Impairment of the TFIIH-associated CDK-activating Kinase Selectively Affects Cell Cycle-regulated Gene Expression in Fission Yeast

Abstract

The fission yeast Mcs6 -Mcs2-Pmh1 complex, homologous to metazoan Cdk7-cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. We analyzed conditional mutants of mcs6 and pmh1, which activate Cdc2 normally but cannot complete cell division at restrictive temperature and arrest with decreased CTD phosphorylation. Transcriptional profiling by microarray hybridization revealed only modest effects on global gene expression: a one-third reduction in a severe mcs6 mutant after prolonged incubation at 36°C. In contrast, a small subset of transcripts (ϳ5%) decreased by more than twofold after Mcs6 complex function was compromised. The signature of repressed genes overlapped significantly with those of cell separation mutants sep10 and sep15. Sep10, a component of the Pol II Mediator complex, becomes essential in mcs6 or pmh1 mutant backgrounds. Moreover, transcripts dependent on the forkhead transcription factor Sep1, which are expressed coordinately during mitosis, were repressed in Mcs6 complex mutants, and Mcs6 also interacts genetically with Sep1. Thus, the Mcs6 complex, a direct activator of Cdc2, also influences the cell cycle transcriptional program, possibly through its TFIIH-associated kinase function. INTRODUCTIONCyclin-dependent kinases (CDKs) play a central role in driving cell division in eukaryotic organisms and also perform essential, conserved functions in the transcription cycle of RNA polymerase (Pol) II (reviewed by Morgan, 1997). Whereas most CDKs can be classified as either cell cycle or transcriptional regulators based on a preeminent physiological function, the metazoan Cdk7 complex is essential in both cell division and gene expression, as the CDK-activating kinase (CAK) and as a component of the general transcription factor IIH (TFIIH) (reviewed by Harper and Elledge, 1998). To execute its dual functions in vivo, Cdk7 has evolved distinct substrate specificities for the activation segment (T-loop) of CDKs and the carboxy-terminal domain (CTD) of the Pol II large subunit, and mechanisms to allow their independent regulation (Garrett et al., 2001;Larochelle et al., 2001). Less clear is how (or whether) the Cdk7 complex serves to coordinate cell division with gene expression.To address this question genetically, we turned to the fission yeast Schizosaccharomyces pombe, which also relies, in part, on a dual-function CDK complex to activate CDKs and phosphorylate Pol II (Buck et al., 1995;Damagnez et al., 1995;Hermand et al., 1998;Lee et al., 1999;Saiz and Fisher, 2002). The orthologue of Cdk7 in S. pombe is Mcs6, which associates with the cyclin Mcs2 and the RING-finger protein Pmh1. Both mcs6 and mcs2 were identified in genetic screens for positive regulators of Cdc2 (S. pombe Cdk1), the major cell cycle CDK (Molz et al., 1989...

Secrets of a double agent: CDK7 in cell-cycle control and transcription

“…The two proteins were later shown to form an active kinase complex (Buck et al, 1995;Damagnez et al, 1995) and to activate fission yeast CDK1-cyclin-B complexes in vitro (Lee et al, 1999). However, mutations in mcs6, mcs2 or pmh1 (which encodes the RING finger subunit) cause neither a defect in CDK activation nor cell-cycle arrest (Molz and Beach, 1993;Buck et al, 1995;Damagnez et al, 1995;Lee et al, 1999;Saiz and Fisher, 2002;Bamps et al, 2004;Lee et al, 2005).…”

“…This assumes, incorrectly as it turns out, that, by mimicking the lack of Mcs6 T-loop phosphorylation in the absence of Csk1, the mcs6 S165A/L238R mutation phenocopies the CAK defect of the mcs6-13 csk1⌬ mutant. The mcs6 S165A/L238R mutant is in fact still capable of CDK1 activation at the restrictive temperature, but shows transcriptional impairment (Saiz and Fisher, 2002;Lee et al, 2005). Moreover, deletion of csk1 + is unconditionally lethal in the mcs6 S165A/L238R background (Saiz and Fisher, 2002).…”

Suppression of the Schizosaccharomyces pombe cut12.1 Cell-Cycle Defect by Mutations in cdc25 and Genes Involved in Transcriptional and Translational Control

“…If a monopolar spindle phenotype is indicative of a compromised commitment to mitosis, it could explain why monopolar spindles occur as a consequence of compromised TFIIH function as it was recently established that TFIIH modulates the cell-cycle levels of transcripts of mitotic inducers prior to mitosis (Lee et al 2005). Compromising TFIIH activity would reduce the ability to regulate mitotic inducers to promote an efficient entrance to mitosis.…”

“…rot 1 genes encode molecules that modulate protein production: The rot 1 genes and corresponding multicopy suppressors were predicted to encode molecules implicated in the control of protein production at the level of transcription, translation, or RNA processing ( Table 2). The rot1/tfb2 1 and rot5 1 /tfb5 1 genes and a suppressor of rot4.1 (tfb1 1 ) encoded conserved components of the TFIIH complex that plays a critical role in promoting cell-cycle-dependent transcription of a subset of S. pombe genes (Lee et al 2005). This predicted functional relationship was supported by genetic interactions as rot1.1 (hereafter referred to as tfb2.rt1) displayed synthetic lethality with rot5.1 (hereafter referred to as tfb5.rt5) and the introduction of a multicopy plasmid harboring the tfb5 1 gene was able to complement the cold sensitivity of tfb2.rt1 ( Figure 1C).…”

Glaucoma Genetics – Regulation of Cell Surviving and Death in the Retina

“…These complexes cyc/cdk become activated or inhibited sequentially in different phases of the cell cycle. Cell cycle progression is the result of the interaction between cyclins and their cdks, and a high variety of inhibitory proteins, the corresponding cdk inhibitors (cdki) (Lee et al, 2005). As shown in the following scheme, the cycD and the corresponding cdk4/6 activities regulate the early progression of the cell cycle, through the G1 phase.…”