Despite recent advances in characterizing the regulation of histone H3 lysine 4 (H3-K4) methylation at the GAL1 gene by the H2B-K123-specific deubiquitinase activity of Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase)-associated Ubp8p, our knowledge on the general role of Ubp8p at the SAGA-dependent genes is lacking. For this study, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay, we have analyzed the role of Ubp8p in the regulation of H3-K4 methylation at three other SAGA-dependent yeast genes, namely, PHO84, ADH1, and CUP1. Like that at GAL1, H3-K4 methylation is increased at the PHO84 core promoter in the UBP8 deletion mutant. We also show that H3-K4 methylation remains invariant at the PHO84 open reading frame in the ⌬ubp8 mutant, demonstrating a highly localized role of Upb8p in regulation of H3-K4 methylation at the promoter in vivo. However, unlike that at PHO84, H3-K4 methylation at the two other SAGA-dependent genes is not controlled by Ubp8p. Interestingly, Ubp8p and H3-K4 methylation are dispensable for preinitiation complex assembly at the core promoters of these genes. Our ChIP assay further demonstrates that the association of Ubp8p with SAGA is mediated by Sgf11p, consistent with recent biochemical data. Collectively, the data show that Ubp8p differentially controls H3-K4 methylation at the SAGA-dependent promoters, revealing a complex regulatory network of histone methylation 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 organis...

Previous biochemical studies have demonstrated that Lys-123 ubiquitination of histone H2B is globally required for up-regulation of mono-, di, and trimethylation of Lys-4 of histone H3. However, recent studies have implicated H2B-Lys-123 ubiquitination in the regulation of di-and trimethylation, but not monomethylation, of H3-Lys-4 in vivo. Using a formaldehydebased cross-linking and chromatin immunoprecipitation assay, we show that H3-Lys-4 trimethylation, but not dimethylation, is up-regulated by H2B-Lys-123 ubiquitination in vivo at the coding sequences of a set of transcriptionally active genes such as ADH1, PHO84, and PYK1. Both the ubiquitination of H2B-Lys-123 and the methylation of H3-Lys-4 are dispensable for recruitment of RNA polymerase II to the coding sequences of these genes, and hence, their transcription is not altered in the absence of these covalent modifications. However, recruitment of RNA polymerase II to the coding sequence of a galactoseinducible gene, GAL1, is significantly reduced in the absence of H2B-Lys-123 ubiquitination but not H3-Lys-4 methylation. Consistently, transcription of GAL1 is altered in the H2B-K123R point mutant strain. Finally, we show that H3-Lys-4 methylation does not regulate H3-Lys-9/14 acetylation. Collectively, our data reveal a "trans-tail" regulation of H3-Lys-4 tribut not dimethylation by H2B-Lys-123 ubiquitination, and these modifications are dispensable for transcription of a certain set of genes in vivo.The eukaryotic genome is packaged into chromatin that is an array of nucleosomes; in each nucleosomes, 146 bp of DNA is wrapped around an octamer of core histone proteins, H2A, H2B, H3, and H4 (1). Chromatin is a dynamic structure that modulates the access of regulatory factors to the genetic material. Thus, transcription and other cellular processes that require access to DNA are regulated by chromatin structure, and therefore, a precise coordination and organization of events in opening and closing the chromatin is crucial for these cellular processes to happen normally.Post-translational modifications (e.g. acetylation, phosphorylation, ubiquitination, and methylation) of the histones affect chromatin structure directly by altering DNA-histone interactions within and between nucleosomes, thus changing higher order chromatin structure (2). Histone modifications are, therefore, the key determinants in defining the active and repressed states of the chromatin (3-8). For example, methylation of H3-Lys-4 is correlated with gene activation, whereas H3-Lys-9 methylation results in repression and heterochromatin formation. Furthermore, histone acetylation is generally correlated with gene activation, although there are exceptions to this rule.Histone methylation on specific Lys or Arg residues is carried out by CARM1, PRMT1, and SET domain-containing enzymes (9, 10). The CARM1 and PRMT1 mediate Arg methylation, whereas Lys is methylated by the SET domain-containing enzymes (9, 10). In Saccharomyces cerevisiae, Arg methylation of histones has not been demonstrated...

The cap-binding complex (CBC) binds to the cap structure of mRNA to protect it from exonucleases as well as to regulate downstream post-transcriptional events, translational initiation and nonsense-mediated mRNA decay. However, its role in regulation of the upstream transcriptional events such as initiation or elongation remains unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with transcriptional, mutational and co-immunoprecipitational analyses, we show that CBC is recruited to the body of yeast gene, and then stimulates the formation of pre-initiation complex (PIC) at several yeast promoters through its interaction with Mot1p (modifier of transcription). Mot1p is recruited to these promoters, and enhances the PIC formation. We find that CBC promotes the recruitment of Mot1p which subsequently stimulates PIC formation at these promoters. Furthermore, the formation of PIC is essential for recruitment of CBC. Thus, our study presents an interesting observation that an mRNA binding factor exhibits a reciprocal synergistic effect on formation of PIC (and hence transcriptional initiation) at the promoter, revealing a new pathway of eukaryotic gene regulation in vivo.