MILESTONES

MILESTONE 20 expression and structure showed that loss of expression was associated with an altered array and reduced accessibility of restriction enzymes Unravelling to the . So, altered chromatin conformation underlies PEV, at least around was unknown. Studies of position-effect the centromeres. variegation (PEV) in fruitflies, and of the Studies of pericentromeric chromatin centromeric and mating-type regions in in fission yeast brought some important fission yeast, have proved key to shedding revelations about heterochromatin light on the mysteries of heterochromatin formation seven years later. The groups structure and function. of Robert Martienssen and Shiv Grewal PEV — a clonally inherited pattern of found that deleting genes that encode active and silent gene states — was first key components of the RNA interference described by Hermann Muller in 1930. Later, (RNAi) machinery resulted in accumulation it was shown that affected silenced genes had of complementary transcripts from been translocated to lie in close proximity centromeric heterochromatic repeats. to heterochromatin. In 1990, Sarah Elgin Expression of centromeric transgenes in and colleagues identified heterochromatin these deletion strains was de-repressed protein 1 (HP1) and showed that mutations and H3K9 methylation — a mark in the gene that encodes this protein in of heterochromatin — was lost. These fruitflies behave as dominant suppressors of results raised the possibility that the PEV, implicating HP1 in generating normal RNAi machinery targets specific histone chromatin structure. modifications to heterochromatic regions. Five years later, the same group showed In a publication that followed two weeks that altered chromatin packaging had a role later, the Grewal group turned to the in PEV. They used a mobile transposable mating-type region of fission yeast to study For a long time, heterochromatin was element — a popular tool among fruitfly the factors that favour heterochromatin considered to be the ‘dark matter’ of geneticists — to insert a marker gene at formation in cis. Little was known about the : highly condensed, late various locations throughout the genome. them, except that repeat elements tended to replicating and associated with lack of PEV was seen for transgenes that inserted be heterochromatic. This was an important . Various lines of evidence near centromeres, and on issue — as was known from PEV, the indicated that heterochromatin could lead to chromosome IV, which is heterochromatic heterochromatic state could spread to transcriptional silencing, but the mechanism in the fruitfly. Careful analysis of transgene inactivate nearby genes. The authors found

MILESTONE 21 — and, therefore, the level of expression — was determined by measuring the intensity of fluo- rescence for each gene. An array of Although this was considered important in terms of the numbers of genes that could opportunities be studied, the fundamental implications for interpreting gene-expression patterns were not Until the mid-1990s, studies of gene expression immediately appreciated. These became clear were limited to measuring transcription from from later studies, such as a paper published one or a few genes. But then a tool arrived that in 1998 that used microarrays to monitor the changed all this, allowing the study of hun- expression of 800 genes throughout the yeast dreds or thousands of transcripts at a time. . Subtle changes in overall gene- This technology — the expression microarray expression patterns over time were revealed — has revolutionized many areas of biology, that could not have been detected by other from basic research to the understanding and methods, even if dozens of genes had been treatment of human disease. studied. The study that brought microarrays to the By allowing different cell types to be accu- huge impact on medical research. In 2000, an attention of most researchers was published rately distinguished on the basis of expres- impressive early example of this was reported, in 1995 by Patrick Brown and colleagues at sion patterns, microarrays have also had a in which microarrays were used to study the Stanford University in California. They used expression of more than 8,000 genes in 65 an automated method to print 45 Arabidopsis human breast tumours. The authors generated thaliana cDNAs onto a glass slide. This ‘array’ “The development of microarray ‘molecular portraits’ of gene expression that was probed with a mixture of fluorescently technology has unequivocally allowed them to distinguish between different labelled cDNAs that were derived from the revolutionized the way we examine and classes of breast tumour and to identify two reverse transcription of mRNAs extracted from interpret gene expression.” new categories that had been overlooked by a tissue sample. The amount of hybridization James Broach traditional classification tools. They were also

S18 | DECEMBER 2005 www.nature.com/milestones/geneexpression © 2005 Nature Publishing Group “The discovery of the first nuclear that cenH, a centromere-homologous repeat that normally lies at the silent mating locus, HAT is a landmark discovery which is sufficient to bring about heterochromatin defines the modern era of chromatin formation at ectopic sites. This ability research.” Thomas Jenuwein is associated with H3K9 methylation and recruitment of Swi6 (a yeast HP1 counterpart) and, importantly, requires the RNAi machinery. The dark matter of the genome was gradually becoming illuminated. The encouraging news was that the findings in fruitflies and fission yeast fitted with MILESTONE 22 as an affinity matrix. This approach, combined results from mammalian cells, in which with the microsequencing of purified proteins, we now know that the targeting of yielded a surprise — one of the proteins had 60% epigenetic modifications to repeats by the Flagging sequence identity to yeast Rpd3, a characterized RNAi machinery also has a central role transcriptional repressor. The two antagonistic in heterochromatin formation and gene enzymatic activities had the opposite func- silencing. Histones undergo a range of post-translational tional effects — activation or repression — on Magdalena Skipper, Chief Editor, modifications, including acetylation, meth- transcription. Nature Reviews ylation, phosphorylation, ubiquitination and So what about ? By 2000, References and links sumoylation. The idea that histone modifica- several histone acetylases and deacetylases had ORIGINAL RESEARCH PAPERS Wallarth, L. L. & Elgin, S. C. R. Position effect variegation in is tions, particularly acetylation and methylation, been identified, but a functional link between associated with an altered chromatin structure. Genes Dev. have fundamental roles in controlling gene histone methylation and chromatin structure 9, 1263–1277 (1995) | Volpe, V. A. et al. Regulation of heterochromatic silencing and lysine-9 transcription is now so firmly established that or gene transcription remained elusive. Genetic methylation by RNAi. Science 297, 1833–1837 (2002) | it is perhaps surprising to realize that this only screens in fruitflies and fission yeast had shown Hall, I. M. et al. Establishment and maintenance of a became apparent during the past 5–10 years. that the fruitfly suppressor of variegation heterochromatin domain. Science 297, 2232–2237 (2002) FUTHER READING Muller, H. J. Types of visible variations Histones had been known to be acetylated 3-9 (Su(var)3-9) and fission yeast clr4 were produced by X-rays in Drosophila melanogaster. J. Genet. and methylated for a long time, and the idea of important for establishing and propagating 22, 299–234 (1930) | Eissenberg, J. C. et al. A mutation in a a function in transcription was considered as heterochromatin — a higher-order chromatin heterochromatin-specific chromosomal protein associated with suppression of position effect variegation in Drosophila early as the 1960s. For example, in their 1964 structure that is repressive for transcription. melanogaster. Proc. Natl Acad. Sci. USA 87, 9923–9927 paper, Allfrey, Faulkner and Mirsky demon- Work on the mammalian homologues had (1990) | Matzke, M. A. & Birchler, J. A. RNAi-mediated pathways in the nucleus. Nature Rev. Genet. 6, 24–35 strated that histone acetylation can reduce extended this link, but a mechanistic under- (2005) their efficacy as inhibitors of transcription, standing of how these proteins controlled het- and thought that this implied “a dynamic and erochromatin formation was lacking. In 2000, reversible mechanism for activation as well as work from Jenuwein and colleagues showed able to identify expression patterns that pre- repression of RNA synthesis.” In subsequent that the mammalian homologue of Su(var)3- dicted the response to chemotherapy. In this decades, enzymes that could acetylate histones 9 was a histone lysine methyltransferase that way, microarrays have paved the way for new were biochemically characterized and cloned. selectively methylated histone H3 at Lys9. methods that allow the accurate classification However, their relevance for regulating gene Together with earlier studies establishing a and diagnosis of disease, and also indicate the transcription was largely ignored. role for histone tails in transcriptional regula- most effective treatment strategies. In 1996, two papers — published within a tion (see Milestone 16), these three studies were Since 1995, the microarray has swiftly month of each other — showed that histone pivotal for focusing the attention of the field on changed from being regarded as a new, cutting- acetylases and deacetylases were, in fact, well- histone modifications and their importance as edge technology to being almost ubiquitous in known transcriptional regulators. These studies epigenetic markers. biological research. The study of expression provided the first clear connection between his- Sowmya Swaminathan, Senior Editor, patterns across an entire genome has moved tone acetylation and transcriptional regulation. Nature Cell Biology from an unattainable dream to an exciting Both papers were hailed as breakthroughs and, References and links reality that has fundamentally altered biology from then on, understanding how histone modi- ORIGINAL RESEARCH PAPERS Brownell, J. E. et al. Tetrahymena histone acetyltransferase A: a homolog to yeast and medicine. fications control chromatin structure and gene Gcn5p linking histone acetylation to gene activation. Cell 84, Louisa Flintoft, Associate Editor, expression became a significant area of enquiry. 843–851 (1996) | Taunton, J., Hassig, C. A. & Schreiber, S. L. Nature Reviews Genetics In the first paper, Allis and colleagues set out A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272, 408–411 (1996) | References and links to clone a histone acetyltransferase (HAT) from Rea, S. et al. Regulation of chromatin structure by site-specific ORIGINAL RESEARCH PAPERS Schena, M., Shalon, D., Tetrahymena thermophila. The cloned HAT histone H3 methyltransferases. Nature 406, 593–599 (2000) Davis, R. W. & Brown, P. O. Quantitative monitoring of gene turned out to have a high level of sequence FURTHER READING Allfrey, V., Faulkner, R. & Mirsky, A. expression patterns with a complementary DNA microarray. Acetylation and methylation of histones and their possible role in Science 270, 467−470 (1995) | Spellman, P. T. et al. relatedness to the yeast Gcn5, a transcriptional the regulation of RNA synthesis. Proc. Natl Acad. Sci. USA 51, Comprehensive identification of cell cycle-regulated activator, with almost 80% sequence identity in 786–794 (1964) | Ruiz-Carillo, A., Wangh, L. J. & Allfrey, V. G. genes of the yeast Saccharomyces cerevisiae by microarray Processing of newly synthesized histone molecules. Science hybridization. Mol. Biol. Cell 9, 3273–3297 (1998) | some domains. Consistent with the sequence 190, 117–128 (1975) | Kleff, S., Andrulis, E. D., Anderson, C. W. Perou, C. M. et al. Molecular portraits of human breast tumours. homology, the authors showed that recombinant & Sternglaz, R. Identification of a gene encoding a yeast histone Nature 406, 747–752 (2000) Gcn5 had HAT activity in vitro. In the second H4 acetyltransferase. J. Biol. Chem. 270, 24674–24677 (1995) | FURTHER READING van ‘t Veer, L. J. et al. Gene expression Hebbes, T. R., Thorne, A. W. & Crane-Robinson, C. A direct link profiling predicts clinical outcome of breast cancer. Nature 415, study, Schreiber and colleagues purified mam- between core histone acetylation and transcriptionally active 530–536 (2002) malian histone deacetylases using an inhibitor chromatin. EMBO J. 5, 1395–1402 (1998)

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