Project description:Chromatin modifications play a pivotal role in cell fate decision. In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis when cells starve. The methylation of H3K4 by Set1-COMPASS and consequent promoter nucleosome deacetylation was shown to repress ste11 induction and cell differentiation but the regulatory steps remain poorly understood. A genetic screen highlighted H2B ubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2B-ub1 at the promoter where it represses expression, and over the transcribed region where it sustains transcription of ste11. By promoting H3K4 methylation at the promoter, H2B-ub1 initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR formation leads to high expression. Therefore, H2B-ub1 represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene. Samples from mononucleosomal DNA from S. pombe strains h-972 and h-972 rsc1::kanR were sequenced (Illumina NextSeq 500 platform) using the pair-end read protocol
Project description:Chromatin modifications play a pivotal role in cell fate decision. In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis when cells starve. The methylation of H3K4 by Set1-COMPASS and consequent promoter nucleosome deacetylation was shown to repress ste11 induction and cell differentiation but the regulatory steps remain poorly understood. A genetic screen highlighted H2B ubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2B-ub1 at the promoter where it represses expression, and over the transcribed region where it sustains transcription of ste11. By promoting H3K4 methylation at the promoter, H2B-ub1 initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR formation leads to high expression. Therefore, H2B-ub1 represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.
Project description:The SAGA complex is a conserved multifunctional coactivator known to play broad roles in eukaryotic transcription. To gain new insights into its functions, we have performed biochemical and genetic analyses of SAGA in the fission yeast, Schizosaccharomyces pombe. Purification of the S. pombe SAGA complex showed that its subunit composition is identical to that of Saccharomyces cerevisiae. Analysis of S. pombe SAGA mutants revealed that SAGA has two opposing roles regulating sexual differentiation. First, in nutrient rich conditions, the SAGA histone acetyltransferase, Gcn5, represses ste11+, which encodes the master regulator of the mating pathway. In contrast, the SAGA subunit Spt8 is required for the induction of ste11+ upon nutrient starvation. Chromatin immunoprecipitation experiments suggest that these regulatory effects are direct, as SAGA is physically associated with the ste11+ promoter independent of nutrient levels. Genetic tests suggest that nutrient levels do cause a switch in SAGA function, as spt8? suppresses gcn5? with respect to ste11+ derepression in rich medium, whereas the opposite relationship, gcn5? suppression of spt8?, occurs during starvation. Thus, SAGA plays distinct roles in the control of the switch from proliferation to differentiation in S. pombe through the dynamic and opposing activities of Gcn5 and Spt8.
Project description:The cell-fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which enodes the central inducer of mating and gametogenesis, know as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates SET3C-dependent deacetylation and repression at the ste11 promoter. Upon starvation, the activation of the ste11 promoter is bidirectional, generating a second lincRNA, rce1, overlapping the promoter of rse1. The transcription of rce1 establishes repressive chromatin at the rse1 promoter, therefore relieving the repression on ste11. Thus, the transcription of two lincRNAs governs the switch from vegetative growth to sexual differentiation in fission yeast. Our data reveals that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differeniation.
Project description:In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123. The SAGA complex component, Ubp8, is one of two proteases that remove this ubiquitin moiety. We analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affects gene expression. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1K123R. This provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Second, microarray analyses of the htb1K123R strain show that loss of histone ubiquitylation results in a two-fold or greater change in expression of ~1.5% of the protein coding genes with greater than two-thirds increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of the methyltransferase components Swd3, Set2 or Dot1 and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1K123R strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of its being a site of ubiquitylation. Keywords: yeast, histone ubiquitylation, Ubp8, gene expression, genetic modification, histone H2B Two dye-swapped, biological replicate experiments were performed for yeast strains CY1272(Htb1_K123R;htb2_delta0), BY10809(ubp8_delta0) and CY1383(Htb1_K123R;htb2_delta0;ubp8_delta0) with reference to BY4742(wt). Three biological replicates, including one dye-swap experiment, were performed comparing CY1272(Htb1_K123R;htb2_delta0) to BY13026(htb2_delta0).
Project description:In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123. The SAGA complex component, Ubp8, is one of two proteases that remove this ubiquitin moiety. We analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affects gene expression. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1K123R. This provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Second, microarray analyses of the htb1K123R strain show that loss of histone ubiquitylation results in a two-fold or greater change in expression of ~1.5% of the protein coding genes with greater than two-thirds increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of the methyltransferase components Swd3, Set2 or Dot1 and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1K123R strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of its being a site of ubiquitylation. Keywords: yeast, histone ubiquitylation, Ubp8, gene expression, genetic modification, histone H2B
Project description:Transcription by RNA polymerase II is regulated by epigenetic modifications to the chromatin template. The mono-ubiquitylation of H2B is established during transcription elongation and broadly impacts chromatin architecture and gene expression. The Polymerase Associated Factor 1 complex (Paf1C) is required for H2B ubiquitylation through an unknown mechanism. Here, we find that a 66-amino acid histone modification domain (HMD) within the Rtf1 subunit of Paf1C promotes H2B ubiquitylation in cells lacking all Paf1C members and present the crystal structure of this domain. Using site-specific in vivo crosslinking, we show that Rtf1 directly interacts with the ubiquitin conjugase Rad6 through a conserved surface on the HMD. Through ChIP-exo analysis, we observe that enrichment of Paf1C correlates with H2B ubiquitylation, and that the HMD, Rad6 and Bre1 localize to H2B. Finally, we demonstrate that the HMD directly stimulates H2B ubiquitylation in a reconstituted system, arguing that Paf1C functions as a cofactor for Rad6-Bre1 mediated catalysis.