The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.
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...
PAX6 is a highly conserved transcription factor and key regulator of several neurogenic processes, including the continuous generation of dopaminergic/GABAergic interneurons in the adult ventricular-subventricular (V-SVZ) neurogenic system in mice. Here we report that PAX6 cooperates with the TALE-homeodomain transcription factor PBX1 in this context. Chromatin-immunoprecipitation showed that PBX1 and PAX6 co-occupy shared genomic binding sites in adult V-SVZ stem- and progenitor cell cultures and mouse embryonic stem cells, while depletion of Pbx1 revealed that association of PAX6 with these sites requires the presence of PBX1. Expression profiling together with viral overexpression or knockdown of Pax6 or Pbx1 identified novel PBX1-PAX6 co-regulated genes, including several transcription factors. Computational modeling of genome wide expression identified novel cross-regulatory networks among these very transcription factors. Taken together, the results presented here highlight the intimate link that exists between PAX6 and TALE-HD family proteins and contribute novel insights into how the orchestrated activity of transcription factors shapes adult V-SVZ neurogenesis.
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