Edited by: Eugene Russo
|Editor's Note: It's an excellent example of how good medicine often relies on the most fundamental, though seemingly obscure, details of hard-core basic research: In March 1996, a laboratory from the University of Rochester announced the discovery of an enzyme integral to unlocking the still-mysterious intricacies of DNA transcription and gene activation, the most basic of cellular processes. A month later, a laboratory at Harvard University, acting completely independently, reported the discovery of a second key enzyme that seemed to have an equally important but opposite effect. Three months ago, researchers from the Memorial Sloan-Kettering Cancer Center reported that they'd treated a 13-year-old girl suffering from acute promyelocytic leukemia with a novel therapy based on the inhibition of one of these enzymes. Within 23 days, the treatment had triggered an almost complete remission of her cancer, a cancer that had been unresponsive to numerous other treatments.1 The first two separate groundbreaking insights of three years ago are directly related to the more recent advent of promising clinical applications. Since the two papers were published in 1996, there's been an explosion of research on the enzymes that regulate and activate histones--the proteins associated with condensed packages of DNA. By better understanding these enzyme interactions, scientists have started to develop cancer therapies that intervene at the DNA level. In the first article below is a discussion of the first identification and cloning of an acetylase, or histone acetyltransferase (HAT). In the second article is a discussion of the first identification and cloning of a histone deacetylase, or HDAC. Both enzymes affect the degree to which chromatin, the mass of genetic material that includes histones, is open or closed to transcription.|
J.E. Brownell, J.X. Zhou, T. Ranalli, R. Kobayashi, D.G. Edmondson, S.Y. Roth, C.D. Allis, "Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation," Cell, 84:843-851, 1996. (Cited in more than 250 papers since publication)
Comments by C. David Allis , Byrd professor of biochemistry and molecular genetics at the University of Virginia
The fact that eukaryotic DNA is packaged into histones, something scientists have known for some time, had always suggested to investigators that the cell must somehow deal with these packages, must somehow decondense them, possibly using enzymes, to make transcription possible. Prior to this paper, there were generally two camps of researchers: one that studied chromatin and one that studied transcription factors. By finding transcription factors with enzymatic activities dedicated to chromatin modification, David Allis's lab--at the University of Rochester at the time--compelled investigators to link the two fields.
Allis's lab succeeded in purifying a nuclear HAT for the first time--an enzyme that, to their surprise, matched a transcriptional regulator called GCN5 that had already been identified in yeast but whose function was poorly understood. "In one swoop," Allis remarks, "we were telling, I think, the world that ... we know how this works. It's an enzyme; it's a HAT. Its job is to 'tickle' the histones in such a way that the transcription process can happen more easily... We made an emotional and intellectual commitment to this enzyme, and went after it." He's quick to note the "Herculean" efforts of lead author Jim Brownell, then a Rochester graduate student, who, Allis says, "broke the project wide open" with the design of a novel assay.
As it turns out, a single-celled protozoan actually held the key to what has blossomed into new cancer therapies in humans. The HAT enzyme that Allis and Brownell purified came from a Tetrahymena, a cousin of the paramecium. They chose the organism because the larger of its two nuclei has very hyperacetylated chromatin, making the enzymes that trigger histone acetylation either more plentiful or more active and hence, more easy to detect. Says Allis of the overlooked protozoan model, "Had we been doing this project [with] any other organism, we would probably not have seen [the HAT]."
Brownell's methodological "trick," as Allis puts it, was to actually put histones from Tetrahymena extracts into a gel as a substrate. When he put the enzyme extracts from the Tetrahymena nuclei into one of these gels, up came a band that clearly indicated that they'd found the protein they'd been looking for. Sufficient amounts of the enzyme were then purified from 200 liters of the organism and its gene cloned and sequenced.
Histone proteins have been known for years to be marked by other modifications besides acetylation, such as methylation, phosphorylation, and ubiquitination. Allis is interested in deciphering how these modifications might be agonistic or antagonistic to one another. He's also intrigued by the possibility that researchers may soon be able to elucidate modules, or motifs, on proteins that recognize the amino acids that get modified in histones upon activation--in much the same way that motifs on some proteins are known to recognize phosphorylated amino acids.
"I feel deeply committed to the fact that it ain't over yet. We've really only seen the tip of the iceberg," maintains Allis. "More and more it seems that the covalent modification of histones is playing a fundamental role in many DNA-templated processes, even those outside transcription." He adds that once more pharmaceutical companies realize the potential for drug design, an abundance of novel therapies could be developed at breakneck speed. "[Our] motivation here was the fundamental aspects of how does a gene fire in the context of chromatin," explains Allis. "It's been incredible what's happened in such a short time."