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Functional analysis of the Cdk7.cyclin H.Mat1 complex in mouse embryonic stem cells and embryos.

Patel SA, Simon MC - J. Biol. Chem. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

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.

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Mat1 depletion does not affect the differentiation of ES cells. A, model of the Mat1·cyclin H·Cdk7 complex. B, QRT-PCR of Mat1 expression after 5 days of lentivirus mediated knockdown. (n = 3 ± 1). C, Western blot of Mat1 knockdown. scram, scramble. D, phase contrast images (left column) and staining for alkaline phosphatase activity (right column) of ES cells after Mat1 knockdown for 5 days. E, QRT-PCR analysis of pluripotency genes and Oct-4 targets after Mat1 knockdown for 5 days (n = 3 ± 1).
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Figure 1: Mat1 depletion does not affect the differentiation of ES cells. A, model of the Mat1·cyclin H·Cdk7 complex. B, QRT-PCR of Mat1 expression after 5 days of lentivirus mediated knockdown. (n = 3 ± 1). C, Western blot of Mat1 knockdown. scram, scramble. D, phase contrast images (left column) and staining for alkaline phosphatase activity (right column) of ES cells after Mat1 knockdown for 5 days. E, QRT-PCR analysis of pluripotency genes and Oct-4 targets after Mat1 knockdown for 5 days (n = 3 ± 1).

Mentions: 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 (1–3). Activation of Cdks requires both binding of cyclin and T-loop phosphorylation. As shown in Fig. 1A, Cdk7 is associated with two regulatory subunits: cyclin H and the RING finger protein Mat1 (ménage-à-trois 1) (4–7). Cdk7 is activated by cyclin H, whereas Mat1 modulates the substrate specificity of the complex (8). Cdk7 has been validated as a functional CAK in vivo using temperature-sensitive alleles in Drosophila and a chemical genetics approach in human cancer cells, where Cdk7 appears to be required for both S phase entry and mitosis (9, 10). However, cyclin H levels and Cdk7 kinase activity do not vary during the cell cycle (11–13).


Functional analysis of the Cdk7.cyclin H.Mat1 complex in mouse embryonic stem cells and embryos.

Patel SA, Simon MC - J. Biol. Chem. (2010)

Mat1 depletion does not affect the differentiation of ES cells. A, model of the Mat1·cyclin H·Cdk7 complex. B, QRT-PCR of Mat1 expression after 5 days of lentivirus mediated knockdown. (n = 3 ± 1). C, Western blot of Mat1 knockdown. scram, scramble. D, phase contrast images (left column) and staining for alkaline phosphatase activity (right column) of ES cells after Mat1 knockdown for 5 days. E, QRT-PCR analysis of pluripotency genes and Oct-4 targets after Mat1 knockdown for 5 days (n = 3 ± 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865308&req=5

Figure 1: Mat1 depletion does not affect the differentiation of ES cells. A, model of the Mat1·cyclin H·Cdk7 complex. B, QRT-PCR of Mat1 expression after 5 days of lentivirus mediated knockdown. (n = 3 ± 1). C, Western blot of Mat1 knockdown. scram, scramble. D, phase contrast images (left column) and staining for alkaline phosphatase activity (right column) of ES cells after Mat1 knockdown for 5 days. E, QRT-PCR analysis of pluripotency genes and Oct-4 targets after Mat1 knockdown for 5 days (n = 3 ± 1).
Mentions: 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 (1–3). Activation of Cdks requires both binding of cyclin and T-loop phosphorylation. As shown in Fig. 1A, Cdk7 is associated with two regulatory subunits: cyclin H and the RING finger protein Mat1 (ménage-à-trois 1) (4–7). Cdk7 is activated by cyclin H, whereas Mat1 modulates the substrate specificity of the complex (8). Cdk7 has been validated as a functional CAK in vivo using temperature-sensitive alleles in Drosophila and a chemical genetics approach in human cancer cells, where Cdk7 appears to be required for both S phase entry and mitosis (9, 10). However, cyclin H levels and Cdk7 kinase activity do not vary during the cell cycle (11–13).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

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.

Show MeSH
Related in: MedlinePlus