A second Wpl1 anti-cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4.

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin.

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response.

The nuclear RNA exosome plays a key role in controlling the levels of multiple protein-coding and non-coding RNAs. Recruitment of the exosome to specific RNA substrates is mediated by RNA-binding co-factors. The transient interaction between co-factors and the exosome as well as the rapid decay of RNA substrates make identification of exosome co-factors challenging. Here, we use comparative poly(A)+ RNA interactome capture in fission yeast expressing three different mutants of the exosome to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identify multiple RNA-binding proteins whose association with RNA is altered in exosome mutants, including the zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc-finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight the importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress.

Transcription and chromatin-based surveillance mechanism controls suppression of cryptic antisense transcription.

Phosphorylation of the RNA polymerase II C-terminal domain Y1S2P3T4S5P6S7 consensus sequence coordinates key events during transcription, and its deregulation leads to defects in transcription and RNA processing. Here, we report that the histone deacetylase activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of antisense transcription when RNA polymerase II phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, although single Hos2 and Set3 mutants have little effect, loss of Hos2 or Set3 combined with ssu72Δ results in a synergistic increase in antisense transcription globally and correlates with elevated sensitivity to genotoxic agents. We demonstrate a key role for the Ssu72/Hos2/Set3 mechanism in the suppression of cryptic antisense transcription at the 3' end of convergent genes that are most susceptible to these defects, ensuring the fidelity of gene expression within dense genomes of simple eukaryotes.

The CDK Pef1 and protein phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes.

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here, we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non-phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4-based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.

Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination.

Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain.