The histone- and PRMT5-associated protein COPR5 is required for myogenic differentiation.

Myogenic differentiation requires the coordination between permanent cell cycle withdrawal, mediated by members of the cyclin-dependent kinase inhibitor (CKI) family, and activation of a cascade of myogenic transcription factors, particularly MYOGENIN (MYOG). Recently, it has been reported that the Protein aRginine Methyl Transferase PRMT5 modulates the early phase of induction of MYOG expression. Here, we show that the histone- and PRMT5-associated protein COPR5 (cooperator of PRMT5) is required for myogenic differentiation. C2C12 cells, in which COPR5 had been silenced, could not irreversibly exit the cell cycle and differentiate into muscle cells. This phenotype might be explained by the finding that, in cells in which COPR5 was downregulated, p21 and MYOG induction was strongly reduced and PRMT5 recruitment to the promoters of these genes was also altered. Moreover, we suggest that COPR5 interaction with the Runt-related transcription factor 1 (RUNX1)-core binding factor-ß (CBFß) complex contributes to targeting the COPR5-PRMT5 complex to these promoters. Finally, we present evidence that COPR5 depletion delayed the in vivo regeneration of cardiotoxin-injured mouse skeletal muscles. Altogether, these data extend the role of COPR5 from an adaptor protein required for nuclear functions of PRMT5 to an essential coordinator of myogenic differentiation.

The LIM-only protein FHL2 is a negative regulator of E4F1.

The E1A-targeted transcription factor E4F1 is a key player in the control of mammalian embryonic and somatic cell proliferation and survival. Mouse embryos lacking E4F die at an early developmental stage, whereas enforced expression of E4F1 in various cell lines inhibits cell cycle progression. E4F1-antiproliferative effects have been shown to depend on its capacity to repress transcription and to interact with pRb and p53. Here we show that full-length E4F1 protein (p120(E4F1)) but not its E1A-activated and truncated form (p50(E4F1)), interacts directly in vitro and in vivo with the LIM-only protein FHL2, the product of the p53-responsive gene FHL2/DRAL (downregulated in rhabdomyosarcoma Lim protein). This E4F1-FHL2 association occurs in the nuclear compartment and inhibits the capacity of E4F1 to block cell proliferation. Consistent with this effect, ectopic expression of FHL2 inhibits E4F1 repressive effects on transcription and correlates with a reduction of nuclear E4F1-p53 complexes. Overall, these results suggest that FHL2/DRAL is an inhibitor of E4F1 activity. Finally, we show that endogenous E4F1-FHL2 complexes form in U2OS cells upon UV-light-induced nuclear accumulation of FHL2.

Three different calcium wave pacemakers in ascidian eggs.

Calcium wave pacemakers in fertilized eggs of ascidians and mouse are associated with accumulations of cortical endoplasmic reticulum in the vegetal hemisphere. In ascidians, two distinct pacemakers (PM1 and PM2) generate two series of calcium waves necessary to drive meiosis I and II. Pacemaker PM2 is stably localized in a cortical ER accumulation situated in the vegetal contraction pole. We now find that pacemaker PM1 is situated in a cortical ER-rich domain that forms around the sperm aster and moves with it during the calcium-dependant cortical contraction triggered by the fertilizing sperm. Global elevations of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels produced by caged Ins(1,4,5)P3 or caged glycero-myo-PtdIns(4,5)P2 photolysis reveal that the cortex of the animal hemisphere, also rich in ER-clusters, is the cellular region most sensitive to Ins(1,4,5)P3 and acts as a third type of pacemaker (PM3). Surprisingly, the artificial pacemaker PM3 predominates over the natural pacemaker PM2, located at the opposite pole. Microtubule depolymerization does not alter the activity nor the location of the three pacemakers. By contrast, blocking the acto-myosin driven cortical contraction with cytochalasin B prevents PM1 migration and inhibits PM2 activity. PM3, however, is insensitive to cytochalasin B. Our experiments suggest that the three distinct calcium wave pacemakers are probably regulated by different spatiotemporal variations in Ins(1,4,5)P3 concentration. In particular, the activity of the natural calcium wave pacemakers PM1 and PM2 depends on the apposition of a cortical ER-rich domain to a source of Ins(1,4,5)P3 production in the cortex. Movies available on-line

The periodic down regulation of Cyclin E gene expression from exit of mitosis to end of G(1) is controlled by a deacetylase- and E2F-associated bipartite repressor element.

The expression of cyclin E and that of a few other bona fide cell cycle regulatory genes periodically oscillates every cycle in proliferating cells. Although numerous experiments have documented the role of E2F sites and E2F activities in the control of these genes as cells exit from G(0) to move through the initial G(1)/S phase transition, almost nothing is known on the role of E2Fs during the subsequent cell cycles. Here we show that a variant E2F-site that is part of the Cyclin E Repressor Module (CERM) (Le Cam et al., 1999b) accounts for the periodic down regulation of the cyclin E promoter observed between the exit from mitosis until the mid/late G(1) phase in exponentially cycling cells. This cell cycle-dependent repression correlates with the periodic binding of an atypical G(1)-specific high molecular weight p107-E2F complex (Cyclin E Repressor Complex: CERC2) that differs in both size and DNA binding behaviors from known p107-E2F complexes. Notably, affinity purified CERC2 displays a TSA-sensitive histone deacetylase activity and, consistent with this, derepression of the cyclin E promoter by trichostatin A depends on the CERM element. Altogether, this shows that the cell cycle-dependent control of cyclin E promoter in cycling cells is embroiled in acetylation pathways via the CERM-like E2F element.

Cyclin A is a mediator of p120E4F-dependent cell cycle arrest in G1.

E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120E4F expression induces cell cycle arrest in G1. Although p21WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120E4F can induce a G1 block in p21(-/-) cells, suggesting that other proteins are essential for the p120E4F-dependent block in G1. We show here that cyclin A promoter activity can be repressed by p120E4F and that this repression correlates with p120E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120E4F-arrested cells from the G1 block. These data identify the cyclin A gene as a cellular target for p120E4F and suggest a mechanism for p120E4F-dependent cell cycle regulation.

pRB binds to and modulates the transrepressing activity of the E1A-regulated transcription factor p120E4F.

The retinoblastoma protein pRB is involved in the transcriptional control of genes essential for cell cycle progression and differentiation. pRB interacts with different transcription factors and thereby modulates their activity by sequestration, corepression, or activation. We report that pRB, but not p107 and p130, binds to and facilitates repression by p120(E4F), a ubiquitously expressed GLI-Kruppel-related protein identified as a cellular target of E1A. The interaction involves two distinct regions of p120(E4F) and the C-terminal part of pRB. In vivo pRB-p120(E4F) complexes can only be detected in growth-arrested cells, and accordingly contain the hypophosphorylated form of pRB. Repression of an E4F-responsive promoter is strongly increased by combined expression of p120(E4F) and pRB, which correlates with pRB-dependent enhancement of p120(E4F) binding activity. Elevated levels of p120(E4F) have been shown to block growth of mouse fibroblasts in G(1). We find this requires pRB, because RB(-/-) fibroblasts are significantly less sensitive to excess p120(E4F).

The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts.

During skeletal muscle differentiation, a subset of myoblasts remains quiescent and undifferentiated but retains the capacity to self-renew and give rise to differentiating myoblasts [1] [2] [3]: this sub-population of muscle cells was recently termed 'reserve cells' [3]. In order to characterise genes that can regulate the ratio between reserve cells and differentiating myoblasts, we examined members of the retinoblastoma tumor suppressor family - Rb, p107 and p130 - an important family of negative regulators of E2F transcription factors and cell cycle progression [4]. Although pRb and p107 positively regulate muscle cell differentiation [5] [6] [7], the role of p130 in muscle cells remains unknown. We show here that p130 (protein and mRNA), but neither pRb nor p107, preferentially accumulates during muscle differentiation in reserve cells. Also, p130 is the major Rb-family protein present in E2F complexes in this sub-population of cells. Although forced expression of either p130 or pRb in mouse C2 myoblasts efficiently blocked cell cycle progression, only p130 inhibited the differentiation program. Furthermore, muscle cells overexpressing p130 had reduced levels of the muscle-promoting factor MyoD. In addition, p130 repressed the transactivation capacity of MyoD, an effect abolished by co-transfection of pRb. Thus, we propose that p130, by blocking cell cycle progression and differentiation, could be part of a specific pathway that defines a pool of reserve cells during terminal differentiation.

A CDE/CHR-like element mediates repression of transcription of the mouse RB2 (p130) gene.

The bipartite repressor elements, termed cell cycle-dependent element (CDE)/cell cycle regulatory element (CCRE)-cell cycle homology region (CHR) control the growth-dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE-CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)-related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up-regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.

Human E2F5 gene is oncogenic in primary rodent cells and is amplified in human breast tumors.

E2F transcription factors (E2F1 to 6) are central players in the control of animal cell proliferation as regulators of genes involved in cell cycle progression and in transformation. In this report, we have investigated the potential involvement of the E2F5 gene in tumorigenesis. We show that E2F5 can promote the formation of morphologically transformed foci in primary baby rat kidney cells (BRK) when it is overexpressed in the presence of its heterodimeric partner DP1 and activated RAS. This suggests that E2F5 behaves like a MYC-type cooperating oncogene in functional assays, prompting us to monitor potential amplifications of the E2F5 gene in primary human tumors. We mapped the human E2F5 gene to 8q21.1-21.3 equidistant from the MOS (8q12) and MYC (8q24) oncogenes. Since the long arm of chromosome 8 is frequently the site of increased gene copy number (ICN) in breast cancer, we screened 442 breast tumor DNAs for gains of E2F5, MOS, and MYC genes. The three genes showed ICN, albeit at variable incidence and levels of amplification, with the ICN of E2F5 occurring concomitantly with those of MOS and/or MYC in almost half of the cases. Moreover, a marked increase of the 2. 5-kb E2F5 transcript was also detected in some tumors and tumor cell lines. In conclusion, the evidence that sustained unregulated expression of E2F5 can cooperate with other oncogenes to promote cell transformation in functional assays, together with the detection of chromosomal amplifications and overexpressions of the E2F5 gene in breast tumors, provides the first indications that E2F5 deregulation may have a role in human tumor development.

Erythroid-specific inhibition of the tal-1 intragenic promoter is due to binding of a repressor to a novel silencer.

The basic helix-loop-helix tal-1 gene plays a key role in hematopoiesis, and its expression is tightly controlled through alternative promoters and complex interactions of cis-acting regulatory elements. tal-1 is not expressed in normal T cells, but its transcription is constitutive in a large proportion of human T cell leukemias. We have previously described a downstream initiation of tal-1 transcription specifically associated with a subset of T cell leukemias that leads to the production of NH(2)-truncated TAL-1 proteins. In this study, we characterize the human promoter (promoter IV), embedded within a GC-rich region in exon IV, responsible for this transcriptional activity. The restriction of promoter IV usage is assured by a novel silencer element in the 3'-untranslated region of the human gene that represses its activity in erythroid but not in T cells. The silencer activity is mediated through binding of a tissue-specific nuclear factor to a novel protein recognition motif (designated tal-RE) in the silencer. Mutation of a single residue within the tal-RE abolishes both specific protein binding and silencing activity. Altogether, our results demonstrate that the tal-1 promoter IV is actively repressed in cells of the erythro-megakaryocytic lineage and that this repression is released in leukemic T cells, resulting in the expression of the tal-1 truncated transcript.

The Rac1- and RhoG-specific GEF domain of Trio targets filamin to remodel cytoskeletal actin.

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively. Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets. Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways.

Inhibition of mammalian cell proliferation by genetically selected peptide aptamers that functionally antagonize E2F activity.

The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This critical regulatory pathway controls the G1/S transition of the mammalian cell cycle by positive and negative regulation of E2F-responsive genes required for DNA replication. To assess the value of the transcription factors E2Fs as targets for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically these proteins in vitro and in vivo. The cellular activity of E2F is the result of the heterodimeric association of two families of proteins, E2Fs and DPs, which then bind DNA. Here, we use a two hybrid approach to isolate from combinatorial libraries peptide aptamers that specifically interact with E2Fs DNA binding and dimerization domains. One of these is a potent inhibitor of E2F binding activity in vitro and in mammalian fibroblasts, blocks cells in G1, and the free variable region from this aptamer has the same effect. Our experiments argue that the variable region of this aptamer is structured, and that it functions by binding E2F with a motif that resembles a DP heterodimerization region, and blocking E2F's association with DP. These results show that cell proliferation can be inhibited using genetically-selected synthetic peptides that specifically target protein-protein interaction motifs within cell cycle regulators. These results also emphasize the critical role of the E2F pathway for cell proliferation and might allow the design of novel antiproliferative agents targeting the cyclin/CDK-pRB-E2F pathway.

Phases of cytoplasmic and cortical reorganizations of the ascidian zygote between fertilization and first division.

Many eggs undergo reorganizations that localize determinants specifying the developmental axes and the differentiation of various cell types. In ascidians, fertilization triggers spectacular reorganizations that result in the formation and localization of distinct cytoplasmic domains that are inherited by early blastomeres that develop autonomously. By applying various imaging techniques to the transparent eggs of Phallusia mammillata, we now define 9 events and phases in the reorganization of the surface, cortex and the cytoplasm between fertilization and first cleavage. We show that two of the domains that preexist in the egg (the ER-rich cortical domain and the mitochondria-rich subcortical myoplasm) are localized successively by a microfilament-driven cortical contraction, a microtubule-driven migration and rotation of the sperm aster with respect to the cortex, and finally, a novel microfilament-dependant relaxation of the vegetal cortex. The phases of reorganization we have observed can best be explained in terms of cell cycle-regulated phases of coupling, uncoupling and recoupling of the motions of cortical and subcortical layers (ER-rich cortical domain and mitochondria-rich domain) with respect to the surface of the zygote. At the end of the meiotic cell cycle we can distinguish up to 5 cortical and cytoplasmic domains (including two novel ones; the vegetal body and a yolk-rich domain) layered against the vegetal cortex. We have also analyzed how the myoplasm is partitioned into distinct blastomeres at the 32-cell stage and the effects on development of the ablation of precisely located small fragments. On the basis of our observations and of the ablation/ transplantation experiments done in the zygotes of Phallusia and several other ascidians, we suggest that the determinants for unequal cleavage, gastrulation and for the differentiation of muscle and endoderm cells may reside in 4 distinct cortical and cytoplasmic domains localized in the egg between fertilization and cleavage.

Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.

Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus p130, p107 and pRb function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in correspondence with the premature expression of cyclin E. Thus, we identify a new regulatory module that controls repression of G1-specific genes in G0/G1.

[Role of the multifunctional Trio protein in the control of the Rac1 and RhoA gtpase signaling pathways]. / Rôle de la protéine multifonctionnelle Trio dans le contrôle des voies de signalisation des GTPases Rac1 et RhoA.

The small GTPases Cdc42, Rac and RhoA have important regulatory roles in mediating cytoskeletal rearrangements, MAP kinase cascades and induction of G1 cell cycle progression. The activity of the GTPases is regulated by guanine nucleotide exchange factors (GEFs) which accelerate their GDP/GTP exchange rate, and thereby activate them. All the GEFs for the Rho-GTPases family share two conserved domains: the DH domain (for Dbl-homology domain) responsible for the enzymatic activity, and the PH domain, probably responsible for the proper localization of the molecule. Trio is a multifunctional protein that is comprised of two functional Rho-GEFs domains and a serine/threonine kinase domain. We have shown in vitro and in vivo that the first GEF domain (GEFD1) activates Rac1, while the second GEF domain (GEFD2) acts on RhoA. Moreover, the co-expression of both domains induces simultaneously the activation of both GTPases. To our knowledge, this is the first example of a member of the Rho-GEF family, that contains two functional exchange factor domains, with restricted and different specificity. We are currently investigating how these GEF domains are activated, by addressing the role of the PH domains in GTPases activation by Trio. We have shown that: 1) the PH1 of Trio is necessary for Rac activation by the GEFD1; 2) the PH1 of Trio targets the molecule to the cytoskeleton; 3) the GEFD1 domain of Trio binds, in a two-hybrid screen, the actin binding protein filamin. These data suggest that the PH1 targets Trio to the cytoskeleton close to Rac and its effectors, probably via interaction with the actin-binding protein filamin, consistent with a role of Trio in actin cytoskeleton remodeling.

Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F.

The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.

Regulation of E2F-1 gene expression in avian cells.

E2F-1 is the prototype of a family of transcription factors playing a central role in the control of cell proliferation and apoptosis. E2F DNA binding activity is down-regulated during cellular differentiation, which is correlated with cell division arrest. We report here that the expression of E2F-1 itself is down-regulated in the developing quail neural retina between embryonic days E8-E10. This event occurs just after the massive arrest of the quail neuroretina cell division (E7-E8). To gain further insight into the regulatory mechanisms monitoring E2F-1 expression in differentiating neurons, we have cloned the quail E2F-1 promoter. In vivo DNA footprintings of this promoter have shown that a number of potential SP-1 and C/EBP response elements are constitutively occupied in the entire quail neuroretina of E5 and E14, whereas the two consensus palindromic E2F binding sites are only protected at E5. This suggests that these E2F elements participate in down-regulation of E2F-1 gene expression during avian neuroretina development. CAT reporter assays have shown that E2F-1 in association with its partner DP-1 transactivates its own promoter, whereas p105Rb inhibits the E2F-1 promoter. Both E2F-1/DP-1 and p105Rh require the presence of the E2F binding sites to mediate their effects. However, experiments performed with deletion mutants of the promoter strongly suggest that other regions located upstream of the E2F binding sites also mediate part of the E2F-1 transactivating effect on its own promoter. Altogether, these results suggest that the down-regulation of E2F-1 gene expression in differentiating neurons could be due, in part, to the E2F/Rb complexes binding to the E2F-1 promoter.

Activation of E2F-mediated transcription by human T-cell leukemia virus type I Tax protein in a p16(INK4A)-negative T-cell line.

The human T-cell leukemia virus type I (HTLV-I) is a causative agent of adult T-cell leukemia. Although the exact mechanism by which HTLV-I contributes to leukemogenesis is still unclear, the Tax protein is thought to play a major role in this process. This 40-kDa polypeptide is able to interact with the tumor suppressor p16(INK4A). Consequently, Tax can activate the signaling pathway that lead to the release of E2F that in turn induces expression of factors required for cell cycle progression. In this paper, we demonstrate that Tax can also activate E2F-mediated transcription independently of p16(INK4A). Indeed, when Tax is coexpressed with the E2F-1 transcription factor in CEM T-cells, which lack expression of p16(INK4A), it strongly potentiates the E2F-dependent activation of a reporter construct driven by a promoter containing E2F binding sites. This stimulation is abrogated by mutations affecting the E2F-binding sites. In addition, Tax also stimulates the transcription of the E2F-1 gene itself. Using Tax mutants that fail to activate either ATF- or NF-kappaB-dependent promoters and different 5' truncation mutants of the E2F-1 promoter, we show that the Tax-dependent transcriptional control of the E2F1 gene involves, at least in part, the ATF binding site located in the E2F-1 promoter.