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• W1544366392 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. 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. Cyclin-dependent kinase 7 (Cdk7) 3The abbreviations used are: Cdkcyclin-dependent kinaseESembryonic stemCAKCdk-activating kinaseCTDcarboxyl-terminal domainICMinner cell massGFPgreen fluorescent proteinshRNAshort hairpin RNAQRTquantitative real timeBrdUrdbromodeoxyuridineGSTglutathione S-transferase. 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 (1Solomon M.J. Harper J.W. Shuttleworth J. EMBO J. 1993; 12: 3133-3142Crossref PubMed Scopus (273) Google Scholar, 2Poon R.Y. Yamashita K. Adamczewski J.P. Hunt T. Shuttleworth J. EMBO J. 1993; 12: 3123-3132Crossref PubMed Scopus (333) Google Scholar, 3Fesquet D. Labbé J.C. Derancourt J. Capony J.P. Galas S. Girard F. Lorca T. Shuttleworth J. Dorée M. Cavadore J.C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (327) Google Scholar). 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) (4Devault A. Martinez A.M. Fesquet D. Labbé J.C. Morin N. Tassan J.P. Nigg E.A. Cavadore J.C. Dorée M. EMBO J. 1995; 14: 5027-5036Crossref PubMed Scopus (201) Google Scholar, 5Mäkelä T.P. Tassan J.P. Nigg E.A. Frutiger S. Hughes G.J. Weinberg R.A. Nature. 1994; 371: 254-257Crossref PubMed Scopus (234) Google Scholar, 6Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (560) Google Scholar, 7Tassan J.P. Jaquenoud M. Fry A.M. Frutiger S. Hughes G.J. Nigg E.A. EMBO J. 1995; 14: 5608-5617Crossref PubMed Scopus (172) Google Scholar). Cdk7 is activated by cyclin H, whereas Mat1 modulates the substrate specificity of the complex (8Yankulov K.Y. Bentley D.L. EMBO J. 1997; 16: 1638-1646Crossref PubMed Scopus (153) Google Scholar). 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 (9Larochelle S. Merrick K.A. Terret M.E. Wohlbold L. Barboza N.M. Zhang C. Shokat K.M. Jallepalli P.V. Fisher R.P. Mol. Cell. 2007; 25: 839-850Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 10Larochelle S. Pandur J. Fisher R.P. Salz H.K. Suter B. Genes Dev. 1998; 12: 370-381Crossref PubMed Scopus (150) Google Scholar). However, cyclin H levels and Cdk7 kinase activity do not vary during the cell cycle (11Tassan J.P. Schultz S.J. Bartek J. Nigg E.A. J. Cell Biol. 1994; 127: 467-478Crossref PubMed Scopus (216) Google Scholar, 12Poon R.Y. Yamashita K. Howell M. Ershler M.A. Belyavsky A. Hunt T. J. Cell Sci. 1994; 107: 2789-2799PubMed Google Scholar, 13Brown A.J. Jones T. Shuttleworth J. Mol. Biol. Cell. 1994; 5: 921-932Crossref PubMed Scopus (53) Google Scholar). cyclin-dependent kinase embryonic stem Cdk-activating kinase carboxyl-terminal domain inner cell mass green fluorescent protein short hairpin RNA quantitative real time bromodeoxyuridine glutathione S-transferase. In addition to these key roles in cell cycle progression, the Cdk7·cyclin H·Mat1 complex is a component of the general transcription factor TFIIH (14Serizawa H. Mäkelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (309) Google Scholar, 15Roy R. Adamczewski J.P. Seroz T. Vermeulen W. Tassan J.P. Schaeffer L. Nigg E.A. Hoeijmakers J.H. Egly J.M. Cell. 1994; 79: 1093-1101Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 16Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 17Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (366) Google Scholar). The CAK complex associates with TFIIH through the Xpd subunit and phosphorylates serines 5 and 7 in the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (18Trigon S. Serizawa H. Conaway J.W. Conaway R.C. Jackson S.P. Morange M. J. Biol. Chem. 1998; 273: 6769-6775Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 6488-6493Crossref PubMed Scopus (140) Google Scholar, 20Akhtar M.S. Heidemann M. Tietjen J.R. Zhang D.W. Chapman R.D. Eick D. Ansari A.Z. Mol. Cell. 2009; 34: 387-393Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 21Glover-Cutter K. Larochelle S. Erickson B. Zhang C. Shokat K. Fisher R.P. Bentley D.L. Mol. Cell Biol. 2009; 20: 5455-5464Crossref Scopus (230) Google Scholar). Serine 5 CTD phosphorylation is detectable in RNA polymerase II complexes localized to the 5′ region of genes and is important for the co-transcriptional recruitment of chromatin-modifying factors and mRNA capping enzymes to the nascent transcript (22Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (302) Google Scholar, 23Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar, 24Viladevall L. St Amour C.V. Rosebrock A. Schneider S. Zhang C. Allen J.J. Shokat K.M. Schwer B. Leatherwood J.K. Fisher R.P. Mol. Cell. 2009; 33: 738-751Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 25Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar). Serine 7 phosphorylation was identified recently and is specifically involved in regulating small nuclear RNA expression (26Egloff S. O'Reilly D. Chapman R.D. Taylor A. Tanzhaus K. Pitts L. Eick D. Murphy S. Science. 2007; 318: 1777-1779Crossref PubMed Scopus (202) Google Scholar, 27Chapman R.D. Heidemann M. Albert T.K. Mailhammer R. Flatley A. Meisterernst M. Kremmer E. Eick D. Science. 2007; 318: 1780-1782Crossref PubMed Scopus (236) Google Scholar). Initial in vitro studies provided conflicting results on the extent to which TFIIH-associated Cdk7 kinase activity is necessary for mRNA transcription (28Akoulitchev S. Mäkelä T.P. Weinberg R.A. Reinberg D. Nature. 1995; 377: 557-560Crossref PubMed Scopus (160) Google Scholar, 29Serizawa H. Conaway J.W. Conaway R.C. Nature. 1993; 363: 371-374Crossref PubMed Scopus (139) Google Scholar). Recent reports from both yeast and human cells using an analog-sensitive kinase indicate that Cdk7 is only required for the expression of a subset of genes (21Glover-Cutter K. Larochelle S. Erickson B. Zhang C. Shokat K. Fisher R.P. Bentley D.L. Mol. Cell Biol. 2009; 20: 5455-5464Crossref Scopus (230) Google Scholar, 24Viladevall L. St Amour C.V. Rosebrock A. Schneider S. Zhang C. Allen J.J. Shokat K.M. Schwer B. Leatherwood J.K. Fisher R.P. Mol. Cell. 2009; 33: 738-751Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 30Kanin E.I. Kipp R.T. Kung C. Slattery M. Viale A. Hahn S. Shokat K.M. Ansari A.Z. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5812-5817Crossref PubMed Scopus (90) Google Scholar). The Cdk7 complex can also regulate gene expression by directly phosphorylating transcription factors, including the retinoic acid receptor and peroxisome proliferator-activated receptor γ, to either enhance or repress their activity (31Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 32Compe E. Drané P. Laurent C. Diderich K. Braun C. Hoeijmakers J.H. Egly J.M. Mol. Cell Biol. 2005; 25: 6065-6076Crossref PubMed Scopus (119) Google Scholar, 33Helenius K. Yang Y. Alasaari J. Mäkelä T.P. Mol. Cell Biol. 2009; 29: 315-323Crossref PubMed Scopus (33) Google Scholar). Thus, the repertoire of Cdk7-responsive genes and functional requirements for the Cdk7·cyclin H·Mat1 complex are likely to vary in a cell type-dependent fashion. Limited functional analysis of the subunits of the CAK complex has been performed in mammalian cells. Mat1 is the only component of the complex that has been mutated in mice. Mat1-deficient mice exhibit peri-implantation lethality, with homozygous mutant blastocysts failing to maintain/expand the inner cell mass (ICM) in culture (34Rossi D.J. Londesborough A. Korsisaari N. Pihlak A. Lehtonen E. Henkemeyer M. Mäkelä T.P. EMBO J. 2001; 20: 2844-2856Crossref PubMed Scopus (83) Google Scholar). Notably, cyclin H and Cdk7 protein levels were also reduced in Mat1 mutant blastocyst explants. Given this early embryonic lethality, conditional alleles have helped to reveal cell type-specific functions of Mat1. Postnatal deletion in the testis results in the loss of spermatagonial stem cells, whereas cardiac-specific Mat1 mutants develop heart failure secondary to mitochondrial dysfunction (35Sano M. Izumi Y. Helenius K. Asakura M. Rossi D.J. Xie M. Taffet G. Hu L. Pautler R.G. Wilson C.R. Boudina S. Abel E.D. Taegtmeyer H. Scaglia F. Graham B.H. Kralli A. Shimizu N. Tanaka H. Mäkelä T.P. Schneider M.D. Cell Metab. 2007; 5: 129-142Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 36Korsisaari N. Rossi D.J. Paetau A. Charnay P. Henkemeyer M. Mäkelä T.P. J. Cell Sci. 2002; 115: 4275-4284Crossref PubMed Scopus (21) Google Scholar). In contrast, loss of Mat1 in mouse embryonic fibroblasts promotes adipogenesis by decreasing inhibitory phosphorylation of proliferator-activated receptor γ (33Helenius K. Yang Y. Alasaari J. Mäkelä T.P. Mol. Cell Biol. 2009; 29: 315-323Crossref PubMed Scopus (33) Google Scholar). These varying phenotypes suggest that Mat1 is not globally required for cell survival or transcription, but rather that it can modulate select target genes in a cell type-specific manner. This is a particularly interesting possibility given the early embryonic lethality of Mat1-null embryos and the requirement for Mat1 in spermatagonial stem cell maintenance, which suggests that Mat1 modulates transcriptional programs that are required for stem cell maintenance. Mat1 was also identified in a yeast two-hybrid screen for proteins that interact with octamer family transcription factors, including the pluripotency factor Oct-4 (37Inamoto S. Segil N. Pan Z.Q. Kimura M. Roeder R.G. J. Biol. Chem. 1997; 272: 29852-29858Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts and represent a unique stem cell population that can self-renew indefinitely while retaining the plasticity to differentiate into all cell types of an adult organism. 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 (38Chambers I. Tomlinson S.R. Development. 2009; 136: 2311-2322Crossref PubMed Scopus (340) Google Scholar). Chromatin binding profiles suggest that these factors work in concert to regulate gene expression (39Loh Y.H. Wu Q. Chew J.L. Vega V.B. Zhang W. Chen X. Bourque G. George J. Leong B. Liu J. Wong K.Y. Sung K.W. Lee C.W. Zhao X.D. Chiu K.P. Lipovich L. Kuznetsov V.A. Robson P. Stanton L.W. Wei C.L. Ruan Y. Lim B. Ng H.H. Nat. Genet. 2006; 38: 431-440Crossref PubMed Scopus (1945) Google Scholar, 40Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3546) Google Scholar). However, genes that are more broadly expressed can also have unique functions in ES cells. For example, the polycomb group proteins interface with the pluripotency network to repress developmental genes in ES cells (41Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. Bell G.W. Otte A.P. Vidal M. Gifford D.K. Young R.A. Jaenisch R. Nature. 2006; 441: 349-353Crossref PubMed Scopus (2055) Google Scholar, 42Endoh M. Endo T.A. Endoh T. Fujimura Y. Ohara O. Toyoda T. Otte A.P. Okano M. Brockdorff N. Vidal M. Koseki H. Development. 2008; 135: 1513-1524Crossref PubMed Scopus (234) Google Scholar). Here we investigate the function of Mat1 and cyclin H in mouse ES cells to understand the mechanism for early embryonic lethality of Mat1-deficient embryos. Surprisingly, we observed that Mat1 down-regulation does not significantly impact ES cell viability or pluripotency. In contrast, depletion of cyclin H, which was also 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 Mat1 mutant embryos. Cyclin H depletion did not alter Cdk phosphorylation but lead to the up-regulation of differentiation-associated genes in ES cells. Of note, down-regulation of the negative elongation factor Spt5, which is phosphorylated by Cdk7 in vitro, also leads to differentiation of ES cells and up-regulation of many of the same developmental genes as regulated by cyclin H. Thus, Spt5 is likely to be an important target for cyclin H-mediated gene repression. The unique phenotypes of Mat1 and cyclin H down-regulation in ES cells underscores how these two subunits have distinct effects on Cdk7 activity and substrate specificity. This study provides, to our knowledge, the first loss-of-function analysis of cyclin H in a mammalian system. E14TG2a (ATCC) and R1 mouse ES cells were cultured on mitomycin C-treated mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium supplemented with 15% Hyclone serum, 2 mm l-glutamine, 0.1 mm nonessential amino acids, 100 units/ml penicillin/streptomycin, 55 μm β-mercaptoethanol, and leukemia inhibitory factor. For experiments, ES cells were passaged onto 0.1% gelatin-coated plates. Low passage (<10) gelatin-adapted ES cells were transduced twice with lentivirus at a multiplicity of infection of 10–20 in ES medium with polybrene (8 μg/ml) and selected with puromycin (1 μg/ml) 24 h after transduction. For proteosome inhibition experiments, the cells were treated with MG-132 (25 μm) or Me2SO for 4 h. Lentiviral stocks were generated by calcium phosphate-based transfection of 293T cells with pLKO vectors (Open Biosystems) and packaging plasmids (Addgene, VSV-G 8454, psPax2 12260). Viral supernatants were concentrated by ultracentrifugation at 25,000 rpm for 2 h, resuspended in Dulbecco's modified Eagle's medium or KSOM + AA (Millipore), aliquoted, and stored at −80 °C. Virus used for embryo transductions was purified a second time by ultracentrifugation over a 20% sucrose cushion. The puromycin resistance cassette of pLKO vectors was also replaced with enhanced GFP for mouse embryo experiments. A scramble shRNA expressing vector was obtained from Addgene (vector 1864). RNA was extracted from cells with TRIzol reagent (Invitrogen) and purified on RNeasy spin columns (Qiagen). The Picopure RNA isolation kit (Fisher) was used to isolate RNA from embryos and blastocyst explants. cDNA was generated by reverse transcription of equivalent quantities of RNA using Superscript II reverse transcriptase (Invitrogen) and random hexamers. QRT-PCR was performed on an Applied Biosystems 7900 HT using either SYBR green or TaqMan probes. β-Actin was used as an endogenous control for ES experiments, whereas Hist2h2aa1 (Mm00501974_s1) and Ubtf (Mm00456972_m1) were used for embryo analysis. A list of SYBR green primers designed for QRT-PCR can be provided upon request. Three biological replicates were averaged for all QRT-PCR experiments, with error bars representing the standard error of the mean. Microarrays were performed by the University of Pennsylvania Microarray Core facility using Mouse Gene ST 1.0 arrays (Affymetrix) with two biological replicates/sample. The data were normalized using the GC-RMA algorithm (Partek). Genes that changed greater than 1.3-fold with both cyclin H-targeting shRNAs and were significant (p < 0.05) with either cyclin H shRNA relative to a scramble control were selected for further analysis. A heat map was generated using the HeatMapImage module in Gene Pattern (43Reich M. Liefeld T. Gould J. Lerner J. Tamayo P. Mesirov J.P. Nat. Genet. 2006; 38: 500-501Crossref PubMed Scopus (1555) Google Scholar). The cell lysates were prepared in radioimmune precipitation assay buffer plus protease inhibitors (Roche Applied Science), sodium vanadate, sodium fluoride, and β-glycerophosphate. Nuclear/cytoplasmic fractionation was performed using the Active Motif kit as per the manufacturer's instructions. Antibodies against cyclin H (C-18), Cdk7 (C-4), Mat1 (F-6), and RNA polymerase II (N-20) were obtained from Santa Cruz Biotechnologies. Xpd, Hsp90, and total Cdk1 were from Abcam, whereas p-Cdk1 (Thr161), p-Cdk2 (Thr160), total Cdk2, cAMP-responsive element-binding protein, and β-tubulin were from Cell Signaling. The Ser2 (H5) and Ser5 (H14) phosphorylation-specific RNA polymerase antibodies were obtained from Covance. For immunoprecipitations, 50–100 μg of lysate was incubated with 1–2 μg of antibody overnight at 4 °C. Protein G-agarose was added for 1 h, immunoprecipitations were washed three times in radioimmune precipitation assay buffer, washed once in kinase buffer, and assayed for kinase activity toward recombinant GST-CTD or histone H1 as previously described (44Pines J. Jackman M. Simpson K. Current Protocols in Cell Biology. 2001; 8: 8.2.1-8.2.11Google Scholar, 45Larochelle S. Chen J. Knights R. Pandur J. Morcillo P. Erdjument-Bromage H. Tempst P. Suter B. Fisher R.P. EMBO J. 2001; 20: 3749-3759Crossref PubMed Scopus (104) Google Scholar). To label proliferating cells, BrdUrd (10 μm) was added to ES cultures for 10 min. The cells were fixed and permeabilized with ethanol, labeled with anti-BrdUrd (Alexa Fluor-488 conjugate; Invitrogen) antibody and propidium iodide, and analyzed by flow cytometry. Female CF-1 mice (4–6 weeks old; Charles River) were superovulated by intraperitoneal injection of pregnant mare serum gonadotropin (5 IU; Sigma) followed 48 h later by human chorionic gonadotropin (5 IU; Calbiochem). The mice were then naturally mated with B6D2F1/J (Jackson Labs) males. One-cell embryos were isolated at 0.5 days post coitus and cultured in microdrops of KSOM + AA in a humidified chamber under 5% O2, 6% CO2, 89% air. To infect one-cell embryos, the zona pellucida was removed by acid Tyrode's solution (Sigma-Aldrich), and individual embryos were placed in 10-μl drops of lentivirus diluted to 2 × 106 particles/ml in KSOM + AA medium as previously described (46Singer O. Tiscornia G. Ikawa M. Verma I.M. Nat. Protoc. 2006; 1: 286-292Crossref PubMed Scopus (29) Google Scholar). Blastocyst explant assays were performed on gelatin coated 96-well plates or 8-well chamber slides as previously described (47Dobles M. Liberal V. Scott M.L. Benezra R. Sorger P.K. Cell. 2000; 101: 635-645Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Given the early embryonic lethality of Mat1-null embryos, we chose to employ ES cells as a model to further elucidate the function of Mat1 in stem cells. To deplete Mat1, we transduced gelatin-adapted E14TG2a ES cells with lentiviruses expressing Mat1-specific shRNAs and selected for virus integration with puromycin. Two shRNAs targeting independent regions of Mat1 transcripts were utilized for all of the experiments. Both shRNAs significantly decreased Mat1 mRNA and protein (Fig. 1, B and C). Mat1 down-regulation did not affect the viability of ES cells or decrease staining for alkaline phosphatase activity, a marker of undifferentiated ES cells (Fig. 1D). Moreover, 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. Alternatively, because Rossi et al. (34Rossi D.J. Londesborough A. Korsisaari N. Pihlak A. Lehtonen E. Henkemeyer M. Mäkelä T.P. EMBO J. 2001; 20: 2844-2856Crossref PubMed Scopus (83) Google Scholar) had previously demonstrated that the levels of Cdk7 and cyclin H were significantly decreased in Mat1 mutant embryo explants, we considered the possibility that decreases in other components of the CAK complex may contribute to the phenotype of these embryos. Because Rossi et al. (34Rossi D.J. Londesborough A. Korsisaari N. Pihlak A. Lehtonen E. Henkemeyer M. Mäkelä T.P. EMBO J. 2001; 20: 2844-2856Crossref PubMed Scopus (83) Google Scholar) demonstrated that cyclin H appeared most dramatically reduced in Mat1-null embryo outgrowths, we first analyzed the functional consequences of cyclin H depletion from ES cells using lentiviral shRNAs. Using two independent cyclin H shRNAs, we significantly reduced the levels of cyclin H mRNA in ES cells (Fig. 2A). Furthermore, a time course of protein abundance shows that cyclin H was reduced at 2 days after transduction and significantly depleted by 3 days (Fig. 2B). The loss of cyclin H in ES cells lead to a dramatic change in cell morphology and a decrease in alkaline phosphatase activity, both consistent with differentiation (Fig. 2C). To more extensively characterize the differentiation phenotype, we performed QRT-PCR analysis for several pluripotency-associated genes and found that Zfp42 (Rex-1) and Tcl1 were considerably reduced, whereas Oct-4 was modestly decreased after 5 days of cyclin H depletion (Fig. 2D). Because Zfp42 rapidly and dramatically decreases upon the loss of pluripotency, these results further suggested that cyclin H down-regulation leads to ES cell differentiation. Moreover, endoderm (FoxA1 and Gata4), ectoderm (Fgf5), mesoderm (Msx1), and trophectoderm (Cdx2 and Hand1) markers were up-regulated in cyclin H shRNA-treated ES cells (Fig. 2E). This increase in multiple lineage-specific genes suggests that the cells were differentiating along several different pathways. 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 (48Aguilar-Fuentes J. Valadez-Graham V. Reynaud E. Zurita M. J. Cell Sci. 2006; 119: 3866-3875Crossref PubMed Scopus (13) Google Scholar). 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. Decreased levels of cyclin H and Cdk7 have been observed in both tissue-specific and global Mat1-deficient mice (34Rossi D.J. Londesborough A. Korsisaari N. Pihlak A. Lehtonen E. Henkemeyer M. Mäkelä T.P. EMBO J. 2001; 20: 2844-2856Crossref PubMed Scopus (83) Google Scholar, 35Sano M. Izumi Y. Helenius K. Asakura M. Rossi D.J. Xie M. Taffet G. Hu L. Pautler R.G. Wilson C.R. Boudina S. Abel E.D. Taegtmeyer H. Scaglia F. Graham B.H. Kralli A. Shimizu N. Tanaka H. Mäkelä T.P. Schneider M.D. Cell Metab. 2007; 5: 129-142Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 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 (49Stead E. White J. Faast R. Conn S. Goldstone S. Rathjen J. Dhingra U. Rathjen P. Walker D. Dalton S. Oncogene. 2002; 21: 8320-8333Crossref PubMed Scopus (305) Google Scholar). To analyze the cell cycle of ES ce" @default.
• W1544366392 created "2016-06-24" @default.
• W1544366392 creator A5057258274 @default.
• W1544366392 creator A5082058259 @default.
• W1544366392 date "2010-05-01" @default.
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• W1544366392 title "Functional Analysis of the Cdk7·Cyclin H·Mat1 Complex in Mouse Embryonic Stem Cells and Embryos" @default.
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