Biology subjects

Fisher, R. P.

Publications and source records attributed to Fisher, R. P..

6 recordsLinked to original sources

bioRxiv2024

Coordinate control of the RNA polymerase II transcription cycle by CDK9-dependent, tripartite phosphorylation of SPT5

The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by reversible protein phosphorylation. The elongation factor SPT5 contains two regions targeted by cyclin-dependent kinase 9 (CDK9) and previously implicated in promoter-proximal pausing and termination: the linker between KOWx-4 and KOW5 domains and carboxy-terminal repeat (CTR) 1, respectively. Here we show that phosphorylations in the KOWx-4/5 linker, CTR1 and a third region, CTR2, coordinately control pause release, elongation speed and RNA processing. Pausing was increased by mutations preventing CTR1 or CTR2 phosphorylation, but attenuated when both CTRs were mutated. Whereas mutating CTR1 alone slowed elongation and repressed nascent transcription, simultaneous mutation of CTR2 partially reversed both effects. Nevertheless, mutating both CTRs led to aberrant splicing, dysregulated termination and diminished steady-state mRNA levels, and impaired cell proliferation more severely than did either single-CTR mutation. Therefore, tripartite SPT5 phosphorylation times pause release and regulates RNAPII elongation rates positively and negatively to ensure productive transcription and cell viability.

molecular biology↗

bioRxiv2024

Structural basis of Cdk7 activation by dual T-loop phosphorylation

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates--the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

biochemistry↗

bioRxiv2020

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3 and 5 ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between KOW motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.

molecular biology↗

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↗