Biology subjects

Parua, P. K.

Publications and source records attributed to Parua, P. K..

3 recordsLinked to original sources

bioRxiv2017

Cdk9, Spt5 and histone H2B mono-ubiquitylation cooperate to ensure antisense suppression by the Clr6-CII/Rpd3S HDAC complex

Cyclin-dependent kinase 9 (Cdk9) and histone H2B monoubiquitylation (H2Bub1) are both implicated in elongation by RNA polymerase II (RNAPII). In fission yeast, Cdk9 and H2Bub1 regulate each other through a feedback loop involving phosphorylation of the elongation factor Spt5. Conversely, genetic interactions suggest opposing functions of H2Bub1 and Cdk9 through an Spt5-independent pathway. To understand these interactions, we performed RNA-seq analysis after H2Bub1 loss, Cdk9 inhibition, or both. Either Cdk9 inhibition or H2Bub1 loss increased levels of antisense transcription initiating within coding regions of distinct subsets of genes; ablation of both pathways led to antisense derepression affecting over half the genome. Cdk9 and H2Bub1 cooperate to suppress antisense transcription by promoting function of the Clr6-CII histone deacetylase (HDAC) complex. H2Bub1 plays a second role, in opposition to Clr6-CII, to promote sense transcription in subtelomeric regions. Therefore, functional genomics revealed both collaborative and antagonistic functions of H2Bub1 and Cdk9.

molecular biology↗

bioRxiv2017

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes1. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination2, but how this chain of events is initiated remains unclear. In a chemical-genetic screen, we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex3. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis24. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with slowing of Pol II5. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq)6, and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5--a bistable switch analogous to a Cdk1-PP1 module that controls mitotic progression4.

molecular biology↗

bioRxiv2017

Cdk9 regulates a promoter-proximal checkpoint to modulate RNA Polymerase II elongation rate

Multiple kinases modify RNA Polymerase II (Pol II) and its associated pausing and elongation factors to regulate Pol II transcription and transcription-coupled mRNA processing1,2. The conserved Cdk9 kinase is essential for regulated eukaryotic transcription3, but its mechanistic role remains incompletely understood. Here, we use altered-specificity kinase mutations and highly-specific inhibitors in fission yeast, Schizosaccharomyces pombe to examine the role of Cdk9, and related Cdk7 and Cdk12 kinases, on transcription at base-pair resolution using Precision Run-On sequencing (PRO-seq). Within a minute, Cdk9 inhibition causes a dramatic reduction in the phosphorylation of Pol II-associated factor, Spt5. The effects of Cdk9 inhibition on transcription are the more severe than inhibition of Cdk7 and Cdk12 and result in a shift of Pol II towards the transcription start site (TSS). A kinetic time course of Cdk9 inhibition reveals that early transcribing Pol II is the most compromised, with a measured rate of only ~400 bp/min, while Pol II that is already well into the gene continues rapidly to the end of genes with a rate > 1 kb/min. Our results indicate that while Pol II in S. pombe can escape promoter-proximal pausing in the absence of Cdk9 activity, it is impaired in elongation, suggesting the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.

molecular biology↗