scientists have already published a draft sequence of our 3-billion-unit jumble of DNA "letters." But the task of thoroughly deciphering the code's protein-making instructions ? something our bodies do with ease all the time ? is a complicated puzzle that will keep researchers busy for decades to come. Dr. C. David Allis of the University of Virginia will be narrating the latest chapter on how our cells translate pieces of DNA into a meaningful message at this year's DeWitt Stetten Jr. Lecture. Allis will describe his research untangling the functions of chromatin, structures inside our cells that package genes. The talk will be held on Wednesday, Oct. 17 at 3 p.m. in Masur Auditorium, Bldg. 10. Allis is a pioneer in research that aims to clarify how cells contain and protect their most precious cargo, DNA, in protein-rich assemblies that are collectively called chromatin. In a sense, chromatin acts as a gatekeeper for our genes, regulating access to DNA by cellular equipment that decodes genes. Dr. C. David Allis His groundbreaking studies have begun to reveal that a key step in how cells interpret their genetic code involves actually finding certain genes inside chromatin. A cell's gene-decoding machinery is drawn to proteins in chromatin that have been "marked" with a variety of natural chemical tags. Putting on these tags and taking them off, Allis has found, is a critical aspect of targeting a cell's gene-reading activities. In recent years, Allis' lab has helped to discover several of the cellular systems that carefully balance the chromatin-marking chemical tags, called acetyl, phosphate and methyl groups. These units get attached to histones, the principal protein building blocks of chromatin that wind DNA into a protective spool. For example, he explains, the "aurora kinase" enzyme puts a tag called a phosphate group at a particular spot on the ends, or "tails," of histone proteins. According to Allis, this tag somehow tells the cell that it is time to bundle up its chromatin in time for cell division. This process helps to ensure that the cell's genetic material is separated precisely in half during cell division. By conveying such a message, genes get turned on ? or in some cases, off ? Allis says. In the case of cancer cells, inappropriate control of certain growth genes can fuel unchecked cell division. According to Allis, the "chromatin link" to cancer is just now beginning to be appreciated. Indeed, his studies have uncovered that several histone-marking enzymes, like the aurora kinase, actually are revved up in cancer cells, making them an important target for developing future cancer drugs. Allis first got interested in chromatin more than 20 years ago, while working as a postdoctoral fellow in the lab of Dr. Martin Gorovsky at the University of Rochester. Then and now, Allis has remained committed to studying chromatin via model systems such as the protozoan Tetrahymena, a single-celled ciliate organism that he refers to as an "offbeat pond-water beast." Offbeat or not, it's also the organism that netted another NIGMS grantee, Dr. Thomas Cech of the University of Colorado at Boulder and the Howard Hughes Medical Institute, the 1989 Nobel prize in chemistry ? for figuring out that the genetic material RNA can work as an enzyme. Scientists like Allis, Cech and countless others use so-called lower organisms like Tetrahymena to address fundamental questions that pervade biology. Such systems are simple, but they retain important similarities to the workings of animal and human cells. "Forget about what you do in the lab, look at what nature did," says Allis, citing the molecules and cellular processes that recur over and over again throughout the biological kingdom. He says he is extremely grateful for years of NIH funding to work with "offbeat" model organisms. "The implications for human biology and disease are far-reaching," he says. Allis earned a bachelor's degree in biology from the University of Cincinnati in 1973 and a doctorate in biology from Indiana University in 1978. He was on the faculty at Baylor College of Medicine from 1981 to 1990, at Syracuse University from 1990 to 1995, and at the University of Rochester from 1995 to 1998. Since 1998, he has been the Harry F. Byrd, Jr. professor of biochemistry and molecular genetics, professor of microbiology, and member of the Center for Cell Signaling at the University of Virginia Health Sciences Center. He has won several awards for both teaching and research, and is an elected member of the American Academy of Arts and Sciences.