Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

Inhibition of proliferation of primary avian fibroblasts through expression of histone H5 depends on the degree of phosphorylation of the protein.

To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.

Myb-Ets fusion oncoprotein inhibits thyroid hormone receptor/c-ErbA and retinoic acid receptor functions: a novel mechanism of action for leukemogenic transformation by E26 avian retrovirus.

The E26 and avian erythroblastosis virus (AEV) avian retroviruses induce acute leukemia in chickens. E26 can block both erythroid and myeloid differentiation at an early multipotent stage. Moreover, E26 can block erythroid differentiation at the erythroid burst-forming unit/erythroid CFU (BFU-E/CFU-E) stage, which also corresponds to the differentiation stage blocked by AEV. AEV carries two oncogenes, v-erbA and v-erbB, whereas E26 encodes a single 135-kDa Gag-Myb-Ets fusion oncoprotein. v-ErbA is responsible for the erythroid differentiation arrest through negative interferences with both the retinoic acid receptor (RAR) and the thyroid hormone receptor (T3R/c-ErbA). We investigated whether Myb-Ets could block erythroid differentiation in a manner similar to v-ErbA. We show here that Myb-Ets inhibits both RAR and c-ErbA activities on specific hormone response elements in transient-expression assays. Moreover, Myb-Ets abrogates the inactivation of transcription factor AP-1 by RAR and T3R, another feature shared with v-ErbA. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to retinoic acid and T3. Analysis of a series of mutants of Myb-Ets reveals that the domains of the oncoprotein involved in these inhibitory activities are the same as those involved in oncogenic transformation of hematopoietic cells. These data demonstrate that the Myb-Ets oncoprotein shares properties with the v-ErbA oncoprotein and that inhibition of ligand-dependent RAR and c-ErbA functions by Myb-Ets is responsible for blocking the differentiation of hematopoietic progenitors.

The chicken c-erbA proto-oncogene is preferentially expressed in erythrocytic cells during late stages of differentiation.

We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.

Functional interference between thyroid hormone receptor alpha (TRalpha) and natural truncated TRDeltaalpha isoforms in the control of intestine development.

Thyroid hormone is known to participate in the control of intestine maturation at weaning. Its action is mediated by the thyroid hormone nuclear receptors, encoded by the TRalpha and TRbeta genes. Since previous studies have shown that TRbeta plays a minor role in the gut, we focused here our analysis on the TRalpha gene. The TRalpha locus generates the TRalpha1 receptor together with the splicing variant TRalpha2 and the truncated products TRDeltaalpha1 and TRDeltaalpha2, which all lack an intact ligand binding domain. The TRDeltaalpha isoforms are transcribed from an internal promoter located in intron 7, and their distribution is restricted to a few tissues including those of the intestine. In order to define the functions of the different isoforms encoded by the TRalpha locus in the intestinal mucosa, we produced mice either lacking all known TRalpha products or harboring a mutation which inactivates the intronic promoter. We performed a detailed analysis of the intestinal phenotypes in these mice and compared it to that of the previously described TRalpha(-/-) mice, in which TRalpha isoforms are abolished but the TRDeltaalpha isoforms remain. This comparative analysis leads us to the following conclusions: (i) the TRalpha1 receptor mediates the T3-dependent functions in the intestine at weaning time and (ii) the TRDeltaalpha products negatively control the responsiveness of the epithelial cells to T3. Moreover, we show that TRDeltaalpha proteins can interfere with the transcription of the intestine-specific homeobox genes cdx1 and cdx2 and that their activity is regulated by TRalpha1. Altogether these data demonstrate that cooperation of TRalpha and TRDeltaalpha products is essential to ensure the normal postnatal development of the intestine and that mutations in the TRalpha locus can generate different phenotypes caused by the disruption of the equilibrium between these products.

Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus.

Thyroid hormone receptors are encoded by the TRalpha (NR1A1) and TRbeta (NR1A2) loci. These genes are transcribed into multiple variants whose functions are unclear. Analysis by gene inactivation in mice has provided new insights into the functional complexity of these products. Different strategies designed to modify the TRalpha locus have led to strikingly different phenotypes. In order to analyze the molecular basis for these alterations, we generated mice devoid of all known isoforms produced from the TRalpha locus (TRalpha(0/0)). These mice are viable and exhibit reduced linear growth, bone maturation delay, moderate hypothermia, and reduced thickness of the intestinal mucosa. Compounding TRalpha(0) and TRbeta(-) mutations produces viable TRalpha(0/0)beta(-/-) mice, which display a more severe linear growth reduction and a more profound hypothermia as well as impaired hearing. A striking phenotypic difference is observed between TRalpha(0/0) and the previously described TRalpha(-/-) mice, which retain truncated TRDeltaalpha isoforms arising from a newly described promoter in intron 7. The lethality and severe impairment of the intestinal maturation in TRalpha(-/-) mice are rescued in TRalpha(0/0) animals. We demonstrate that the TRDeltaalpha protein isoforms, which are natural products of the TRalpha locus, are the key determinants of these phenotypical differences. These data reveal the functional importance of the non-T3-binding variants encoded by the TRalpha locus in vertebrate postnatal development and homeostasis.

Thyroid hormone regulates heparan sulfate proteoglycan expression in the growth plate.

Thyroid hormone is essential for normal skeletal development. Hypothyroidism is associated with growth arrest, failure of chondrocyte differentiation, and abnormal matrix synthesis. Thyroid hormone modulates the Indian hedgehog/PTHrP feedback loop and regulates fibroblast growth factor (FGF)/FGF receptor signaling. Because heparan sulfate (HS) proteoglycans (Prgs) (HSPGs) are absolutely required by these signaling pathways, we have investigated whether thyroid status affects HSPG expression within the growth plate. Tibial growth plate sections were obtained from 12-wk-old rats rendered euthyroid, thyrotoxic, or hypothyroid at 6 wk of age, 14-d-old congenitally hypothyroid Pax8-null mice, and TRalpha/TRbeta double-null mice lacking all thyroid hormone receptors. HS and chondroitin sulfate Prg expression was determined by immunohistochemistry using three monoclonal antibodies. There was increased HS staining in growth plates from hypothyroid animals predominantly within the extracellular matrix of reserve and proliferative zones. Cellular HS staining was also increased particularly in prehypertrophic chondrocytes. T3 regulation of HSPG core protein and HS synthetic and modification enzyme expression was studied in ATDC5 cells using semiquantitative RT-PCR. Thyroid hormone negatively regulated expression of the core protein Gpc6, the polymerase Ext1, and the modification enzyme Hs6st2. These studies demonstrate that the expression and distribution of growth plate Prgs are regulated by thyroid hormone, and the regulation of HSPG expression provides an important additional link between FGF and Indian hedgehog signaling and T3. These novel observations suggest that the cartilage matrix and especially HSPGs are critical mediators of the skeletal response to thyroid hormone.

Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor.

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Role of the different RAR isoforms in controlling the erythrocytic differentiation sequence. Interference with the v-erbA and p135gag-myb-ets nuclear oncogenes.

Little is known as to how the nuclear oncogenes v-erbA and p135gag-myb-ets do transform cells. The elucidation of their molecular mechanisms of action requires the identification of relevant target genes. We analysed the possibility for the RARbeta gene to represent such a target gene. We first show that the RARbeta gene induction is a specific and direct process, requiring the continuous presence of retinoids and under the control of the RARalpha isoform exclusively. We then show that the expression of either the v-erbA or the p135gag-myb-ets oncogene is not sufficient to block the RARbeta gene induction. We confirmed the loss of RARbeta gene response in certain cell lines but we discarded the possibility that this loss might represent a necessary step for cell lines immortalization. We further show that the RARalpha isoform activation is necessary and sufficient to induce the growth inhibition and the differentiation stimulation characteristic for the commitment-inducing ability of retinoids in chicken erythrocytic progenitor cells. We therefore propose a model showing that RARalpha but not RARbeta is the key mediator for commitment to differentiation and that it should control two different set of genes whose expression is differentially affected by the v-erbA and the p135gag-myb-ets oncogenes.

c-ErbA, but not v-ErbA, competes with a putative erythroid repressor for binding to the carbonic anhydrase II promoter.

The carbonic anhydrase II (CAII) gene is the only known gene identified as direct target for v-ErbA-mediated repression in avian erythroleukemic cells transformed by Avian Erythroblastosis Virus (AEV). This gene is transcriptionally activated by thyroid hormone (T3) in normal erythrocytic cells. In this work we have analysed the molecular basis of the transcriptional control of the CAII gene by c-ErbA and v-ErbA. We show that several domains in the promoter control hormonal regulation of transcription. One domain proximal to the TATA box mediates T3 response but contains no identified binding site for c-ErbA. An other domain termed PAL2 is approximately 600 bp upstream the transcription initiation site and contains a c-ErbA binding site. We show that when it is associated to a heterologous promoter this site mediates transcriptional repression in erythrocytic cells but not in HeLa cells. Moreover, this site binds a nuclear erythrocyte-specific factor that we called NFX, which is different from c-ErbA. heterodimers between c-ErbA and the 9-cis retinoic acid receptor (RXR) compete with NFX for binding to PAL2. In contrast, v-ErbA alone or in association with RXR is a very poor competitor and is unable to chase NFX out of the PAL2 site. We propose that NFX is a transcription repressor whose activity is inhibited by c-ErbA but not v-ErbA. This mechanism might contribute to the overall regulation of the carbonic anhydrase II promoter. These data illustrate another possible mechanism through which v-ErbA might antagonize the function of c-ErbA in controlling gene expression.

EGF promotes in vivo tumorigenic growth of primary chicken embryo fibroblasts expressing v-myc and enhances in vitro transformation by the v-erbA oncogene.

We report that the activation of the endogenous chicken EGF receptor leads to the tumorigenic growth in vivo of early passage chicken embryo fibroblasts (CEFs) that express a nonsarcomagenic oncogene, v-myc. To provide a continuous paracrine source of this growth factor in vivo, we employed irradiated Rat-1 cells which had been stably transfected with a synthetic cDNA to human EGF. Expression of another non-sarcomagenic nuclear oncogene, v-erbA, prones the CEFs to in vitro transformation by EGF, but does not cause EGF dependent tumorigenicity in vivo. The short period of incubation in the in vivo assay employed by our study (10 days), together with the genetic stability of primary chicken embryo fibroblasts, make it very likely that the reported alterations in cellular behaviour are a direct and primary effect of the expression of the relevant oncogenes and their cooperation with the EGF induced response. Dose response and ligand binding assays suggest that the EGF response is transmitted via the chicken c-erbB molecule, which by virtue of its preference for TGF-alfa is distinct from the mammalian EGF receptors studied so far. The level of expression of the endogenous chicken EGF receptor is within the same range as that reported for primary human fibroblasts (5-7 x 10(3) per cell). The cooperative effect of v-myc with chicken c-erbB probably takes place at a post receptor level, as its expression did not affect the steady state level or affinity for ligand of the chicken EGF receptor.

Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.

The expression of the retinoblastoma susceptibility (RB-1) gene was investigated in highly proliferating mouse embryonic stem (ES) cells and in slowly proliferating mouse embryonic fibroblasts. The RB protein was expressed at the same level in these two cell types. Mainly hyperphosphorylated RB was detected in exponentially-growing ES cells. Embryonic fibroblasts and embryonic stem cells were synchronized by colcemid block followed by mitotic shake-off. In embryonic fibroblasts, DNA replication started 10-15 h after exit from mitosis and RB was transiently dephosphorylated during the G1 phase as previously described. In ES cells, DNA replication started 2 h after release from the colcemid block but virtually no hypophosphorylated RB was observed after the release. Instead, there was a dramatic decrease in the total RB protein level between exit from mitosis and entry into S phase. These observations were made by using two different monoclonal antibodies, both in immunoblotting and immunoprecipitation experiments. Absence of hypophosphorylated RB and cell cycle-dependent change in total RB protein level may be relevant to the high proliferation rate and to the tumorigenic nature of mouse embryonic stem cells.

A truncated RAR alpha co-operates with the v-erbB oncogene to transform early haematopoietic progenitors in vitro and in vivo.

We have shown recently that a retrovirus vector expressing a natural mutant form of the PML-RAR alpha protein characteristic of human acute promyelocytic leukaemia can transform early chicken hematopoietic progenitors (Altabef et al., 1996). Neither truncated PML nor truncated RAR alpha alone could induce transformation which suggest that the two domains should cooperate for the oncogenicity of the fusion product. To further investigate the mechanisms of this co-operation, we have tested whether a truncated RAR alpha could cooperate with the v-erbB oncogene. This oncogene has previously been shown to co-operate with the rearranged thyroid hormone receptor, v-erbA, to transform erythrocytic progenitors. We show that v-erbB and a truncated RAR alpha co-operate when expressed simultaneously as independent products to transform very early chicken haematopoietic cells close to pluripotent stage. In addition, we show that v-erbB alters transcriptional abilities of RAR alpha by both enhancing its effects on RARE and reducing those on AP-1. Therefore, RAR alpha is able to co-operate with different kinds of proteins to induce transformation of early haematopoietic cells. This strongly suggests that RAR alpha are involved in the differentiation commitment of early haematopoietic progenitors during the normal process of haematopoietic differentiation. These data bring new insights in the mechanisms of oncogenic transformation by rearranged RAR alpha.

The 12S adenoviral E1A protein immortalizes avian cells and interacts with the avian RB product.

Quail cells were immortalized for the first time by using retroviruses expressing the 12S adenoviral E1A gene. In these cells, interaction between the 12S E1A product and the quail RB protein was shown, suggesting that the 12S adenoviral E1A product works in avian cells through similar biochemical pathways as in mammalian cells by interacting and inactivating host cellular proteins, including the RB product. These results confirm that the RB product exhibits a universal function among higher vertebrates in controlling cellular growth and tumor progression.

Leukemogenicity of v-myb-transformed monoblasts cells can be modulated by normal bone marrow environment.

The avian myeloblastosis virus (AMV) causes monoblastic leukemia in the chick. Two non-producer clones of AMV-transformed monoblasts, BM2/C3A and BM2L/A2B5, have been described (see Bottazzi et al., this issue). They differ in their growth requirements and in their ability to induce leukemia when injected into the chick embryo. We first genetically tagged these clones by retroviral infection with a vector expressing the bacterial lacZ gene. Then, we injected the lacZ-positive cells via the chorioallantoic vein into chick embryos. With BM2L/A2B5 cells, the bone marrow of the injected birds was rapidly invaded by lacZ-positive cells. In addition, these cells rapidly overgrew cultures of bone marrow cells derived from injected animals. Conversely, the growth of BM2/C3A was inhibited in the injected animals and only a few blue cells, with the morphology of macrophages, were detected in cultures of bone marrow cells. We developed an in vitro assay to mimic in vitro the differential growth of BM2/C3A and BM2L/A2B5 observed in vivo. These data strongly suggest that BM2/C3A cells retain their ability to differentiate into macrophages in the normal bone marrow environment and that BM2L/A2B5 cells differ from BMC/C3A in the loss of this capacity.

Overexpression of avian or mouse c-jun in primary chick embryo fibroblasts confers a partially transformed phenotype.

The coding sequences of avian (quail) or murine c-jun proto-oncogenes were introduced into a non-defective retroviral vector derived from Rous sarcoma virus (RSV) in which c-jun replaces v-src. Primary avian fibroblasts chronically infected with either one of these viruses exhibit some phenotypic traits characteristic of RSV-transformed cells, including sustained growth in low serum medium and ability to develop colonies from single cells in agar, even though they are still of normal morphology and contact inhibited. This altered growth control correlates with enhanced AP1-specific DNA binding activity as well as with higher levels of c-Jun products. Unexpectedly, repression of the endogenous c-Jun product is observed in cells overexpressing murine c-Jun. Cells expressing the avian and the murine c-Jun products display qualitatively similar phenotypes; nevertheless, for every transformed trait considered, the murine c-jun seemed more potent than its quail homologue. These data suggest that the avian or murine c-jun proto-oncogenes may trigger a subset of the 'transforming functions' normally induced by v-src, and which are more specifically related to growth in low serum and in the absence of solid support.

The various domains of v-myb and v-ets oncogenes of E26 retrovirus contribute differently, but cooperatively, in transformation of hematopoietic lineages.

The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.

v-erbA oncogene abrogates growth inhibition of chicken embryo fibroblasts induced by retinoic acid.

Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.

Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.

The expression of E and D-type cyclins, Cyclin-Dependent Kinase (CDK) 2 and 4, as well as CDK inhibitors p21Cip1 and p27Kip1 were examined during in vitro differentiation of mouse embryonic stem (ES) cells. ES cells cultured in presence of Differentiation Inhibitory Activity/Leukemia Inhibitory Factor (DIA/LIF) express very low levels of cyclin E/CDK2 complexes, p21Cip1 and p27Kip1 CDK inhibitors, while cyclin D/CDK4-associated kinase activity is undetectable. Withdrawal of DIA/LIF, which induces differentiation, results in the progressive up-regulation of all. Up-regulation of D cyclins occurs through an increase in the steady-state levels of mRNA, concomitantly with the activation of Brachyury and Goosecoid, two early markers of mesoderm differentiation. Similarly, cells from the epiblast of the early postimplantation mouse embryo do not express any cyclin D/CDK4 complexes. These are progressively upregulated at gastrulation and early organogenesis. DIA/LIF-stimulated ES cells are not growth-arrested by overexpression of p16Ink4a, a specific inhibitor of CDK4 and CDK6. We propose that the G1/S transition may be regulated by a minimal mechanism in mouse embryonic stem cells. Induction of differentiation triggers the establishment of a more sophisticated mechanism involving both cyclin D/CDK4- and CDK inhibitor-associated control of G1-phase progression.