Ubp8p, a Histone Deubiquitinase Whose Association with SAGA Is Mediated by Sgf11p, Differentially Regulates Lysine 4 Methylation of Histone H3 In Vivo

Transcription is a highly coordinated and orchestrated process. It takes place on the DNA template that is packaged into chromatin in the nucleus. The chromatin is an array of nucleosomes in which 146 base pairs of DNA (in each nucleosome) are wrapped around the histone octamer (27). Each octamer consists of two molecules each of the histones H2A, H2B, H3, and H4 (27). Nucleosomal arrays fold into a 30-nm fiber upon incorporation of the linker histone H1 (19, 22). Histones are subject to posttranslational modifications such as acetylation, phosphorylation, ubiquitination, and methylation, and these modifications are known to play important roles in epigenetic regulation of transcription (33, 44). Most modifications were originally observed on the N-terminal tails of histones, with the exception of ubiquitination, which occurs on the C-terminal tails of H2A and H2B (13). However, several novel posttranslational modifications have been identified recently in the core region of the histones (45). Histone modifications alter DNA-histone interactions within and between nucleosomes, and thus they affect the higher-order chromatin structure. Alternatively, combinations of histone modifications present an interaction surface for other proteins that translate the so-called “histone code” into a gene expression pattern (42), explaining how the same chemical modification can have different functional consequences depending on the target amino acid residue. Methylation of histone H3 at lysine 4 (K4) is associated with active chromatin in a wide range of eukaryotic organisms (5, 15, 25, 30, 32, 41) and is correlated with gene activation (38) as well as rRNA gene silencing (7, 9). Lysine methylation of histones in vivo occurs in the following three states: mono-, di-, and trimethyl. Histone H3 has been found to be dimethylated at K4 in active euchromatic regions but not in silent heterochromatic sites, whereas trimethylated H3-K4 is mainly found associated with the 5′ regions of the actively transcribing genes (28) and, thus, unlike dimethyl-H3-K4, is typically not detected at a locus prior to transcription activation (38). Interestingly, H3-K4 methylation is up-regulated in a “trans-tail” process (11, 43) by Rad6p-mediated ubiquitination of H2B at K123 (36), thus suggesting roles of H2B-K123 ubiquitination and deubiquitination in the regulation of gene activation. A histone deubiquitinase, Ubp8p, has recently been implicated as a new component of the Saccharomyces cerevisiae SAGA complex (10, 20, 37). Ubp8p has consistently been shown to hydrolyze the ubiquitin moiety of K123-ubiquitinated H2B at the SAGA-dependent GAL1 promoter (20). Such H2B-K123 deubiquitination by Ubp8p modulates the level of H3-K4 methylation at the GAL1 promoter in a “trans-tail” process, hence altering the expression of the GAL1 gene (20). Although Ubp8p is known to regulate H3-K4 methylation at the SAGA-regulated GAL1 promoter (20), its general role at other SAGA-dependent yeast promoters is not known. To understand in greater detail the role of Ubp8p in the regulation of H3-K4 methylation and, hence, transcription, we have analyzed its contribution to regulation of H3-K4 methylation and formation of the preinitiation complex (PIC) assembly at three other SAGA-dependent yeast promoters (i.e., PHO84, ADH1, and CUP1) by a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay. Furthermore, we have analyzed the role of Sgf11p (a newly identified component of SAGA) in the association of Ubp8p with SAGA in vivo. Our results reveal differences in the regulation of H3-K4 methylation by Ubp8p. We also show that Ubp8p and Sgf11p constitute a discrete structural module within SAGA in vivo.