Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast

Abstract

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe. H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2 +-a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2 + antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 proteincoding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycledependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

“…5A-B, top rows). The observed increase in antisense transcripts among the H2BK123ub-defective mutants is in agreement with reports of increased antisense transcription in S. pombe H2B ubiquitylation mutants (8,52), although to our knowledge this is the first time this effect has been reported for an S. cerevisiae integrated H2B-K123R double mutant. To assess the extent of overlap between their transcriptomes, we next performed direct mutant-to-mutant comparisons (Fig.…”

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription

“…To uncover changes in RNAPII transcription that might illuminate previously described genetic interactions between the Cdk9 and H2Bub1 pathways, we performed strand-specific RNA-seq analysis, comparing wild-type cells and ones with 1) an analog-sensitive ( as ) mutation ( cdk9 as ) that confers sensitivity to inhibition by the bulky adenine analog 3-MB-PP1 ( 36 ), 2) htb1-K119R or 3) both mutations ( 41 ). Cdk9 inhibition or H2Bub1 ablation had specific rather than global effects on steady-state mRNA abundance (Figure 1A ), consistent with previous analyses ( 9 , 36 ).…”

Opposing functions of the Hda1 complex and histone H2B mono-ubiquitylation in regulating cryptic transcription inSaccharomyces cerevisiae

“…This suggests that H2Bub functions independently of these downstream modifications in preventing cryptic initiation. H2Bub has been shown to stabilize nucleosomes, particularly in the middle and ends of genes ( Chandrasekharan et al 2009 ; Batta et al 2011 ), and loss of H2Bub in S. pombe up-regulates noncoding RNAs, particularly antisense transcripts ( Pagé et al 2019 ; Murawska et al 2020 ; Sansó et al 2020 ). Recent work using optical tweezers has confirmed the increase in stability of nucleosomes containing H2Bub, and this increase in stability is an energetic barrier to passage by Pol II ( Chen et al 2019 ).…”