Biomedical subjects

D Trouche

Publications and source records attributed to D Trouche.

At least 19 recordsLinked to original sources

PubMed1998

Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A.

Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator. These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase. Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes. Here we show that CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity. This enzymatic activation is mimicked by E1A.

3T3 Cells↗

PubMed1998

The three members of the pocket proteins family share the ability to repress E2F activity through recruitment of a histone deacetylase.

The transcription factor E2F plays a major role in cell cycle control in mammalian cells. E2F binding sites, which are present in the promoters of a variety of genes required for S phase, shift from a negative to a positive role in transcription at the commitment point, a crucial point in G1 that precedes the G1/S transition. Before the commitment point, E2F activity is repressed by members of the pocket proteins family. This repression is believed to be crucial for the proper control of cell growth. We have previously shown that Rb, the founding member of the pocket proteins family, represses E2F1 activity by recruiting the histone deacetylase HDAC1. Here, we show that the two other members of the pocket proteins family, p107 and p130, also are able to interact physically with HDAC1 in live cells. HDAC1 interacts with p107 and Rb through an "LXCXE"-like motif, similar to that used by viral transforming proteins to bind and inactivate pocket proteins. Indeed, we find that the viral transforming protein E1A competes with HDAC1 for p107 interaction. We also demonstrate that p107 is able to interact simultaneously with HDAC1 and E2F4, suggesting a model in which p107 recruits HDAC1 to repress E2F sites. Indeed, we demonstrate that histone deacetylase activity is involved in the p107- or p130-induced repression of E2F4. Taken together, our data suggest that all members of the E2F family are regulated in early G1 by similar complexes, containing a pocket protein and the histone deacetylase HDAC1.

Carrier Proteins↗

PubMed1998

A rapid and sensitive assay for histone acetyl-transferase activity.

Histone acetyl-transferases (HATs) seem to be key elements in the regulation of transcription. We have designed an enzymatic assay to quantify HAT enzymatic activity. In this assay, the substrate is a peptide corresponding to the 24 first amino acids of histone H4 which is coupled to biotin. After acetylation using [14C]acetyl-CoA, the peptide is purified on streptavidin beads and the associated radioactivity is measured. This assay is sensitive, rapid and convenient.

3T3 Cells↗

PubMed1998

Retinoblastoma protein represses transcription by recruiting a histone deacetylase.

The retinoblastoma tumour-suppressor protein Rb inhibits cell proliferation by repressing a subset of genes that are controlled by the E2F family of transcription factors and which are involved in progression from the G1 to the S phase of the cell cycle. Rb, which is recruited to target promoters by E2F1, represses transcription by masking the E2F1 transactivation domain and by inhibiting surrounding enhancer elements, an active repression that could be crucial for the proper control of progression through the cell cycle. Some transcriptional regulators act by acetylating or deacetylating the tails protruding from the core histones, thereby modulating the local structure of chromatin: for example, some transcriptional repressors function through the recruitment of histone deacetylases. We show here that the histone deacetylase HDAC1 physically interacts and cooperates with Rb. In HDAC1, the sequence involved is an LXCXE motif, similar to that used by viral transforming proteins to contact Rb. Our results strongly suggest that the Rb/HDAC1 complex is a key element in the control of cell proliferation and differentiation and that it is a likely target for transforming viruses.

Amino Acid Sequence↗

PubMed1998

[Histone deacetylase and retinoblastoma protein].

The balance between cellular proliferation and differentiation is strictly controlled in the cell and the deregulation of this balance can lead to tumour formation. The tumour suppressor protein Rb plays a key role in this balance essentially by repressing progression through the cell cycle and thereby it blocks the cell in G1 phase. Rb represses S phase genes through the recruitment of an enzyme which modifies DNA structure, the histone deacetylase HDAC1. The Rb/HDAC1 complex is a key element in the control of cell proliferation and differentiation. Moreover, this complex is likely to be a target for transforming viral proteins.

Adenovirus E1A Proteins↗

PubMed1997

The CREB-binding protein (CBP) cooperates with the serum response factor for transactivation of the c-fos serum response element.

The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62(TCF), and the CArG box, which binds p67(SRF). These two proteins are direct targets for signal transduction pathways; p62(TCF) is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67(SRF) is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62(TCF) activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67(SRF). Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67(SRF) in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67(SRF) could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67(SRF) and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67(SRF) and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67(SRF), and p62(TCF), with multiple interactions between the components of the complex.

3T3 Cells↗

PubMed1997

RB and hbrm cooperate to repress the activation functions of E2F1.

Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture. A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical interaction between RB and hbrm/BRG-1. We show here that in transfected cells RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found within the same complex in vivo. Furthermore, RB and hbrm cooperate to repress E2F1 activity in transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site. However, the bromodomain seems dispensable for this activity. Taken together, our results point out an unexpected role of corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G1 arrest. Finally, we demonstrate that the domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity.

Amino Acid Sequence↗

PubMed1997

[Control of cell proliferation by the retinoblastoma gene product].

Transformed cells proliferate abnormally, due to the unregulated activation of oncogenes and the inactivation of anti-oncogenes. The molecular mechanisms by which the product of the RB anti-oncogene, the RB protein, regulates cell proliferation begin to be understood. Major targets of RB include proteins involved in cell cycle entry, like the E2F transcription factor, and effectors of terminal differentiation. The effect of RB is thus to block cells into the G1 phase of the cell cycle and to induce them to terminally differentiate. Recently, a new role has been shown for RB. RB is able to repress the activity of the RNA polymerases I and III, thereby modulating the protein biosynthesis capacities of the cell. RB appears thus to control directly the balance between DNA and protein synthesis during the cell cycle.

Carrier Proteins↗

PubMed1996

The CBP co-activator stimulates E2F1/DP1 activity.

The cell cycle-regulating transcription factors E2F1/DP1 activate genes whose products are required for S phase progression. During most of the G1 phase, E2F1/DP1 activity is repressed by the retinoblastoma gene product RB, which directly contacts the E2F1 activation domain and silences it. The E2F1 activation domain has sequence similarity to the N-terminal activation domain of E1A(12S), which contains binding sites for CBP as well as RB. Here, we present evidence that the CBP protein directly contacts E2F1/DP1 and stimulates its activation capacity. We show that CBP interacts with the activation domain of E2F1 both in vitro and in vivo. Deletion of four residues from the E2F1 activation domain reduces CBP binding as well as transcriptional activation, but still allows the binding of RB and MDM2. This deletion removes residues which are conserved in the N-terminal activation domain of E1A and which are required for the binding of CBP to E1A. When the E1A N-terminus is used as a competitor in squelshing experiments it abolishes CBP-induced activation of E2F1/DP1, whereas an E1A mutant lacking CBP binding ability fails to do so. These results indicate that CBP can act as a coactivator for E2F1 and suggest that CBP recognises a similar motif within the E1A and E2F1 activation domains. The convergence of the RB and CBP pathways on the regulation of E2F1 activity may explain the cooperativity displayed by these proteins in mediating the biological functions of E1A. We propose a model in which E1A activates E2F not only by removing the RB repression but also by providing the CBP co-activator.

Amino Acid Sequence↗

PubMed1996

Repression of RNA polymerase III transcription by the retinoblastoma protein.

Transcription by RNA polymerase (pol) III is under cell-cycle control, being higher in S and G2 than in G0 and early G1 phases. Many transformed cell types have elevated pol III activity, presumably to sustain sufficient protein synthesis for unrestrained growth. The retinoblastoma tumour-suppressor protein (Rb) restricts cellular proliferation, and is often found mutated in transformed cells. Here we demonstrate that Rb can repress the level of transcription from pol III templates both in vitro and vivo. Analysis of Rb-deficient SAOS2 cells and primary fibroblasts from Rb-/- mice demonstrates elevated levels of pol III activity in the absence of functional Rb protein. Rb-induced repression of pol III activity is alleviated by mutations in the Rb pocket domain that occur naturally in tumours, and by viral transforming proteins that bind and inactivate Rb. These results implicate repression of pol III transcription as a mechanism for Rb-induced growth arrest, and suggest that restraining protein biosynthesis may be important in the prevention of tumour development.

Adenovirus E1A Proteins↗

PubMed1996

Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins.

Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.

3T3 Cells↗

PubMed1996

E2F1 and E1A(12S) have a homologous activation domain regulated by RB and CBP.

The E2F1 transcription factor has a well-characterized activation domain at its C terminus and the E1A protein has a recently defined activation domain at its N terminus. Here we show that these activation domains are highly related in sequence. The sequence homology reflects, at least partly, the conservation of common binding sites for the RB and CBP/p300 proteins, which are preserved in the same relative order along E2F1 and E1A. Furthermore, the interaction of RB and CBP with these two activation domains results in the same functional consequences: RB represses both activation domains, whereas CBP stimulates them. We conclude that the activation domains of E1A(12s) and E2F1 belong to a novel functional class, characterized by specific protein binding sites. The implication of this conservation with respect to E1A-induced stimulation of E2F activity is discussed.

Adenovirus E1A Proteins↗

PubMed1995

Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein.

The MDM2 proto-oncogene is found amplified in a variety of tumours. The oncogenic capacity of the MDM2 protein is attributed to its ability to bind the p53 tumour-suppressor protein and mask its transcriptional activation potential. Here we show that MDM2 makes a functional contact with two cooperating transcription factors, E2F1 and DP1 (refs 4,5), which are involved in S-phase progression. MDM2 contacts the activation domain of E2F1 using residues conserved in the activation domain of p53. However, in contrast to its repression of p53 activity, MDM2 stimulates the activation capacity of E2F1/DP1. These results indicate that MDM2 not only releases a proliferative block by silencing the tumour suppressor p53, it also positively augments proliferation by stimulating the S-phase inducing transcription factors E2F1/DP1.

3T3 Cells↗

PubMed1995

Myogenin binds to and represses c-fos promoter.

Myogenin (a member of the myogenic basic helix-loop-helix transcription factor family) seems to be the main effector of proliferation repression, a crucial step which precedes muscle cell terminal differentiation during muscle development. Proliferation repression most likely occurs through inhibition of proliferation-associated genes such as the proto-oncogene, c-fos. Here, we demonstrate that myogenin binds to an E-box located in the main element of the c-fos promoter, the serum response element (SRE). Results from co-transfection experiments indicate that myogenin acts as a repressor for the SRE. Our data suggest that myogenin could play a role in c-fos inhibition at the onset of muscle cell terminal differentiation.

3T3 Cells↗

PubMed1995

Regulation of transcription by E2F1/DP1.

The E2F1 transcription factor, in co-operation with DP1, controls the expression of several S-phase specific genes. This activity is most likely responsible for the oncogenic and S-phase inducing properties of E2F1, suggesting that this transcription factor plays a key role in regulating the cell cycle. The transcriptional activation functions of E2F1 are resident in a small C-terminal domain which can independently activate transcription. Here we review the protein-protein interactions which impinge upon and regulate this activation domain and put forward some models on their mechanism of action.

Animals↗

PubMed1993

The serum unresponsive Rous sarcoma virus promoter sustains a high serum response factor-dependent transcription in vitro.

CArG boxes are cis-regulatory elements which are represented both in serum responsive and unresponsive promoters. Here we show that the RSV Long Terminal Repeat contains two CArG boxes, which were efficiently recognised by purified Serum Response Factor, although they remained unresponsive to serum in transient transfection assays. However, RSV CArG boxes were as efficient as c-fos Serum Response Element in mediating a Serum Response Factor-dependent transcription in vitro. Thus, the fact that a CArG box is able to bind Serum Response Factor in an active form is insufficient for serum responsiveness in vivo.

3T3 Cells↗

PubMed1993

Repression of c-fos promoter by MyoD on muscle cell differentiation.

Terminal differentiation and cell proliferation are in many cases, as in muscle cells, mutually exclusive processes. While differentiating myoblasts are withdrawn from the cell cycle, myogenesis is inhibited by some mitogens and overexpression of some oncogenes, including proto-oncogene c-fos (which expresses a growth-associated protein constituting the regulatory factor AP-1 in conjunction with c-Jun). MyoD, a muscle-specific transcription factor of the basic helix-loop-helix family, acts at both levels because it triggers a muscle differentiation programme in non-muscle cells, and induces a complete block of cell proliferation. Antagonistic interaction between MyoD and c-Jun has been demonstrated. We here show that c-fos expression greatly decreases upon muscle cell differentiation, concomitant with MyoD-induced activity. We have identified a MyoD-binding site overlapping with the serum-responsive element in the c-fos promoter. We demonstrate that MyoD can act as a negative regulator for c-fos transcription by blocking serum responsiveness through this binding site. These data suggest that the MyoD negative effect on cell growth could be partly mediated by transcriptional inactivation of growth-responsive genes.

3T3 Cells↗

PubMed1991

Universality of c-fos transcriptional regulation: the Dyad Symmetry Element mediates activation by PMA in T lymphocytes.

We here have delineated the regulatory sequences responsible for c-fos transcriptional activation in human primary T lymphoblasts and in a human tumor T cell line (Jurkat), using transient transfection assays. Our results indicate that, as it has been demonstrated for fibroblastic or epithelial cells, the Dyad Symmetry Element is necessary and sufficient to confer responsiveness to an heterologous promoter in both cell types. Protein binding to this element was constitutive, as assessed by gel shift assays. These results suggest that c-fos transcriptional regulation occurs through a widely conserved mechanism in highly differentiated tissues.

Base Sequence↗