Cell Cycle and Developmental Regulations of Replication Factors in Mouse Embryonic Stem Cells*

Embryonic stem (ES) cells can grow rapidly and permanently while maintaining their differentiation capacity. To gain insight into how the cell cycle progression of undifferentiated murine ES cells is regulated, we have examined the expression patterns of various replication and cell cycle regulators. Most factors including cyclins, Cdc6, and geminin are rather constitutively expressed during the cell cycle of ES cells. Furthermore, the transcript levels of almost all the cell cycle regulators we investigated except for p21 and p27 are higher in undifferentiated ES cells than in murine embryonic fibroblasts (MEFs), and the increased stability of mRNA in ES cells may be partially responsible for this at least with some of the factors. More strikingly, the transcriptional levels of these factors are strongly correlated with the acetylated state of histone H3 at their promoter regions. However, the methylation state of histone or CpG methylation of the promoter region is not generally correlated significantly with the expression pattern of these factors in both cell types. On the protein level, Cdc6, ASK, cyclin A2, and cyclin B1 are extremely abundant in ES cells compared with MEFs. Furthermore, they are rapidly down-regulated upon induction of differentiation of ES cells. The significance of these findings is discussed in relation to the unusual proliferative properties of ES cells in an undifferentiated state.

[1]  C. Sherr.  G1 phase progression: Cycling on cue , 1994, Cell.

[2]  K. Arai,et al.  A 63-Base Pair DNA Segment Containing an Sp1 Site but Not a Canonical E2F Site Can Confer Growth-dependent and E2F-mediated Transcriptional Stimulation of the Human ASKGene Encoding the Regulatory Subunit for Human Cdc7-related Kinase* , 2002, The Journal of Biological Chemistry.

[3]  Kristian Helin,et al.  Cell Cycle-Regulated Expression of MammalianCDC6 Is Dependent on E2F , 1998, Molecular and Cellular Biology.

[4]  J. A. Castillejo,et al.  5' CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia. , 2002, Blood.

[5]  Tony Kouzarides,et al.  Retinoblastoma protein recruits histone deacetylase to repress transcription , 1998, Nature.

[6]  J. Mattick.  RNA regulation: a new genetics? , 2004, Nature Reviews Genetics.

[7]  G. Pfeifer,et al.  In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression , 1995, Molecular and cellular biology.

[8]  E. Lees,et al.  A Novel Growth- and Cell Cycle-Regulated Protein, ASK, Activates Human Cdc7-Related Kinase and Is Essential for G1/S Transition in Mammalian Cells , 1999, Molecular and Cellular Biology.

[9]  L. Stivala,et al.  Proliferating cell nuclear antigen bound to DNA synthesis sites: phosphorylation and association with cyclin D1 and cyclin A. , 1994, Experimental cell research.

[10]  A. Gartel,et al.  Activation and repression of p21WAF1/CIP1 transcription by RB binding proteins , 1998, Oncogene.

[11]  R. Sclafani.  Cdc7p-Dbf4p becomes famous in the cell cycle. , 2000, Journal of cell science.

[12]  Jonathan R Pollack,et al.  A transcriptional response to Wnt protein in human embryonic carcinoma cells , 2002, BMC Developmental Biology.

[13]  E. Li.  Chromatin modification and epigenetic reprogramming in mammalian development , 2002, Nature Reviews Genetics.

[14]  Satoshi Tanaka,et al.  Epigenetic Control of Mouse Oct-4 Gene Expression in Embryonic Stem Cells and Trophoblast Stem Cells* , 2004, Journal of Biological Chemistry.

[15]  A. Hatzigeorgiou,et al.  Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter , 1996, Molecular and cellular biology.

[16]  D. Skalnik,et al.  Histone deacetylase activity is required for embryonic stem cell differentiation , 2004, Genesis.

[17]  A. Sachs,et al.  Messenger RNA degradation in eukaryotes , 1993, Cell.

[18]  H. Masai,et al.  Genetic Dissection of Mammalian Cdc7 Kinase: Cell Cycle and Developmental Roles , 2004, Cell cycle.

[19]  M. Inagaki,et al.  Cell Cycle- and Chromatin Binding State-dependent Phosphorylation of Human MCM Heterohexameric Complexes , 1998, The Journal of Biological Chemistry.

[20]  Myriam Gorospe,et al.  Loss of HuR Is Linked to Reduced Expression of Proliferative Genes during Replicative Senescence , 2001, Molecular and Cellular Biology.

[21]  R. Weinberg,et al.  Regulation of cyclin E transcription by E2Fs and retinoblastoma protein. , 1996, Oncogene.

[22]  Charles J. Sherr,et al.  Mammalian G1 cyclins , 1993, Cell.

[23]  J. Nevins,et al.  Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  H. Nishitani,et al.  Control of DNA replication licensing in a cell cycle , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[25]  Kiyoshi Ohtani,et al.  Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F , 1999, Oncogene.

[26]  S. Cawley,et al.  Unbiased Mapping of Transcription Factor Binding Sites along Human Chromosomes 21 and 22 Points to Widespread Regulation of Noncoding RNAs , 2004, Cell.

[27]  C. Allis,et al.  Translating the Histone Code , 2001, Science.

[28]  A. Harel-Bellan,et al.  A Suv39h‐dependent mechanism for silencing S‐phase genes in differentiating but not in cycling cells , 2004, The EMBO journal.

[29]  D. O’Carroll,et al.  Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression , 2002, The EMBO journal.

[30]  Stephen Dalton,et al.  Cdk6–cyclin D3 activity in murine ES cells is resistant to inhibition by p16INK4a , 2004, Oncogene.

[31]  J. Peters,et al.  Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1. , 2000, Genes & development.

[32]  Y. Tomita,et al.  Methylation of promoter region in p27 gene plays a role in the development of lymphoid malignancies. , 2003, International journal of oncology.

[33]  Y. Bergman,et al.  A Unique Developmental Pattern of Oct-3/4DNA Methylation Is Controlled by a cis-demodification Element* , 2002, The Journal of Biological Chemistry.

[34]  J. Ross,et al.  mRNA stability in mammalian cells. , 1995, Microbiological reviews.

[35]  Myriam Gorospe,et al.  HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation , 2000, The EMBO journal.

[36]  J. Celis,et al.  S‐phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis , 1985, FEBS letters.

[37]  M. Inagaki,et al.  Cell Cycle Regulation of Human CDC6 Protein , 1999, The Journal of Biological Chemistry.

[38]  D. Marshak,et al.  Stem Cell Biology , 2001 .

[39]  Duncan Walker,et al.  Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities , 2002, Oncogene.

[40]  A. Schulze,et al.  Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Nevins,et al.  Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes , 1995, Molecular and cellular biology.

[42]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.

[43]  Paul Tempst,et al.  MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex , 1999, Nature Genetics.

[44]  J. A. Steitz,et al.  HuR and mRNA stability , 2001, Cellular and Molecular Life Sciences CMLS.

[45]  Jeffrey M. Trimarchi,et al.  Transcription: Sibling rivalry in the E2F family , 2002, Nature Reviews Molecular Cell Biology.

[46]  M. Fujita,et al.  Cdk2-dependent and -independent Pathways in E2F-mediated S Phase Induction* , 2000, The Journal of Biological Chemistry.

[47]  Myriam Gorospe,et al.  HuR Regulates p21 mRNA Stabilization by UV Light , 2000, Molecular and Cellular Biology.

[48]  H. Schöler,et al.  Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation , 1998, Mechanisms of Development.

[49]  Kiyoshi Ohtani,et al.  Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle transcription factor E2F , 1998, Oncogene.

[50]  John K. Heath,et al.  Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides , 1988, Nature.

[51]  M. Fujita,et al.  In Vivo Interaction of Human MCM Heterohexameric Complexes with Chromatin , 1997, The Journal of Biological Chemistry.

[52]  Donald Metcalf,et al.  Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells , 1988, Nature.

[53]  L. Szekely,et al.  Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts. , 1994, Oncogene.

[54]  P. Fernandez,et al.  Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. , 2001, Genes & development.

[55]  C. Plass,et al.  Methylation of Adjacent CpG Sites Affects Sp1/Sp3 Binding and Activity in the p21Cip1 Promoter , 2003, Molecular and Cellular Biology.

[56]  J. Strouboulis,et al.  Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription , 1998, Nature Genetics.

[57]  K. Arai,et al.  Cdc7 kinase complex: A key regulator in the initiation of DNA replication , 2002, Journal of cellular physiology.

[58]  D. Dean,et al.  Rb Interacts with Histone Deacetylase to Repress Transcription , 1998, Cell.

[59]  W. Dean,et al.  Epigenetic reprogramming during early development in mammals. , 2004, Reproduction.

[60]  S. Mittnacht,et al.  Control of pRB phosphorylation. , 1998, Current opinion in genetics & development.

[61]  Bruce Stillman,et al.  Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis , 2000, Molecular and Cellular Biology.

[62]  A. Bird,et al.  Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals , 2003, Nature Genetics.

[63]  Peter L. Jones,et al.  DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters , 2000, Nature Genetics.

[64]  Marielle Afanassieff,et al.  Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells , 2002, Oncogene.

[65]  S. Lowe,et al.  Rb-Mediated Heterochromatin Formation and Silencing of E2F Target Genes during Cellular Senescence , 2003, Cell.

[66]  J. Nevins,et al.  Regulation of the cyclin E gene by transcription factor E2F1. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[67]  Antonio Iavarone,et al.  Regulation of the cdk inhibitor p21 gene during cell cycle progression is under the control of the transcription factor E2F , 1998, Oncogene.

[68]  J. Blow,et al.  Replication licensing — Origin licensing: defining the proliferative state? , 2002 .

[69]  P. Greengard,et al.  Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor , 2004, Nature Medicine.

[70]  H. Masai,et al.  Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development. , 2003, Mutation research.