Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

Abstract

Cyclin-dependent kinases (Cdk) coordinate timely progression through the cell cycle. Cdk activity is modulated by the association of regulatory subunits (cyclins, inhibitors, and assembly factors) and by activating and inhibiting phosphorylation at conserved residues (1). Phosphorylation within the activating segment, referred to as the T loop, is essential for maximal activity and is catalyzed by a Cdk-activating kinase (CAK) (2, 3). Genetic and biochemical studies in several model organisms pointed to Cdk7-cyclin H-Mat1 as the in vivo CAK in metazoans. Cdk7 specifically phosphorylates both Cdk1 and Cdk2 in human cells (4-6), and elegant experiments using chemical genetics showed that Cdk7 is required for the assembly of Cdk1-cyclin B (7). CAK activity is decreased in cdk7 mutants in either Drosophila (8) or worms (9). However, correlations between biochemical data and phenotypes have always been complicated by the additional role of Cdk7 in transcription (10). Indeed, the trimeric CAK is also a component of the RNA polymerase II (Pol II) general transcription factor TFIIH, where it phosphorylates the C-terminal domain (CTD) of Rpb1, the largest subunit of the Pol II enzyme (11,12). Within the Cdk family, Cdk7 also is distinct in being activated by either a phosphorylation on its T loop or by the assembly factor Mat1 (13). These functions and regulations of Cdk7 are conserved in the budding yeast Saccharomyces cerevisiae, where its ortholog, Kin28, is well described as a CTD kinase regulating transcription (14, 15). Intriguingly, Kin28 is devoid of CAK activity (16), and Cak1, a divergent, single-subunit kinase distantly related to Cdk (17, 18), instead catalyzes Cdk activation at both transitions of the budding yeast cell cycle.The fission yeast Schizosaccharomyces pombe possesses two CAKs, the nonessential Csk1 and the essential Mcs6 kinases, corresponding to the yeast Cak1 and the metazoan Cdk7, respectively (19-25). Csk1 activates Mcs6 by phosphorylation of the T loop on serine 165 (20), and a widely held view is that the two CAKs act redundantly for Cdc2 activation (22,24), implying that inactivation of Mcs6 reveals the sole role of Cdk7 in transcription. However, strong genetic data argue against the idea that Csk1 contributes significantly to Cdc2 activation in vivo. First, the original mutant alleles of both the mcs6 (mcs6-13) and mcs2 (mcs2-75) genes, which, respectively, encode Cdk7 and cyclin H, were independently identified in a screen for suppressor of mitotic catastrophe, a phenotype resulting from hyperactive Cdc2, and both show allele-specific interaction with cdc2 (23). This is inconsistent with a functional redundancy with Csk1, as the strain was otherwise wild type for csk1. Moreover, another allele of fission yeast cdk7 (mcs6 S165A L238R [the S-to-A change at position 165 and the L-to-R change at position 238 encoded by mcs6]) leads to thermosensitivity associated with decreased Cdc2 phosphorylation (24), and the growth defect is suppressed by Cdc2 activators or by budding yeast Cak1 but not...

A conserved role of the RSC chromatin remodeler in the establishment of nucleosome-depleted regions

“…Our recent work identified RSC as a key downstream effector of a cascade controlling the level of acetylation around the NDR of ste11 , which encodes the master regulator of gametogenesis in S. pombe (Anandhakumar et al 2013; Cassart et al 2012; Coudreuse et al 2010; Devos et al 2015). Deletion of non-essential subunits of RSC or transcriptional switch-off of snf21 both impede ste11 expression and correlate with higher nucleosome occupancy at the ste11 NDR (Materne et al 2015, 2016).…”

Promoter nucleosome dynamics regulated by signalling through the CTD code

“…Despite discrepancies, genome-wide analyses of CTD phosphorylation support that the CTD cycle is uniform across genes ( Kim et al, 2010 ; Mayer et al, 2010 ; Tietjen et al, 2010 ; Bataille et al, 2012 ), and a broad body of evidence supports the seminal role of CTD S5P and S2P in transcriptional regulation and chromatin metabolism ( Buratowski, 2009 ). However, recent works showed that contrary to S5P, S2P is dispensable in both fission yeast and budding yeast ( Cassart et al, 2012 ; Devos et al, 2015 ) and only affects the steady-state level of a subset of mRNAs ( Coudreuse et al, 2010 ; Saberianfar et al, 2011 ; Sukegawa et al, 2011 ). Therefore, a disconnect exists between the apparent uniform occupancy of the phosphorylated PolII and the gene-specific defects resulting from the disappearance of a phosphorylation; a similar case occurring with chromatin regulators ( Drogat and Hermand, 2012 ; Egloff et al, 2012 ; Weiner et al, 2012 ).…”

Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

“…When unbound of TFIIH, TFIIK plays a pivotal role in cell cycle control through activation of cell cycle kinases by T‐loop phosphorylation. Cell cycle kinase‐activating kinase (CAK) activity of human CDK7 is mediated by autophosphorylation and CYCH binding (Martinez et al ., ; Fisher, , ), whereas for binding of Msc2/cyclin H Msc6 requires its T‐loop phosphorylation by the Csk1 CAK‐kinase (CAKAK), which has no CAK activity (Hermand et al ., ; Devos et al ., ). In contrast, budding yeast Kin28 has no CAK activity (Cismowski et al ., ) and its activating T‐loop phosphorylation is mediated by the CDC28 cell cycle kinase‐activating kinase Cak1/Cvi1 (Kaldis et al ., ).…”