The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation. Here we report that Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. We also demonstrate that lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly.

Newly synthesized histone H4 is deposited in a diacetylated isoform in a wide variety of organisms. In Tetrahymena a specific pair of residues, lysines 4 and 11, have been shown to undergo this modification in vivo. In this report, we demonstrate that the analogous residues, lysines 5 and 12, are acetylated in Drosophila and HeLa 114. These data strongly suggest that deposition-related acetylation sites in H4 have been highly, perhaps absolutely, conserved. In Tetrahymena and Drosophila newly synthesized histone H3 is also deposited in several modified forms. Using pulse-labeled H3 we have determined that, like H4, a specific, but distinct, subset of lysines is acetylated in these organisms. In Tetrahymena, lysines 9 and 14 are highly preferred sites of acetylation in new H3 while in Drosophila, lysines 14 and 23 are strongly preferred. No evidence has been obtained for acetylation of newly synthesized H3 in HeLa cells. Thus, although the pattern and sites of deposition-related acetylation appear to be highly conserved in H4, the same does not appear to be the case for histone H3.Modification of histones by acetylation of the E-amino group of specific lysine residues in the N-terminal domain of all four core histones is an active metabolic process whose exact function remains controversial. The primary focus of much current research is on how histone acetylation relates to the regulation of gene expression (1, 2). Less attention is being placed on understanding the biological function of depositionrelated acetylation, a reaction first described to affect specific histones during synthesis and deposition onto replicating chromatin (3,4).Biochemical analyses of deposition-related acetylation is hampered by the fact that only a fraction of the total histone is affected and once newly synthesized histone is deposited into nuclei, the pattern of acetylation is remodeled to fulfill transcription-related functions. In most systems, depositionrelated acetylation is witnessed only by administering a short pulse of label to preferentially label newly synthesized histones. Using this approach, numerous studies have reported that newly synthesized H4 is deposited as a modified isoform (3-9). This modification, although poorly understood, occurs in organisms ranging from protozoa to humans and thus appears to be highly conserved.In the ciliated protozoan Tetrahymena, deposition-related acetylation is particularly clear because of the separation between germ-line and somatic nuclei. Each vegetative cell contains a transcriptionally active, somatic macronucleus that governs the phenotype of the cell and a transcriptionally inert, germinal micronucleus that is responsible for genetic continuity. During conjugation, the sexual stage of the life cycle, essentially all of the newly synthesized H3 and H4 is selectively deposited into micronuclei; little if any new H3 or H4 is deposited into macronuclei (4). By stain and by label, histone extracted from these micronuclei is greatly enriched in diacetylated H4 and mono-and di...

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Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.Heterochromatin, defined cytologically as regions of the genome that remain condensed throughout the cell cycle, can exert transcriptional repression (32). In Drosophila melanogaster, translocation of a euchromatic region of the genome to a site adjacent to heterochromatin often yields variable repression of the translocated genes. This repression results from heterochromatin spreading into the euchromatic domain, a process referred to as position effect variegation (17,25,75). In female mammalian cells, one of the two X chromosomes becomes heterochromatic early in development, which leads to heritable repression of most of its genes (11,58,60). The hallmark of both these cases of heterochromatin-induced regulation is that repression is position specific but gene nonspecific.Certain loci in Saccharomyces cerevisiae are subject to longterm repression that is similar to repression associated with metazoan heterochromatin. Three separate loci on chromosome III-MAT, HML, and HMR-contain either a or ␣ mating-type genes. At MAT these genes are expressed to specify the corresponding a or ␣ cell type (27, 28). However, despite the fact that the promoters, coding sequences, and even...