Abstract
The trimeric Cdk7·cyclin H·Mat1 complex functions in cell cycle regulation, as the Cdk-activating kinase, and in transcription, as a module of the general transcription factor TFIIH. As a component of TFIIH, Cdk7 phosphorylates serines 5 and 7 of the carboxyl-terminal domain of RNA polymerase II and can also directly phosphorylate transcription factors to regulate gene expression. Here we have investigated the function of the Cdk7·cyclin H·Mat1 complex in murine embryonic stem (ES) cells and preimplantation embryos to determine whether it regulates the unique cell cycle structure and transcriptional network of pluripotent cells. We demonstrate that depletion of cyclin H leads to differentiation of ES cells independent of changes in cell cycle progression. In contrast, we observed that developmental genes are acutely up-regulated after cyclin H down-regulation, likely perturbing normal ES self-renewal pathways. We further demonstrate that Spt5, a known phosphorylation target of Cdk7, similarly regulates ES pluripotency and gene expression. Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass of blastocysts, a transient pluripotent stem cell population in vivo. Our findings indicate that cyclin H has an essential function in promoting the self-renewal of the pluripotent stem cells of blastocyst stage embryos. Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development.
Highlights
Cyclin-dependent kinase 7 (Cdk7)3 was initially isolated as a Cdk-activating kinase (CAK) through biochemical studies that showed it could phosphorylate a key threonine residue in the activation segment (T-loop) of other Cdks [1,2,3]
We investigate the function of Mat1 and cyclin H in mouse Embryonic stem (ES) cells to understand the mechanism for early embryonic lethality of Mat1-deficient embryos
The levels of Oct-4 and Sox-2 protein and the expression of a number of well characterized Oct-4 target genes were not significantly altered by decreased Mat1 (Fig. 1, C and E). These results were surprising considering the dramatic defect in expansion of the inner cell mass from Mat1-null embryos. One explanation for these observations is that there is a unique requirement for Mat1 in early embryonic development, which does not manifest in an established ES cell line
Summary
For cell survival or transcription, but rather that it can modulate select target genes in a cell type-specific manner. A network of transcription factors that are uniquely expressed in pluripotent cells and critical for self-renewal, such as Oct-4 and Nanog, have been identified in recent studies [38]. Depletion of cyclin H, which was decreased in Mat1-deficient embryos, leads to differentiation of ES cells and defects in expansion of the ICM of explanted blastocysts. This suggests that decreased cyclin H protein levels could contribute to the early embryonic lethality of Mat mutant embryos. This study provides, to our knowledge, the first loss-of-function analysis of cyclin H in a mammalian system
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