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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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Differentiation of ES cells does not alter the levels or subcellular localization of the CAK complex. Western blots of nuclear and cytoplasmic fractions of ES cells differentiated by retinoic acid (RA) treatment. cAMP-responsive element-binding protein (Creb) and β-tubulin are nuclear and cytoplasmic controls, respectively, for cell fractionation.
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Figure 3: Differentiation of ES cells does not alter the levels or subcellular localization of the CAK complex. Western blots of nuclear and cytoplasmic fractions of ES cells differentiated by retinoic acid (RA) treatment. cAMP-responsive element-binding protein (Creb) and β-tubulin are nuclear and cytoplasmic controls, respectively, for cell fractionation.

Mentions: Given these effects on cell fate, we next determined whether changes in either the levels or localization of components of the CAK complex occur during ES differentiation. We analyzed the subcellular localization of the CAK complex because it has been shown to shift from the cytoplasm to the nucleus during Drosophila development (48). ES cells were differentiated by withdrawal of leukemia inhibitory factor combined with retinoic acid treatment, which leads to rapid loss of Oct-4 expression (Fig. 3). Fractionation of differentiating cells collected over an 8-day time course revealed that neither the levels nor subcellular localization of the CAK complex is altered by differentiation. Thus, we conclude that although cyclin H is not uniquely or more highly expressed in ES cells, it is critical to prevent precocious differentiation. Although Cdk7 was thought to be predominantly nuclear, we observed a significant fraction of the Cdk7 complex in the cytoplasm of ES cells and their differentiated progeny. Cytoplasmic functions for this complex have not been described; therefore the significance of this localization is unclear.


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

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

Differentiation of ES cells does not alter the levels or subcellular localization of the CAK complex. Western blots of nuclear and cytoplasmic fractions of ES cells differentiated by retinoic acid (RA) treatment. cAMP-responsive element-binding protein (Creb) and β-tubulin are nuclear and cytoplasmic controls, respectively, for cell fractionation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Differentiation of ES cells does not alter the levels or subcellular localization of the CAK complex. Western blots of nuclear and cytoplasmic fractions of ES cells differentiated by retinoic acid (RA) treatment. cAMP-responsive element-binding protein (Creb) and β-tubulin are nuclear and cytoplasmic controls, respectively, for cell fractionation.
Mentions: Given these effects on cell fate, we next determined whether changes in either the levels or localization of components of the CAK complex occur during ES differentiation. We analyzed the subcellular localization of the CAK complex because it has been shown to shift from the cytoplasm to the nucleus during Drosophila development (48). ES cells were differentiated by withdrawal of leukemia inhibitory factor combined with retinoic acid treatment, which leads to rapid loss of Oct-4 expression (Fig. 3). Fractionation of differentiating cells collected over an 8-day time course revealed that neither the levels nor subcellular localization of the CAK complex is altered by differentiation. Thus, we conclude that although cyclin H is not uniquely or more highly expressed in ES cells, it is critical to prevent precocious differentiation. Although Cdk7 was thought to be predominantly nuclear, we observed a significant fraction of the Cdk7 complex in the cytoplasm of ES cells and their differentiated progeny. Cytoplasmic functions for this complex have not been described; therefore the significance of this localization is unclear.

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