Proc Natl Acad Sci U S A 97(25): 13708-13713

Genomewide studies of histone deacetylase function in yeast

^{Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and Center for Genomics Research, Harvard University, 12 Oxford Street, Cambridge, MA 02138; and}^{Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115}

^{To whom reprint requests should be addressed. E-mail:}ude.dravrah.sirisls@sls.

Contributed by Stuart L. Schreiber

Accepted 2000 Oct 9.

Abstract

The trichostatin A (TSA)-sensitive histone deacetylase (HDAC) Rpd3p exists in a complex with Sin3p and Sap30p in yeast that is recruited to target promoters by transcription factors including Ume6p. Sir2p is a TSA-resistant HDAC that mediates yeast silencing. The transcription profile of rpd3 is similar to the profiles of sin3, sap30, ume6, and TSA-treated wild-type yeast. A Ume6p-binding site was identified in the promoters of genes up-regulated in the sin3 strain. Two genes appear to participate in feedback loops that modulate HDAC activity: ZRT1 encodes a zinc transporter and is repressed by RPD3 (Rpd3p is zinc-dependent); BNA1 encodes a nicotinamide adenine dinucleotide (NAD)-biosynthesis enzyme and is repressed by SIR2 (Sir2p is NAD-dependent). Although HDACs are transcriptional repressors, deletion of RPD3 down-regulates certain genes. Many of these are down-regulated rapidly by TSA, indicating that Rpd3p may also activate transcription. Deletion of RPD3 previously has been shown to repress (“silence”) reporter genes inserted near telomeres. The profiles demonstrate that 40% of endogenous genes located within 20 kb of telomeres are down-regulated by RPD3 deletion. Rpd3p appears to activate telomeric genes sensitive to histone depletion indirectly by repressing transcription of histone genes. Rpd3p also appears to activate telomeric genes repressed by the silent information regulator (SIR) proteins directly, possibly by deacetylating lysine 12 of histone H4. Finally, bioinformatic analyses indicate that the yeast HDACs RPD3, SIR2, and HDA1 play distinct roles in regulating genes involved in cell cycle progression, amino acid biosynthesis, and carbohydrate transport and utilization, respectively.

Abstract

Reverse genetic or reverse chemical genetic approaches to the analysis of protein function require a broad search for phenotypes resulting from targeted (often deletion) mutations or from small molecules, respectively. Global mRNA expression monitoring (transcription profiling) has emerged as a useful method for searching broadly for phenotypes resulting from mutations or small molecules (1). The study of mutation-based perturbations (reverse genetics) is an inherently steady-state process because the phenotypic analysis is performed after a cell adapts to its altered genetic composition. In contrast, the study of small-molecule-based perturbations (reverse chemical genetics) allows the immediate effects of the perturbation to be assessed. Small molecules modulate function in cells rapidly, and global expression can be monitored at selected time points, allowing the time course of change to be assessed.

In this study, we combined reverse genetic and reverse chemical genetic experiments involving transcription profiling to study the function of histone deacetylases (HDACs) in Saccharomyces cerevisiae. HDACs are transcriptional repressors that reduce histone acetylation levels to create localized regions of repressed chromatin. The small molecules trapoxin and trichostatin A (TSA) were instrumental in the initial characterization of an HDAC, the mammalian HDAC1, which was found to be related in sequence to yeast Rpd3p (2). Consistent with its deacetylase function, deletion of RPD3 results in increased cellular histone acetylation (3). TSA treatment also has been shown to induce a hyperacetylated state in yeast (4). It is now known that at least six HDACs exist in yeast, encoded by the yeast genes RPD3, HDA1, HOS1, HOS2, HOS3, and SIR2. Of these, Rpd3p and Hda1p are sensitive to the HDAC inhibitor TSA. Hos3p and Sir2p are TSA-insensitive, Sir2p is activated by nicotinamide adenine dinucleotide (NAD), and little is known about Hos1p and Hos2p (5, 6). Rpd3p forms a complex with Sin3p and Sap30p that is recruited to DNA by the Ume6p transcription factor (7, 8). SIR2 is one of the silent information regulator (SIR) genes that mediate silencing (repression) at telomeres, mating type loci and ribosomal DNA (8, 9). In an apparent paradox, RPD3 deletion increases silencing of reporter genes inserted at these loci (3, 10, 11).

Although transcription profiles have been reported for a few HDAC deletions in yeast, analysis has been limited (12, 13). Here, we present transcription profiles of the yeast deletion strains rpd3, sin3, sap30, ume6, hda1, hos2, and hos3. In addition, we present profiles of wild-type yeast treated with TSA in concentration- and time-dependent manners. Bioinformatic analyses of these data in the context of existing profiling databases (12–16) yield a cohesive, global view of HDAC function.

Acknowledgments

The rpd3, sin3, hda1, and TSA-titration arrays were produced by Matthew Marton, Chris Roberts, and Stephen Friend of Rosetta Inpharmatics. Technical assistance with Affymetrix GeneChips was provided by the Howard Hughes Medical Institute–Massachusetts Institute of Technology Biopolymers facility. We thank Finny Kuruvilla, Christina Grozinger, James Hardwick, and Julie Sneddon for helpful discussions. We are especially grateful to Chris Hassig and Dan Gottschling for critical readings of the manuscript. This research was supported by a grant from the National Institute of General Medical Sciences. J.K.T. was the recipient of a Fellowship from the National Science Foundation. S.L.S. is an investigator at the Howard Hughes Medical Institute.

Acknowledgments

Abbreviations

HDAC	histone deacetylase
TSA	trichostatin
SIR	silent information regulator
NAD	nicotinamide adenine dinucleotide

Abbreviations

Footnotes

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.250477697.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.250477697

Footnotes

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