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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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Cyclin H is critical for the stability of Mat1 and Cdk7 and Cdk7 kinase activity. A, Western blot of CAK components after 5 days of Mat1 knockdown. B, Western blots of the subunits of the CAK complex 3 and 5 days after cyclin H knockdown. C, QRT-PCR of Mat1 and Cdk7 after 5 days of cyclin H knockdown. (n = 3 ± 1). D, Western blot of Mat1, Cdk7, and HIF1α 5 days after cyclin H depletion, comparing cells treated with dimethyl sulfoxide (DMSO) or MG-132 (4 h). E, Western blot of core TFIIH component Xpd after cyclin H knockdown for 5 days. F, kinase assay of immunoprecipitated (IP) Cdk7 after 3 days of cyclin H knockdown with GST-CTD as a substrate (upper panel). Equivalent quantities of Cdk7 were immunoprecipitated (lower panel). scram, scramble; cycH-1, cyclin H shRNA 1; cycH-2, cyclin H shRNA 2.
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Figure 4: Cyclin H is critical for the stability of Mat1 and Cdk7 and Cdk7 kinase activity. A, Western blot of CAK components after 5 days of Mat1 knockdown. B, Western blots of the subunits of the CAK complex 3 and 5 days after cyclin H knockdown. C, QRT-PCR of Mat1 and Cdk7 after 5 days of cyclin H knockdown. (n = 3 ± 1). D, Western blot of Mat1, Cdk7, and HIF1α 5 days after cyclin H depletion, comparing cells treated with dimethyl sulfoxide (DMSO) or MG-132 (4 h). E, Western blot of core TFIIH component Xpd after cyclin H knockdown for 5 days. F, kinase assay of immunoprecipitated (IP) Cdk7 after 3 days of cyclin H knockdown with GST-CTD as a substrate (upper panel). Equivalent quantities of Cdk7 were immunoprecipitated (lower panel). scram, scramble; cycH-1, cyclin H shRNA 1; cycH-2, cyclin H shRNA 2.

Mentions: Decreased levels of cyclin H and Cdk7 have been observed in both tissue-specific and global Mat1-deficient mice (34, 35). In contrast, after 5 days of Mat1 down-regulation in ES cells, we did not observe changes in the levels of either cyclin H or Cdk7 protein (Fig. 4A). If decreased levels of cyclin H contribute to the early embryonic lethality of Mat1 knockout embryos, this difference may explain why Mat1 depletion in ES cells does not significantly influence viability or pluripotency. In contrast, when cyclin H was down-regulated, the levels of Mat1 protein also decreased dramatically within 3 days, and the abundance of Cdk7 was significantly reduced by 5 days after lentiviral transduction (Fig. 4B). These changes in Mat1 and Cdk7 protein are post-transcriptional, because the levels of Mat1 and Cdk7 transcripts were not significantly decreased after cyclin H depletion (Fig. 4C). To determine whether the decrease in Mat1 and Cdk7 protein was proteosome-dependent, scramble and cyclin H shRNA-infected cells were treated with a proteosome inhibitor (MG-132, 4 h) or vehicle control (Me2SO). Treatment with MG-132 did not increase the levels of Mat1 or Cdk7 protein in cyclin H-depleted cells; however, we were able to detect stabilization of HIF1α, indicating that the inhibitor treatment was effective (Fig. 4D). This suggests that proteosomal degradation does not contribute significantly to the decreased levels of Mat1 and Cdk7 protein in cyclin H-depleted cells. Of note, we did not observe changes in the levels of the TFIIH subunit Xpd after cyclin H down-regulation, indicating that core TFIIH stability was not disrupted. (Fig. 4E). To confirm that cyclin H down-regulation does indeed reduce Cdk7 kinase activity, we immunoprecipitated Cdk7 from cyclin H-depleted cells and measured kinase activity toward a GST fusion with the CTD of RNA polymerase II (GST-CTD). At the 3-day time point, before the total cellular levels of Cdk7 had decreased, kinase activity was reduced with both cyclin H shRNAs, confirming that cyclin H is required for maximal Cdk7 activity and that other cellular cyclins cannot compensate for the loss of cyclin H (Fig. 4F). Because ES cells have a unique cell cycle structure, characterized by rapid proliferation and constitutively high Cdk2 activity, we next wanted to determine how this decrease in Cdk7 activity affects cell cycle progression (49).


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

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

Cyclin H is critical for the stability of Mat1 and Cdk7 and Cdk7 kinase activity. A, Western blot of CAK components after 5 days of Mat1 knockdown. B, Western blots of the subunits of the CAK complex 3 and 5 days after cyclin H knockdown. C, QRT-PCR of Mat1 and Cdk7 after 5 days of cyclin H knockdown. (n = 3 ± 1). D, Western blot of Mat1, Cdk7, and HIF1α 5 days after cyclin H depletion, comparing cells treated with dimethyl sulfoxide (DMSO) or MG-132 (4 h). E, Western blot of core TFIIH component Xpd after cyclin H knockdown for 5 days. F, kinase assay of immunoprecipitated (IP) Cdk7 after 3 days of cyclin H knockdown with GST-CTD as a substrate (upper panel). Equivalent quantities of Cdk7 were immunoprecipitated (lower panel). scram, scramble; cycH-1, cyclin H shRNA 1; cycH-2, cyclin H shRNA 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Cyclin H is critical for the stability of Mat1 and Cdk7 and Cdk7 kinase activity. A, Western blot of CAK components after 5 days of Mat1 knockdown. B, Western blots of the subunits of the CAK complex 3 and 5 days after cyclin H knockdown. C, QRT-PCR of Mat1 and Cdk7 after 5 days of cyclin H knockdown. (n = 3 ± 1). D, Western blot of Mat1, Cdk7, and HIF1α 5 days after cyclin H depletion, comparing cells treated with dimethyl sulfoxide (DMSO) or MG-132 (4 h). E, Western blot of core TFIIH component Xpd after cyclin H knockdown for 5 days. F, kinase assay of immunoprecipitated (IP) Cdk7 after 3 days of cyclin H knockdown with GST-CTD as a substrate (upper panel). Equivalent quantities of Cdk7 were immunoprecipitated (lower panel). scram, scramble; cycH-1, cyclin H shRNA 1; cycH-2, cyclin H shRNA 2.
Mentions: Decreased levels of cyclin H and Cdk7 have been observed in both tissue-specific and global Mat1-deficient mice (34, 35). In contrast, after 5 days of Mat1 down-regulation in ES cells, we did not observe changes in the levels of either cyclin H or Cdk7 protein (Fig. 4A). If decreased levels of cyclin H contribute to the early embryonic lethality of Mat1 knockout embryos, this difference may explain why Mat1 depletion in ES cells does not significantly influence viability or pluripotency. In contrast, when cyclin H was down-regulated, the levels of Mat1 protein also decreased dramatically within 3 days, and the abundance of Cdk7 was significantly reduced by 5 days after lentiviral transduction (Fig. 4B). These changes in Mat1 and Cdk7 protein are post-transcriptional, because the levels of Mat1 and Cdk7 transcripts were not significantly decreased after cyclin H depletion (Fig. 4C). To determine whether the decrease in Mat1 and Cdk7 protein was proteosome-dependent, scramble and cyclin H shRNA-infected cells were treated with a proteosome inhibitor (MG-132, 4 h) or vehicle control (Me2SO). Treatment with MG-132 did not increase the levels of Mat1 or Cdk7 protein in cyclin H-depleted cells; however, we were able to detect stabilization of HIF1α, indicating that the inhibitor treatment was effective (Fig. 4D). This suggests that proteosomal degradation does not contribute significantly to the decreased levels of Mat1 and Cdk7 protein in cyclin H-depleted cells. Of note, we did not observe changes in the levels of the TFIIH subunit Xpd after cyclin H down-regulation, indicating that core TFIIH stability was not disrupted. (Fig. 4E). To confirm that cyclin H down-regulation does indeed reduce Cdk7 kinase activity, we immunoprecipitated Cdk7 from cyclin H-depleted cells and measured kinase activity toward a GST fusion with the CTD of RNA polymerase II (GST-CTD). At the 3-day time point, before the total cellular levels of Cdk7 had decreased, kinase activity was reduced with both cyclin H shRNAs, confirming that cyclin H is required for maximal Cdk7 activity and that other cellular cyclins cannot compensate for the loss of cyclin H (Fig. 4F). Because ES cells have a unique cell cycle structure, characterized by rapid proliferation and constitutively high Cdk2 activity, we next wanted to determine how this decrease in Cdk7 activity affects cell cycle progression (49).

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