Advertisement

Cyclin-dependent Kinase 2-associating Protein 1 Commits Murine Embryonic Stem Cell Differentiation through Retinoblastoma Protein Regulation*

  • Yong Kim
    Correspondence
    To whom correspondence may be addressed: UCLA School of Dentistry and Dental Research Institute, 10833 Le Conte Ave., 73-017 CHS, Los Angeles, CA 90095. Tel.: 310-825-7210; Fax: 310-825-7609;
    Affiliations
    School of Dentistry and Dental Research Institute

    Jonsson Comprehensive Cancer Center
    Search for articles by this author
  • Amit Deshpande
    Affiliations
    School of Dentistry and Dental Research Institute
    Search for articles by this author
  • Yanshan Dai
    Affiliations
    School of Dentistry and Dental Research Institute
    Search for articles by this author
  • Jeffrey J. Kim
    Affiliations
    School of Dentistry and Dental Research Institute
    Search for articles by this author
  • Anne Lindgren
    Affiliations
    Department of Molecular Cell and Developmental Biology, College of Letters and Science, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California 90095
    Search for articles by this author
  • Anne Conway
    Affiliations
    Department of Molecular Cell and Developmental Biology, College of Letters and Science, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California 90095
    Search for articles by this author
  • Amander T. Clark
    Affiliations
    Department of Molecular Cell and Developmental Biology, College of Letters and Science, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California 90095
    Search for articles by this author
  • David T. Wong
    Correspondence
    To whom correspondence may be addressed: UCLA School of Dentistry and Dental Research Institute, 10833 Le Conte Ave., 73-017 CHS, Los Angeles, CA 90095. Tel.: 310-206-3048; Fax: 310-825-0921;
    Affiliations
    School of Dentistry and Dental Research Institute

    Jonsson Comprehensive Cancer Center

    Molecular Biology Institute

    Division of Head and Neck Surgery/Otolaryngology
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Public Service Grants T32 DE 007296-08 (to Y. K.) and R01 DE 14857 (to D. T. W.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7.
Open AccessPublished:June 29, 2009DOI:https://doi.org/10.1074/jbc.M109.026088
      Mouse embryonic stem cells (mESCs) maintain pluripotency and indefinite self-renewal through yet to be defined molecular mechanisms. Leukemia inhibitory factor has been utilized to maintain the symmetrical self-renewal and pluripotency of mESCs in culture. It has been suggested that molecules with significant cellular effects on retinoblastoma protein (pRb) or its related pathways should have functional impact on mESC proliferation and differentiation. However, the involvement of pRb in pluripotent differentiation of mESCs has not been extensively elaborated. In this paper, we present novel experimental data indicating that Cdk2ap1 (cyclin-dependent kinase 2-associating protein 1), an inhibitor of G1/S transition through down-regulation of CDK2 and an essential gene for early embryonic development, confers competency for mESC differentiation. Targeted disruption of Cdk2ap1 in mESCs resulted in abrogation of leukemia inhibitory factor withdrawal-induced differentiation, along with altered pRb phosphorylation. The differentiation competency of the Cdk2ap1−/− mESCs was restored upon the ectopic expression of Cdk2ap1 or a nonphosphorylatable pRb mutant (mouse Ser788 → Ala), suggesting that the CDK2AP1-mediated differentiation of mESCs was elicited through the regulation of pRb. Further analysis on mESC maintenance or differentiation-related gene expression supports the phosphorylation at serine 788 in pRb plays a significant role for the CDK2AP1-mediated differentiation of mESCs. These data clearly demonstrate that CDK2AP1 is a competency factor in the proper differentiation of mESCs by modulating the phosphorylation level of pRb. This sheds light on the role of the establishment of the proper somatic cell type cell cycle regulation for mESCs to enter into the differentiation process.
      Maintenance of pluripotency is essential to guarantee proper differentiation of cells and embryo development. ES
      The abbreviations used are: ES
      embryonic stem
      ESC
      embryonic stem cell
      LIF
      leukemia inhibitory factor
      mESC
      mouse embryonic stem cell
      LIF-R
      LIF receptor
      pRb
      retinoblastoma protein
      AP
      alkaline phosphatase
      GFP
      green fluorescent protein
      WT
      wild type
      HA
      hemagglutinin
      tTA
      tetracycline-controlled transactivator.
      3The abbreviations used are: ES
      embryonic stem
      ESC
      embryonic stem cell
      LIF
      leukemia inhibitory factor
      mESC
      mouse embryonic stem cell
      LIF-R
      LIF receptor
      pRb
      retinoblastoma protein
      AP
      alkaline phosphatase
      GFP
      green fluorescent protein
      WT
      wild type
      HA
      hemagglutinin
      tTA
      tetracycline-controlled transactivator.
      cells can be maintained pluripotent in culture, and leukemia inhibitory factor (LIF) has been utilized to maintain the symmetrical self-renewal of mESCs (
      • Smith A.G.
      • Heath J.K.
      • Donaldson D.D.
      • Wong G.G.
      • Moreau J.
      • Stahl M.
      • Rogers D.
      ,
      • Williams R.L.
      • Hilton D.J.
      • Pease S.
      • Willson T.A.
      • Stewart C.L.
      • Gearing D.P.
      • Wagner E.F.
      • Metcalf D.
      • Nicola N.A.
      • Gough N.M.
      ). Binding of LIF to its receptor, LIF-R, causes heterodimerization of LIF-R and gp130 and triggers downstream activation of Jak (
      • Ernst M.
      • Oates A.
      • Dunn A.R.
      ). Several lines of evidence show that Stat3 (signal transducer and activator of transcription 3) is the key downstream mediator of the LIF/gp130 signaling pathway, leading to the maintenance of self-renewal and pluripotency (
      • Niwa H.
      • Burdon T.
      • Chambers I.
      • Smith A.
      ,
      • Ernst M.
      • Novak U.
      • Nicholson S.E.
      • Layton J.E.
      • Dunn A.R.
      ,
      • Raz R.
      • Lee C.K.
      • Cannizzaro L.A.
      • d'Eustachio P.
      • Levy D.E.
      ). However, other lines of evidence show that LIF/gp130/Stat3 are not essential for pluripotency, and the existence of unidentified novel pathways that maintain pluripotency has been suggested (
      • Mitsui K.
      • Tokuzawa Y.
      • Itoh H.
      • Segawa K.
      • Murakami M.
      • Takahashi K.
      • Maruyama M.
      • Maeda M.
      • Yamanaka S.
      ). In undifferentiated embryonic stem cells, pRb is known to remain as an inactive form, and CDK2 and E2F1 remain constitutively active, allowing rapid self-regeneration of cells. When the cells differentiate, the cell cycle regulatory machinery becomes active and initiates pRb-dependent cell cycle control (
      • Savatier P.
      • Huang S.
      • Szekely L.
      • Wiman K.G.
      • Samarut J.
      ,
      • White J.
      • Stead E.
      • Faast R.
      • Conn S.
      • Cartwright P.
      • Dalton S.
      ).
      p12CDK2AP1 (CDK2AP1) is a highly conserved, ubiquitously expressed gene that is down-regulated in ∼70% of oral cancers (
      • Shintani S.
      • Mihara M.
      • Terakado N.
      • Nakahara Y.
      • Matsumura T.
      • Kohno Y.
      • Ohyama H.
      • McBride J.
      • Kent R.
      • Todd R.
      • Tsuji T.
      • Wong D.T.
      ,
      • Tsuji T.
      • Duh F.M.
      • Latif F.
      • Popescu N.C.
      • Zimonjic D.B.
      • McBride J.
      • Matsuo K.
      • Ohyama H.
      • Todd R.
      • Nagata E.
      • Terakado N.
      • Sasaki A.
      • Matsumura T.
      • Lerman M.I.
      • Wong D.T.
      ). Murine Cdk2ap1 with only three amino acid deviations from the human CDK2AP1 is located at chromosome 5 (
      • Kim Y.
      • Tsuji T.
      • Elovic A.
      • Shintani S.
      • Mihara M.
      • Salih E.
      • Kohno Y.
      • Chin B.R.
      • Patel V.
      • Wong D.T.W.
      • Todd R.
      ,
      • Kim Y.
      • McBride J.
      • Zhang R.
      • Zhou X.
      • Wong D.T.
      ). CDK2AP1 has been shown to be an S-phase regulator through two important cellular partners: CDK2 and DNA polymerase-α/primase (
      • Matsuo K.
      • Shintani S.
      • Tsuji T.
      • Nagata E.
      • Lerman M.
      • McBride J.
      • Nakahara Y.
      • Ohyama H.
      • Todd R.
      • Wong D.T.
      ,
      • Shintani S.
      • Ohyama H.
      • Zhang X.
      • McBride J.
      • Matsuo K.
      • Tsuji T.
      • Hu M.G.
      • Hu G.
      • Kohno Y.
      • Lerman M.
      • Todd R.
      • Wong D.T.
      ). Murine embryonic stem cells with disrupted expression of Cdk2ap1 showed an increased proliferation and an altered cell cycle profile with a reduced G2/M phase along with an increased CDK2 activity (
      • Kim Y.
      • McBride J.
      • Zhang R.
      • Zhou X.
      • Wong D.T.
      ). Recently, Cdk2ap1 has been identified as one of the stem cell-specific genes that are enriched in both embryonic and adult stem cells (
      • Ramalho-Santos M.
      • Yoon S.
      • Matsuzaki Y.
      • Mulligan R.C.
      • Melton D.A.
      ). Cdk2ap1 has been categorized as one of genes that are expressed in early stage preimplantation embryos (
      • Sharov A.A.
      • Piao Y.
      • Matoba R.
      • Dudekula D.B.
      • Qian Y.
      • VanBuren V.
      • Falco G.
      • Martin P.R.
      • Stagg C.A.
      • Bassey U.C.
      • Wang Y.
      • Carter M.G.
      • Hamatani T.
      • Aiba K.
      • Akutsu H.
      • Sharova L.
      • Tanaka T.S.
      • Kimber W.L.
      • Yoshikawa T.
      • Jaradat S.A.
      • Pantano S.
      • Nagaraja R.
      • Boheler K.R.
      • Taub D.
      • Hodes R.J.
      • Longo D.L.
      • Schlessinger D.
      • Keller J.
      • Klotz E.
      • Kelsoe G.
      • Umezawa A.
      • Vescovi A.L.
      • Rossant J.
      • Kunath T.
      • Hogan B.L.
      • Curci A.
      • D'Urso M.
      • Kelso J.
      • Hide W.
      • Ko M.S.
      ). In addition, Cdk2ap1 mRNA has been found to be elevated upon estrogen treatment during early implantation process, suggesting its role in uterine decidualization (
      • Lee S.
      • Lee S.A.
      • Shim C.
      • Khang I.
      • Lee K.A.
      • Park Y.M.
      • Kang B.M.
      • Kim K.
      ).
      In this paper, we analyzed phenotypic characteristics of Cdk2ap1/ mESCs. Upon withdrawal of LIF, Cdk2ap1/ mESCs showed significantly abrogated differentiation phenotype and hyperphosphorylation of pRb. The differentiation competency of the Cdk2ap1/ mESCs was restored upon the ectopic expression of Cdk2ap1 or a pRb phosphorylation mutant, S788A (equivalent to human pRb S795A (
      • Connell-Crowley L.
      • Harper J.W.
      • Goodrich D.W.
      ,
      • Adams P.D.
      • Li X.
      • Sellers W.R.
      • Baker K.B.
      • Leng X.
      • Harper J.W.
      • Taya Y.
      • Kaelin Jr., W.G.
      )). It links pRb phosphorylation to the regulation of differentiation competency in mESCs. Further analysis on mESC maintenance or differentiation-related gene expression suggests that the phosphorylation at serine 788 in pRb plays a significant role for the CDK2AP1-mediated differentiation of mESCs. Taken together, we conclude that CDK2AP1 is a competency factor in mESC differentiation through the regulation of pRb phosphorylation. Our current data support the unique role of CDK2AP1 in the proper regulation of cellular differentiation of mESCs during early embryonic development.

      Discussion

      Embryonic stem (ES) cells undergo unique self-renewal process and maintain pluripotent competency for specification into different cell lineages (
      • O'Shea K.S.
      ,
      • Chambers I.
      ). Recent findings suggest potential molecular mechanisms for how ES cells maintain pluripotency and control the self-renewal process, but their details are still largely unknown (
      • Raz R.
      • Lee C.K.
      • Cannizzaro L.A.
      • d'Eustachio P.
      • Levy D.E.
      ,
      • Mitsui K.
      • Tokuzawa Y.
      • Itoh H.
      • Segawa K.
      • Murakami M.
      • Takahashi K.
      • Maruyama M.
      • Maeda M.
      • Yamanaka S.
      ,
      • Chambers I.
      ,
      • Hanna L.A.
      • Foreman R.K.
      • Tarasenko I.A.
      • Kessler D.S.
      • Labosky P.A.
      ,
      • Avilion A.A.
      • Nicolis S.K.
      • Pevny L.H.
      • Perez L.
      • Vivian N.
      • Lovell-Badge R.
      ,
      • Sato N.
      • Meijer L.
      • Skaltsounis L.
      • Greengard P.
      • Brivanlou A.H.
      ). One of the prominent features of cell cycle regulation in ESCs is the lack of the pRb pathway. Embryonic stem cells have a short G1 phase in which hypophosphorylated pRb is virtually undetectable (
      • Savatier P.
      • Huang S.
      • Szekely L.
      • Wiman K.G.
      • Samarut J.
      ). It is likely that Rb is rephosphorylated immediately after mitosis in ES cells. ESCs express Rb and p107, but not p130 (
      • Robanus-Maandag E.
      • Dekker M.
      • van der Valk M.
      • Carrozza M.L.
      • Jeanny J.C.
      • Dannenberg J.H.
      • Berns A.
      • te Riele H.
      ,
      • LeCouter J.E.
      • Whyte P.F.
      • Rudnicki M.A.
      ). ESCs fail to arrest in G1 after DNA damage but arrest at the Rb-independent G2/M checkpoint (
      • Aladjem M.I.
      • Spike B.T.
      • Rodewald L.W.
      • Hope T.J.
      • Klemm M.
      • Jaenisch R.
      • Wahl G.M.
      ,
      • Prost S.
      • Bellamy C.O.
      • Clarke A.R.
      • Wyllie A.H.
      • Harrison D.J.
      ). Evidence suggests that ESCs are not controlled by pRb in G1 (
      • Dannenberg J.H.
      • van Rossum A.
      • Schuijff L.
      • te Riele H.
      ,
      • Sage J.
      • Mulligan G.J.
      • Attardi L.D.
      • Miller A.
      • Chen S.
      • Williams B.
      • Theodorou E.
      • Jacks T.
      ). The cellular mechanism underlying functional inactivation of pRb is being elucidated. ES cells show a low level of cyclin D, and Cdk4-associated kinase activity is virtually undetectable. In contrast, ESCs show constitutive cyclin E/CDK2 kinase activity. During differentiation, there is a robust expression of D-type cyclins and Cdk4-associated kinase activity along with a reduction of cyclin E/CDK2 kinase activity, resulting in G1/S control by pRb pathway (
      • Stead E.
      • White J.
      • Faast R.
      • Conn S.
      • Goldstone S.
      • Rathjen J.
      • Dhingra U.
      • Rathjen P.
      • Walker D.
      • Dalton S.
      ,
      • Savatier P.
      • Lapillonne H.
      • van Grunsven L.A.
      • Rudkin B.B.
      • Samarut J.
      ). From these findings, it is evident that molecules with significant cellular effects on pRb or its related pathways should have functional impact on mESC proliferation and differentiation. However, the involvement of pRb in pluripotent differentiation of mESCs has not been extensively elucidated.
      The biological role of the specific phosphorylation site for pRb regulation still remains elusive. Especially, its contribution to the differentiation of mESCs is largely unknown. The differential expression of stem cell maintenance or differentiation-related genes mediated by the preferential phosphorylation of pRb at serine 788 implicates that the knock-out of Cdk2ap1 in mESCs leads to the collective alteration of differentiation potential, leading to the observed differentiation blockade in the Cdk2ap1/ mESCs (Fig. 5). The most significant and interesting molecular changes observed upon the inducible expression of pRb S788A in Cdk2ap1/ mESCs were the down-regulation of Oct3/4 and up-regulation of Hand1 (Fig. 5). The regulation of these two genes showed consistent changes before and after the restoration of CDK2AP1 and the pRb S788A mutant. Although we convincingly show that CDK2AP1 plays a role in the regulation of CDK2 activity and further in pRb phosphorylation, it must be one of many mechanisms regulating pRb phosphorylation. It is also speculated that the activation of CDK2 itself in mESCs may have different cellular effects from expressing S788A, one of its downstream targets. The restoration of Cdk2ap1 in Cdk2ap1/ mESCs should be more specific to the pathway involving CDK2AP1-mediated pRb phosphorylation. Since CDK2 itself has many downstream targets and also Ser788 is a target of multiple effectors, it is expected that the manipulation of these two will have somewhat different downstream molecular effects. One way to appreciate their concurrent effects would be by identifying any overlapping events elicited by the two.
      Although ectopic expression of Cdk2ap1 did not elicit spontaneous differentiation in WT and Cdk2ap1/ mESCs in the presence of LIF, it induced differentiation in Cdk2ap1/ mESCs only in the absence of LIF. This demonstrates that CDK2AP1 is a downstream regulator of LIF-dependent self-renewal/differentiation of mESCs. In contrast, the ectopic expression of pRb S788A mutant resulted in the spontaneous differentiation of Cdk2ap1/, but not WT mESCs, regardless of LIF treatment. This suggests that the deletion of Cdk2ap1 in mESCs drives the cells toward an LIF-independent cascade. We speculate that expression of the pRb S788A mutant in WT mESC has no phenotypic effect, because the cells maintain normal LIF-dependent signaling in the presence of WT Cdk2ap1. Deletion of Cdk2ap1 results in the mESCs becoming capable of self-renewing even in the absence of LIF signaling, with the phosphorylation of pRb at Ser788 as one of the downstream events. This is in addition to other possible molecular alterations in the absence of Cdk2ap1 that could mediate the function of Cdk2ap1 in LIF-dependent mESC self-renewal/differentiation, such as the epigenetic control of Oct3/4 promoter, as demonstrated by our group (
      • Deshpande A.M.
      • Dai Y.S.
      • Kim Y.
      • Kim J.
      • Kimlin L.
      • Gao K.
      • Wong D.T.
      ).
      Further study is required to unveil how Cdk2ap1 responds to a differentiation signal and transmits it further downstream. It is possible that Cdk2ap1 itself is under the regulation of mESC differentiation through either transcriptional or post-translational regulation, such as molecular dimerization that has been shown to influence the activity of CDK2AP1 (
      • Kim Y.
      • Ohyama H.
      • Patel V.
      • Figueiredo M.
      • Wong D.T.
      ). Also the signaling and molecular mechanism needs to be delineated to understand how pRb mediates or triggers differentiation of mESCs through modulation of downstream genes. It is speculated that the activation of pRb should elicit its effect on gene regulation through either the regulation of a certain transcription factor or via epigenetic regulation, such as DNA methylation and histone acetylation. Our preliminary data suggest that the hyperphosphorylation of pRb in Cdk2ap1/ mESCs is accompanied by the activation of E2F1 promoter (data not shown). Further detailed examination of E2F1 activation should reveal the consequence and significance of this event in stem cell maintenance and differentiation. As an extension of this study, it will be meritorious and highly translational to examine if CDK2AP1 functions as a competency factor in human embryonic stem cell differentiation.

      Acknowledgments

      We thank Dr. Brenda Gallie (University of Toronto) and Dr. David Goodrich (Rosewell Park Cancer Institute) for kindly providing murine pRb plasmids. We thank Dr. J. Wade Harper (Harvard Medical School) and Dr. Philip Hinds (Tufts University) for human pRb WT and mutant plasmids. We also thank Dr. Philip Hinds and Dr. Cun-Yu Wang (UCLA) for valuable comments and suggestions regarding our work.

      References

        • Smith A.G.
        • Heath J.K.
        • Donaldson D.D.
        • Wong G.G.
        • Moreau J.
        • Stahl M.
        • Rogers D.
        Nature. 1988; 336: 688-690
        • Williams R.L.
        • Hilton D.J.
        • Pease S.
        • Willson T.A.
        • Stewart C.L.
        • Gearing D.P.
        • Wagner E.F.
        • Metcalf D.
        • Nicola N.A.
        • Gough N.M.
        Nature. 1988; 336: 684-687
        • Ernst M.
        • Oates A.
        • Dunn A.R.
        J. Biol. Chem. 1996; 271: 30136-30143
        • Niwa H.
        • Burdon T.
        • Chambers I.
        • Smith A.
        Genes Dev. 1998; 12: 2048-2060
        • Ernst M.
        • Novak U.
        • Nicholson S.E.
        • Layton J.E.
        • Dunn A.R.
        J. Biol. Chem. 1999; 274: 9729-9737
        • Raz R.
        • Lee C.K.
        • Cannizzaro L.A.
        • d'Eustachio P.
        • Levy D.E.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 2846-2851
        • Mitsui K.
        • Tokuzawa Y.
        • Itoh H.
        • Segawa K.
        • Murakami M.
        • Takahashi K.
        • Maruyama M.
        • Maeda M.
        • Yamanaka S.
        Cell. 2003; 113: 631-642
        • Savatier P.
        • Huang S.
        • Szekely L.
        • Wiman K.G.
        • Samarut J.
        Oncogene. 1994; 9: 809-818
        • White J.
        • Stead E.
        • Faast R.
        • Conn S.
        • Cartwright P.
        • Dalton S.
        Mol. Biol. Cell. 2005; 16: 2018-2027
        • Shintani S.
        • Mihara M.
        • Terakado N.
        • Nakahara Y.
        • Matsumura T.
        • Kohno Y.
        • Ohyama H.
        • McBride J.
        • Kent R.
        • Todd R.
        • Tsuji T.
        • Wong D.T.
        Clin. Cancer Res. 2001; 7: 2776-2782
        • Tsuji T.
        • Duh F.M.
        • Latif F.
        • Popescu N.C.
        • Zimonjic D.B.
        • McBride J.
        • Matsuo K.
        • Ohyama H.
        • Todd R.
        • Nagata E.
        • Terakado N.
        • Sasaki A.
        • Matsumura T.
        • Lerman M.I.
        • Wong D.T.
        J. Biol. Chem. 1998; 273: 6704-6709
        • Kim Y.
        • Tsuji T.
        • Elovic A.
        • Shintani S.
        • Mihara M.
        • Salih E.
        • Kohno Y.
        • Chin B.R.
        • Patel V.
        • Wong D.T.W.
        • Todd R.
        Int. J. Oral Biol. 2001; (December 2001): 87-91
        • Kim Y.
        • McBride J.
        • Zhang R.
        • Zhou X.
        • Wong D.T.
        Oncogene. 2005; 24: 407-418
        • Matsuo K.
        • Shintani S.
        • Tsuji T.
        • Nagata E.
        • Lerman M.
        • McBride J.
        • Nakahara Y.
        • Ohyama H.
        • Todd R.
        • Wong D.T.
        FASEB J. 2000; 14: 1318-1324
        • Shintani S.
        • Ohyama H.
        • Zhang X.
        • McBride J.
        • Matsuo K.
        • Tsuji T.
        • Hu M.G.
        • Hu G.
        • Kohno Y.
        • Lerman M.
        • Todd R.
        • Wong D.T.
        Mol. Cell. Biol. 2000; 20: 6300-6307
        • Ramalho-Santos M.
        • Yoon S.
        • Matsuzaki Y.
        • Mulligan R.C.
        • Melton D.A.
        Science. 2002; 298: 597-600
        • Sharov A.A.
        • Piao Y.
        • Matoba R.
        • Dudekula D.B.
        • Qian Y.
        • VanBuren V.
        • Falco G.
        • Martin P.R.
        • Stagg C.A.
        • Bassey U.C.
        • Wang Y.
        • Carter M.G.
        • Hamatani T.
        • Aiba K.
        • Akutsu H.
        • Sharova L.
        • Tanaka T.S.
        • Kimber W.L.
        • Yoshikawa T.
        • Jaradat S.A.
        • Pantano S.
        • Nagaraja R.
        • Boheler K.R.
        • Taub D.
        • Hodes R.J.
        • Longo D.L.
        • Schlessinger D.
        • Keller J.
        • Klotz E.
        • Kelsoe G.
        • Umezawa A.
        • Vescovi A.L.
        • Rossant J.
        • Kunath T.
        • Hogan B.L.
        • Curci A.
        • D'Urso M.
        • Kelso J.
        • Hide W.
        • Ko M.S.
        PLoS Biol. 2003; 1: E74
        • Lee S.
        • Lee S.A.
        • Shim C.
        • Khang I.
        • Lee K.A.
        • Park Y.M.
        • Kang B.M.
        • Kim K.
        Mol. Reprod. Dev. 2003; 64: 405-413
        • Connell-Crowley L.
        • Harper J.W.
        • Goodrich D.W.
        Mol. Biol. Cell. 1997; 8: 287-301
        • Adams P.D.
        • Li X.
        • Sellers W.R.
        • Baker K.B.
        • Leng X.
        • Harper J.W.
        • Taya Y.
        • Kaelin Jr., W.G.
        Mol. Cell. Biol. 1999; 19: 1068-1080
        • Keller G.M.
        Curr. Opin. Cell Biol. 1995; 7: 862-869
        • Kim Y.
        • McBride J.
        • Kimlin L.
        • Pae E.K.
        • Deshpande A.
        • Wong D.T.
        PLoS ONE. 2009; 4: e4518
        • Hu M.G.
        • Hu G.F.
        • Kim Y.
        • Tsuji T.
        • McBride J.
        • Hinds P.
        • Wong D.T.
        Cancer Res. 2004; 64: 490-499
        • Deshpande A.M.
        • Dai Y.S.
        • Kim Y.
        • Kim J.
        • Kimlin L.
        • Gao K.
        • Wong D.T.
        J. Biol. Chem. 2009; 284: 6043-6047
        • Benevolenskaya E.V.
        • Murray H.L.
        • Branton P.
        • Young R.A.
        • Kaelin Jr., W.G.
        Mol. Cell. 2005; 18: 623-635
        • Sidle A.
        • Palaty C.
        • Dirks P.
        • Wiggan O.
        • Kiess M.
        • Gill R.M.
        • Wong A.K.
        • Hamel P.A.
        Crit. Rev. Biochem. Mol. Biol. 1996; 31: 237-271
        • Slack R.S.
        • El-Bizri H.
        • Wong J.
        • Belliveau D.J.
        • Miller F.D.
        J. Cell Biol. 1998; 140: 1497-1509
        • Stead E.
        • White J.
        • Faast R.
        • Conn S.
        • Goldstone S.
        • Rathjen J.
        • Dhingra U.
        • Rathjen P.
        • Walker D.
        • Dalton S.
        Oncogene. 2002; 21: 8320-8333
        • McLear J.A.
        • Garcia-Fresco G.
        • Bhat M.A.
        • Van Dyke T.A.
        Mol. Cell Neurosci. 2006; 33: 260-273
        • Jori F.P.
        • Galderisi U.
        • Napolitano M.A.
        • Cipollaro M.
        • Cascino A.
        • Giordano A.
        • Melone M.A.
        Mol. Cell Neurosci. 2007; 34: 299-309
        • Kotake Y.
        • Cao R.
        • Viatour P.
        • Sage J.
        • Zhang Y.
        • Xiong Y.
        Genes Dev. 2007; 21: 49-54
        • O'Shea K.S.
        Anat. Rec. 1999; 257: 32-41
        • Chambers I.
        Cloning Stem Cells. 2004; 6: 386-391
        • Hanna L.A.
        • Foreman R.K.
        • Tarasenko I.A.
        • Kessler D.S.
        • Labosky P.A.
        Genes Dev. 2002; 16: 2650-2661
        • Avilion A.A.
        • Nicolis S.K.
        • Pevny L.H.
        • Perez L.
        • Vivian N.
        • Lovell-Badge R.
        Genes Dev. 2003; 17: 126-140
        • Sato N.
        • Meijer L.
        • Skaltsounis L.
        • Greengard P.
        • Brivanlou A.H.
        Nat. Med. 2004; 10: 55-63
        • Robanus-Maandag E.
        • Dekker M.
        • van der Valk M.
        • Carrozza M.L.
        • Jeanny J.C.
        • Dannenberg J.H.
        • Berns A.
        • te Riele H.
        Genes Dev. 1998; 12: 1599-1609
        • LeCouter J.E.
        • Whyte P.F.
        • Rudnicki M.A.
        Oncogene. 1996; 12: 1433-1440
        • Aladjem M.I.
        • Spike B.T.
        • Rodewald L.W.
        • Hope T.J.
        • Klemm M.
        • Jaenisch R.
        • Wahl G.M.
        Curr. Biol. 1998; 8: 145-155
        • Prost S.
        • Bellamy C.O.
        • Clarke A.R.
        • Wyllie A.H.
        • Harrison D.J.
        FEBS Lett. 1998; 425: 499-504
        • Dannenberg J.H.
        • van Rossum A.
        • Schuijff L.
        • te Riele H.
        Genes Dev. 2000; 14: 3051-3064
        • Sage J.
        • Mulligan G.J.
        • Attardi L.D.
        • Miller A.
        • Chen S.
        • Williams B.
        • Theodorou E.
        • Jacks T.
        Genes Dev. 2000; 14: 3037-3050
        • Savatier P.
        • Lapillonne H.
        • van Grunsven L.A.
        • Rudkin B.B.
        • Samarut J.
        Oncogene. 1996; 12: 309-322
        • Kim Y.
        • Ohyama H.
        • Patel V.
        • Figueiredo M.
        • Wong D.T.
        J. Biol. Chem. 2005; 280: 23273-23279