In the late 1800s, Albrecht Kossel discovered proteins called histones in goose blood cells. These abundant proteins, he showed, associate with nucleic acid to form a conglomerate called chromatin. Until the 1940s, many scientists thought that histones, not DNA, constitute the inherited material in eukaryotes, organisms whose cells contain nuclei.
By the 1960s, DNA had stolen the genetic-code limelight. Still, histones were plentiful and their partnership with the all-important genes intrigued investigators. Perhaps, evidence suggested, the proteins stifle the production of RNA from DNA, a process called transcription. In this view, stripping histones from eukaryotic DNA would allow the molecular apparatus that synthesizes RNA to adhere to its template and do its job.
In 1964, Vincent Allfrey (1921-2002), a biochemist at Rockefeller University, provided the first hints about events that might trigger such a process. Allfrey observed that histones from calf thymus nuclei that were adorned with acetyl chemical groups impair RNA synthesis poorly even though they bind DNA. On the basis of this finding, Allfrey made a bold hypothesis: The acetyl modifications create “presumably reversible changes in histone structure” that offer “a means of switching on or off RNA synthesis.”
Allfrey’s idea was provocative, but it rested largely on correlations between acetylation and gene activity. Rather than promoting transcription by fastening to histones, acetyl groups might instead affix to the proteins as a consequence of transcription. Although subsequent experiments from numerous investigators showed that histone acetylation characterizes active genes, the lack of cause-and-effect evidence left the idea without momentum.
In 1974, Roger Kornberg (Stanford University) proposed a theoretical model, now accepted, to explain how chromatin is organized. Two copies each of four distinct histone molecules combine to make a core around which DNA winds. Repeating units of this so-called nucleosome appear across the genome, like beads on a string.
By the late 1980s, elegant experiments by Yahli Lorch and Kornberg with purified cellular components suggested that nucleosomes can prevent the transcription machinery from accessing DNA and initiating RNA synthesis. But many scientists who studied transcription resisted the idea that nucleosomes inhibit gene activity. Because the evidence came from mixing reaction components in a test tube, no one knew whether these observations reflected events inside living cells. Furthermore, researchers had absorbed a paradigm from experiments on bacteria, which lack histones: Gene activators and repressors attach to specific spots in bacterial DNA to turn genes on and off. From this perspective, histones were not necessary to explain gene regulation. Instead, they were thought to function as inert structures that simply package DNA. Widespread thinking at the time held that nucleosomes were invisible to the transcriptional apparatus.
Histone-based gene control
In 1988, Grunstein catapulted the field forward. He genetically engineered yeast strains to shut down production of a particular histone, H4. This manipulation reduced the number of nucleosomes and boosted the amount of RNA synthesis from several test genes. The observation indicated that nucleosome loss in a living cell triggers gene activation and suggested that nucleosomes foil the transcription machinery’s ability to begin copying DNA into RNA. Grunstein had demonstrated that nucleosomes in intact eukaryotic cells do not serve merely as static spools that hold DNA; rather, they help regulate genes.
He discovered that one of the histone ends is needed to curb gene activity, and a specific amino acid in that N-terminal “tail”—a lysine that can be acetylated—plays a crucial role. Grunstein exploited the key lysine to identify molecules with which it collaborates, and showed that it interacts with a protein called SIR3, which was known to silence genes near chromosomal ends, where histones carry few acetyl groups. This finding, together with work from other labs, suggested a link between histone acetylation state and factors that regulate gene activity.
Grunstein went on to discover that the histone tail is required not just to quiet genes, but to activate them as well, and amino acid-substitution experiments indicated that acetylation of particular lysines is necessary for these effects. By counteracting the lysines’ positive charges, the acetyl groups presumably perturb chromatin structure in a way that promotes transcription (see Figure for one scenario).
In one view of transcriptional activation by histone acetylation, unacetylated lysines, which carry a positive charge, allow the histones (light blue) to tightly bind DNA (dark blue), which carries a negative charge. Consequently, the transcriptional machinery cannot access the DNA, and genes remain inactive (top). Addition of acetyl groups (orange) to particular lysines in histone tails (box, middle) neutralizes the positive charge and loosens the nucleosome’s grip on DNA. This process allows the transcriptional machinery (yellow) to access the DNA, and genes are active (bottom). Other modifications (pink and green) affect transcription in different ways.
Although these experiments suggested that the acetylation state of histones contributes to gene control, acetylation per se had not been directly demonstrated in biochemical experiments. Allis had been seeking enzymes that perform this reaction, and in 1996, he reported a discovery that galvanized the field.
He was working with Tetrahymena thermophila, a single-celled pond-dwelling critter that has two nuclei, one of which percolates with transcription. Because histones in this nucleus are generously acetylated, Allis reasoned, they must contain an enzyme that adds acetyls to histones. He set out to find it.
Using 200 liters of Tetrahymena and an ingenious experimental system he devised to identify proteins that tack acetyl groups onto histones, he isolated such a protein. Sequence analysis of its gene revealed that it closely resembles a yeast protein called GCN5, a well-established co-activator of transcription. How GCN5 functions biochemically to turn on genes, however, was unknown. In a now-classic experiment, Allis showed that purified GCN5 was an enzyme that can acetylate particular lysines in histone tails—including some that Grunstein had pinpointed in his studies of transcriptional activation. The demonstration that a bona fide gene regulator acetylates histones provided another mechanistic tie between chromatin structure and gene control. A key question then arose: What property of GCN5 promotes gene activation? Is it the intrinsic catalytic activity of GCN5 or some other feature of the enzyme?
In 1998, Allis and, independently, Shelley Berger (University of Pennsylvania), showed that histone acetylation and gene activation in yeast fell if the catalytic activity of GCN5 was selectively abolished. This crucial demonstration cemented the principle that remodeling histone structure by enzymatic acetylation can determine where and when genes are triggered or subdued.
In the meantime, other researchers had been approaching the question from the other side—by pursuing enzymes that eliminate acetyl groups from histones. In 1990, Teruhiko Beppu (University of Tokyo) reported that a particular chemical inhibits removal of acetyl groups from histones in mammalian cells, and he began illuminating the biological effects exerted by this compound. Stuart Schreiber (Harvard University) followed up and found an enzyme that the inhibitor blocks. He showed that the enzyme bears sequence similarity to a yeast protein called RPD3, which previously had been shown to repress transcription of certain genes.
The field had now exploded. Enzymes could increase gene activity by adding acetyl groups to histones (GCN5) or decrease gene activity by removing them (RPD3). Moreover, by the end of 1996, Allis had demonstrated that a core component of the mammalian transcription machinery can acetylate histones, and a flurry of papers from other researchers were reporting similar findings that firmly extended the yeast and tetrahymena findings to mammals. Cells were bubbling with histone acetylase activity among proteins known to govern gene activity.
In 2000, Thomas Jenuwein (Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany), in collaboration with Allis, discovered that an established transcriptional regulator was an enzyme that adds a different chemical group—a methyl—to a particular histone lysine. Others similarly connected gene activity with addition and subtraction of other chemical embellishments, including phosphate and ubiquitin moieties, to histones.
We now know that mistakes in histone modification touch a tremendous array of physiological pathways. Defects in the process underlie numerous inherited disorders of development—Kabuki syndrome and Rubinstein-Taybi syndrome, for instance—and these conditions affect multiple organ systems. Pharmaceutical companies are targeting the enzymes that attach or sever histone modifications with the aim of ameliorating human illnesses, especially certain forms of cancer.
By reshaping chromatin structure, chemical modifications of histones guide decisions about which stretches of DNA are read by conventional transcription activators and repressors. Grunstein and Allis have transformed our view of the histone proteins. Histones are not passive participants in DNA packaging, but rather, are key contributors to biological responses.
by Evelyn Strauss
Key publications of C. David Allis
Brownell, J.E., Zhou, J., Ranalli, T., Kobayashi, R., Edmondson, D.G., Roth, S.Y., and Allis, C.D. (1996) Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell. 84, 843-851.
Kuo, M.-H., Brownell, J.E., Sobel, R.E., Ranalli, T.A., Cook, R.G., Edmondson, D.G., Roth, S.Y., and Allis, C.D. (1996). Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature. 383, 269-272.
Mizzen, C.A., Yang, X.J., Kokubo, T., Brownell, J.E., Bannister, A.J., Owen-Hughes, T., Workman, J., Wang, L., Berger, S.L., Kouzarides, T., Nakatani, Y., and Allis, C.D. (1996). The TAFII250 subunit of TFIID has histone acetyltransferase activity. Cell. 87, 1261-1270.
Kuo, M.-H., Zhou, J., Jambeck, P., Churchill, M., and Allis, C.D. (1998). Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes Dev. 12, 627-639.
Rea, S., Eisenhaber, F., O’Carroll, D., Strahl, B., Opravil, S., Schmid, M., Mechtler, K., Pontig, C., Allis, C.D., and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone methyltransferases. Nature. 406, 593-599.
Lewis, P.W., Mueller, M.M., Koletsky, M.S., Cordero, F., Lin, S., Banaszynski, L.A., Garcia, B.A., Muir, T.W., Becher, O.J., and Allis, C.D. (2013). Inhibition of PRC2 activity by gain-of-function mutations in pediatric gliobastoma. Science. 340, 857-861.
Maze, I., Noh, K.M., Soshnev, A.A., and Allis, C.D. (2014). Every amino acid matters: essential contributions of histone variants to mammalian development and disease. Nat. Rev. Genet. 15, 259-271.
Key publications of Michael Grunstein
Kayne, P.S., Kim, U.-J., Han, M., Mullen, J.R., Yoshizaki, F., and Grunstein, M. (1988). Extremely conserved histone H4 N-terminus is dispensable for growth but essential for repressing the silent mating loci in yeast. Cell. 55, 27-39.
Han, M., and Grunstein, M. (1988). Nucleosome loss activates yeast downstream promoters in vivo. Cell. 55, 1137-1145.
Johnson, L.M., Kayne, P.S., Kahn, E.S. and Grunstein, M. (1990). Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 87, 6286-6290.
Durrin, L.K., Mann, R.K., Kayne, P.S. and Grunstein, M. (1991). Yeast histone H4 N-terminal sequence is required for promoter activation in vivo. Cell. 65, 1023-1031.
Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S., and Grunstein, M. (1995). Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell. 80, 583-592.
Kurdistani, S.K., Tavazoie, S., and Grunstein, M. (2004). Mapping global histone acetylation patterns to gene expression. Cell. 117, 721-733.
Shahbazian, M.D., and Grunstein, M. (2007). Functions of site-specific histone acetylation and deacetylation. Annu. Rev. Biochem. 76, 75-100.