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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 required for the maintenance of pluripotent cells in blastocyst explants. A, QRT-PCR analysis of gene expression in one-cell to blastocyst stage embryos. B, QRT-PCR and Western blot showing cyclin H knockdown in transduced embryos. The QRT-PCR results are the averages of three independent experiments with pools of three to five embryos/time point. (± 1 S.E.; *, p < 0.05; **, p < 0.01). Western blot was performed on 10 blastocysts (96 h)/sample. C, the percentage of scramble (n = 83) and cyclin H shRNA2 (n = 82) transduced one-cell embryos that reached the blastocyst stage. Averages of four independent experiments with 15–30 embryos/experiment are shown. D, development of scramble and cyclin H transduced embryos at 48, 72, and 96 h after transduction. GFP expression in blastocyst stage embryos is apparent in both the ICM (arrow) and trophectoderm. E, phase contrast images of blastocyst explants from embryos transduced at the one-cell stage with scramble or cyclin H shRNA2. Representative images for varying levels of cyclin H depletion (the fold change relative to scramble and the n value are noted below each set of micrographs). scram, scramble; cycH-2, cyclin H shRNA 2.
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Figure 8: Cyclin H is required for the maintenance of pluripotent cells in blastocyst explants. A, QRT-PCR analysis of gene expression in one-cell to blastocyst stage embryos. B, QRT-PCR and Western blot showing cyclin H knockdown in transduced embryos. The QRT-PCR results are the averages of three independent experiments with pools of three to five embryos/time point. (± 1 S.E.; *, p < 0.05; **, p < 0.01). Western blot was performed on 10 blastocysts (96 h)/sample. C, the percentage of scramble (n = 83) and cyclin H shRNA2 (n = 82) transduced one-cell embryos that reached the blastocyst stage. Averages of four independent experiments with 15–30 embryos/experiment are shown. D, development of scramble and cyclin H transduced embryos at 48, 72, and 96 h after transduction. GFP expression in blastocyst stage embryos is apparent in both the ICM (arrow) and trophectoderm. E, phase contrast images of blastocyst explants from embryos transduced at the one-cell stage with scramble or cyclin H shRNA2. Representative images for varying levels of cyclin H depletion (the fold change relative to scramble and the n value are noted below each set of micrographs). scram, scramble; cycH-2, cyclin H shRNA 2.

Mentions: Given the importance of cyclin H function in maintaining ES cell identity, we next determined whether cyclin H had a role in either the initial establishment or expansion of pluripotent cells of the inner cell mass. Gene expression analysis showed that cyclin H transcript levels increase in four-cell embryos and then remain high through the blastocyst stage relative to one-cell embryos (Fig. 8A). To deplete cyclin H in early embryogenesis, we infected one-cell embryos with lentiviruses expressing cyclin H shRNA 2, also used in our previous studies. To monitor infection, the puromycin cassette of the lentivirus was replaced with enhanced GFP. The embryos were cultured in individual microdrops to the blastocyst stage. GFP expression was first apparent in morulae, ∼72 h after embryo transduction, and QRT-PCR showed that cyclin H mRNA was significantly reduced at this time point (Fig. 8B; *, p < 0.05). Cyclin H protein was also reduced by the blastocyst stage, along with a further decrease in mRNA (**, p < 0.01).


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 required for the maintenance of pluripotent cells in blastocyst explants. A, QRT-PCR analysis of gene expression in one-cell to blastocyst stage embryos. B, QRT-PCR and Western blot showing cyclin H knockdown in transduced embryos. The QRT-PCR results are the averages of three independent experiments with pools of three to five embryos/time point. (± 1 S.E.; *, p < 0.05; **, p < 0.01). Western blot was performed on 10 blastocysts (96 h)/sample. C, the percentage of scramble (n = 83) and cyclin H shRNA2 (n = 82) transduced one-cell embryos that reached the blastocyst stage. Averages of four independent experiments with 15–30 embryos/experiment are shown. D, development of scramble and cyclin H transduced embryos at 48, 72, and 96 h after transduction. GFP expression in blastocyst stage embryos is apparent in both the ICM (arrow) and trophectoderm. E, phase contrast images of blastocyst explants from embryos transduced at the one-cell stage with scramble or cyclin H shRNA2. Representative images for varying levels of cyclin H depletion (the fold change relative to scramble and the n value are noted below each set of micrographs). scram, scramble; cycH-2, cyclin H shRNA 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Cyclin H is required for the maintenance of pluripotent cells in blastocyst explants. A, QRT-PCR analysis of gene expression in one-cell to blastocyst stage embryos. B, QRT-PCR and Western blot showing cyclin H knockdown in transduced embryos. The QRT-PCR results are the averages of three independent experiments with pools of three to five embryos/time point. (± 1 S.E.; *, p < 0.05; **, p < 0.01). Western blot was performed on 10 blastocysts (96 h)/sample. C, the percentage of scramble (n = 83) and cyclin H shRNA2 (n = 82) transduced one-cell embryos that reached the blastocyst stage. Averages of four independent experiments with 15–30 embryos/experiment are shown. D, development of scramble and cyclin H transduced embryos at 48, 72, and 96 h after transduction. GFP expression in blastocyst stage embryos is apparent in both the ICM (arrow) and trophectoderm. E, phase contrast images of blastocyst explants from embryos transduced at the one-cell stage with scramble or cyclin H shRNA2. Representative images for varying levels of cyclin H depletion (the fold change relative to scramble and the n value are noted below each set of micrographs). scram, scramble; cycH-2, cyclin H shRNA 2.
Mentions: Given the importance of cyclin H function in maintaining ES cell identity, we next determined whether cyclin H had a role in either the initial establishment or expansion of pluripotent cells of the inner cell mass. Gene expression analysis showed that cyclin H transcript levels increase in four-cell embryos and then remain high through the blastocyst stage relative to one-cell embryos (Fig. 8A). To deplete cyclin H in early embryogenesis, we infected one-cell embryos with lentiviruses expressing cyclin H shRNA 2, also used in our previous studies. To monitor infection, the puromycin cassette of the lentivirus was replaced with enhanced GFP. The embryos were cultured in individual microdrops to the blastocyst stage. GFP expression was first apparent in morulae, ∼72 h after embryo transduction, and QRT-PCR showed that cyclin H mRNA was significantly reduced at this time point (Fig. 8B; *, p < 0.05). Cyclin H protein was also reduced by the blastocyst stage, along with a further decrease in mRNA (**, p < 0.01).

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