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Biomedical subjects

A Harel-Bellan

Publications and source records attributed to A Harel-Bellan.

At least 19 recordsLinked to original sources
PubMed2000

CBP/p300 histone acetyl-transferase activity is important for the G1/S transition.

Transforming viral proteins such as E1A which force quiescent cells into S phase have two essential cellular target proteins, Rb and CBP/p300. Rb regulates the G1/S transition by controlling the transcription factor E2F. CBP/p300 is a transcriptional co-activator with intrinsic histone acetyl-transferase activity. This activity is regulated in a cell cycle dependent manner and shows a peak at the G1/S transition, suggesting a function for CBP/p300 in this crucial step of the cell cycle. Here, we have artificially modulated CBP/p300 levels in individual cells through microinjection of specific antibodies and expression vectors. We show that CBP/p300 is required for cell proliferation and has an essential function during the G1/S transition. Using the same microinjection system and GFP-reporter vectors, we demonstrate that CBP/p300 is essential for the activity of E2F, a transcription factor that controls the G1/S transition. In addition, our results suggest that CBP HAT activity is required both for the G1/S transition and for E2F activity. Thus CBP/p300 seems to be a versatile protein involved in opposing cellular processes, which raises the question of how its multiple activities are regulated.

3T3 Cells↗
PubMed2000

RbAp48 belongs to the histone deacetylase complex that associates with the retinoblastoma protein.

The retinoblastoma susceptibility gene product, the Rb protein, is a key regulator of mammalian cell proliferation. One of the major targets of Rb is the S phase inducing E2F transcription factor. Once bound to E2F, Rb represses the expression of E2F-regulated genes. Transcriptional repression by Rb is believed to be crucial for the proper control of cell growth. Recently, we and others showed that Rb represses transcription through the recruitment of a histone deacetylase. Interestingly, we show here that the Rb-associated histone deacetylase complex could deacetylate polynucleosomal substrates, indicating that other proteins could be present within this complex. The Rb-associated protein RbAp48 belongs to many histone deacetylase complexes. We show here that the histone deacetylase HDAC1 is able to mediate the formation of a ternary complex containing Rb and RbAp48. Moreover, less deacetylase activity was found associated with Rb in cell extracts depleted for RbAp48 containing complexes, demonstrating that Rb, histone deacetylase, and RbAp48 are physically associated in live cells. Taken together, these data indicate that RbAp48 is a component of the histone deacetylase complex recruited by Rb. Finally, we found that E2F1 and RbAp48 are physically associated in the presence of Rb and HDAC1, suggesting that RbAp48 could be involved in transcriptional repression of E2F-responsive genes.

Amino Acid Sequence↗
PubMed2000

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Base Sequence↗
PubMed2000

Histone acetylation and the control of the cell cycle.

The critical steps of the cell cycle are generally controlled through the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to acetylation or deacetylation of core histone tails: acetylated histone tails are generally associated with active chromatin, whereas deacetylated histone tails are associated with silent parts of the genome. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss some of the critical cell cycle steps in which these enzymes are involved.

Acetylation↗
PubMed1999

Gene activation by triplex-forming oligonucleotide coupled to the activating domain of protein VP16.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chimera (TFO-P) can specifically recognise its DNA target at physiological salt and pH conditions. Bound to the double-stranded target DNA in a promoter region, the TFO-P is able to activate gene expression. Our results suggest that this type of molecule may prove useful in the design of new tools for artificial modulation of gene expression.

Amino Acid Sequence↗
PubMed1999

Phosphorylation by p44 MAP Kinase/ERK1 stimulates CBP histone acetyl transferase activity in vitro.

The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure. CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways. In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP. CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases. Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity. The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain. These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule. Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways.

3T3 Cells↗
PubMed1999

Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways.

Psoralen is an asymmetric photoreactive intercalator with a furane and a pyrone side. When intercalated at 5'-TpA-3' sites and upon UVA irradiation, the psoralen can react with the thymine residues on both strands, introducing an interstrand crosslink. Using psoralen-coupled triple-helix-forming oligonucleotides, psoralen interstrand crosslinks can be site-specifically introduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors. In addition, crosslinks introduced via a triple-helix-forming oligonuleotide are oriented with the furane side of the psoralen associated with a specific strand of the target sequence. Here, the transformation efficiency, the mutation frequency and the mutational spectra of site-specifically placed and oriented crosslinks were examined in yeast cells. We found that the nature of the targeted mutations depended on the crosslink orientation: bypass of the pyrone-adducted thymine yielded T-->A or T-->C substitutions and A insertions, while bypass of the furane-adducted thymine yielded T-->G substitutions and G insertions. Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis. In addition, the observed pattern of mutagenesis suggests a coupling to transcription, similar to the one observed in mammalian cells. Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed strand, suggesting that the incision preference of psoralen crosslinks, which has been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells.

Base Sequence↗
PubMed1999

Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.

Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity. In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen. The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial. Here we show, using psoralen-coupled TFOs and the yeast as a convenient cellular test system, that interstrand crosslinks are quantitatively poorly repaired, resulting in an efficient modification of target gene activity. In addition, these lesions result in the introduction of mutations in a high proportion of cells. We show that these mutations are generated by the Error-Prone Repair pathway, alone or in combination with Nucleotide Excision Repair. Taken together, these results suggest that TFOs coupled to psoralen could be used to inactivate a gene with significant efficiency.

Adenosine Triphosphatases↗
PubMed1999

Histone acetylation in signal transduction by growth regulatory signals.

Cell fate is determined by extracellular signals which are transmitted to the nucleus and result in the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to enzymatic activities which are able to acetylate or deacetylate core histone tails. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss the involvement of these enzymes in critical steps of cell proliferation or cell differentiation control

Acetylation↗
PubMed1999

Autonomously binding protein detected on ets box of c-fos serum response element in proliferating cells.

The serum response element (SRE) in the c-fos promoter contains an ets box whose integrity is required for full activation of this proto-oncogene by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. Electrophoretic mobility shift assays (EMSA) detect a protein in nuclear extracts that binds to the wild-type SRE, but not to an SRE containing a mutated ets box. Competition studies using unlabeled probes, and supershift experiments using antibodies and in vitro translated core serum response factor (SRF) indicate that the protein in question is not YY1, SAP-1, nor Elk-1 and that it does not exhibit ternary complex factor (TCF) activity, so that it may correspond to an autonomously binding Ets family protein. The complete disappearance of this "Ets-like autonomous binding factor" upon terminal differentiation of both L6alpha2 myoblastic and PC12 pheochromocytoma cells points to a possible role in the proliferation/differentiation process.

3T3 Cells↗
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

Activation of the c-fos SRE through SAP-1a.

TCFs, which are members of the Ets family of transcription factors, are recruited to the Serum Response Element (SRE) in the c-fos promoter by SRF. These Ets proteins, which are substrates for the MAP kinases, are direct targets of the Ras/MAP kinase signal transduction pathway. In this paper, we demonstrate that one of the TCFs, SAP-1a, displays a significant level of autonomous binding to the SRE Ets box. In contrast to previous observations, deletion of the SRF binding domain did not modulate the autonomous binding of SAP-1a. Also, the autonomous binding was not modulated by the phosphorylation of SAP-1a by MAP kinases. The autonomous binding was also detected in live cells: transfected SAP-1a was able to restore the response of a CArG-less SRE in PC12 cells. The response occurred in the absence of SRF recruitment since a mutant of SAP-1a in which the B-box, a domain required for interaction with SRF, had been deleted was still able to transactivate the CArG-less SRE. The transactivation was repressed by a Ras transdominant negative mutant, indicating the involvement of the Ras/MAP kinase pathway. Taken together, these data demonstrate that SAP-1a is capable of binding to the c-fos SRE in the absence of SRF.

Animals↗