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R Benezra

Publications and source records attributed to R Benezra.

At least 37 records · Page 2Linked to original sources
PubMed1996

Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1.

The expression of Id1, a helix-loop-helix protein which inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Idl expression, we have isolated a 210-bp enhancer element in the upstream region of the Id1 gene whose activity recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, we have identified two functional elements within the 210-bp enhancer which are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, we propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early-growth-response transcription factors and dominant-negative helix-loop-helix proteins.

Animals↗
PubMed1995

Chromosomal assignment of human ID1 and ID2 genes.

The Id (inhibitor of DNA binding) proteins regulate transcription during development by interacting with transcription factors. Three human genes, ID1, ID2, and ID3, have been identified that belong to this family of transcription regulators. We show, by somatic cell hybridization and fluorescence in situ hybridization experiments, that ID1 and ID2 are localized at 20q11 and 2p25, respectively.

Blotting, Southern↗
PubMed1994

An intermolecular disulfide bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures.

It is demonstrated in this report that purified E2A helix-loop-helix (HLH) proteins spontaneously form homodimers that are linked by an intermolecular disulfide bond. These homodimers bind DNA at physiological temperatures but fail to associate with either Id or MyoD. When the disulfide bond is reduced by an activity present in muscle cell lysates or disrupted by site-directed mutagenesis, the monomeric form of the protein is strongly favored at 37 degrees C. These E2A monomers cannot bind DNA but heterodimerize efficiently with Id and MyoD. It is also shown that an intermolecular disulfide bond cross-links E2A homodimers in B cells but not in muscle cells in which only heterodimers have been detected. These results suggest a novel mechanism for regulating the dimerization status and DNA binding properties of E2A HLH transcription factors.

Animals↗
PubMed1993

Bone morphogenetic protein 2 transiently enhances expression of a gene, Id (inhibitor of differentiation), encoding a helix-loop-helix molecule in osteoblast-like cells.

Bone morphogenetic protein 2 (BMP-2) is a potent inducer of differentiation of osteoblasts both in vivo and in vitro. We examined the action of BMP-2 on expression of a helix-loop-helix-type transcription factor, Id (inhibitor of differentiation), in osteoblast-like cells, as well as in osteoblast-enriched cells and possible precursor cells. To our surprise, BMP-2 enhanced Id gene expression in the cell types of osteoblastic lineage we examined. The maximal BMP-2 enhancement was observed within 24 hr in early proliferating cultures and the enhancement lasted up to 96 hr. The BMP-2 effect was not blocked by actinomycin D, while it was blocked by cycloheximide, suggesting that BMP-2 regulates Id gene expression at least in part via posttranscriptional events, which require protein synthesis. Other experiments indicated that BMP-2 did not further enhance Id mRNA levels promoted by dexamethasone, while BMP-2 did not resume the Id mRNA levels suppressed by 1,25-dihydroxyvitamin D3. Similar BMP-2 enhancement of Id message expression was also observed in osteoblast-enriched fetal rat calvaria cells as well as C3H10T1/2 cells. These results indicate that BMP-2 enhances expression of Id in early cultures of osteoblastic cells and suggest that enhancement of Id expression may somehow be involved in the promotion of differentiation by this cytokine in these osteoblastic cells and in their precursor cells.

3T3 Cells↗
PubMed1993

The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.

Amino Acid Sequence↗
PubMed1992

Inhibition of myeloid differentiation by the helix-loop-helix protein Id.

Id is a helix-loop-helix (HLH) protein that represses activity of several basic helix-loop-helix (bHLH) proteins involved in cell type--specific transcription and cell lineage commitment. The myeloid precursor cell line 32DC13(G) expressed Id messenger RNA, which was transiently decreased when cells were induced to terminally differentiate with granulocyte--colony-stimulating factor. Concomitant with the decrease of Id messenger RNA was the appearance in nuclear extracts of DNA binding proteins that recognized a canonical E-box motif, a DNA binding site for some bHLH proteins. Constitutive expression of an Id complementary DNA in 32DC13(G) cells blocked their ability to differentiate and to induce E-box-binding activity. These results suggest that Id and, hence, bHLH proteins function in the process of myeloid differentiation.

Cell Differentiation↗
PubMed1992

Id expression during mouse development: a role in morphogenesis.

We have characterized the spatial and temporal pattern of Id transcription during mouse embryogenesis. The Id gene encodes a helix-loop-helix (HLH) protein which can heterodimerize with the ubiquitously expressed HLH protein products of the E2A gene, and prevent them from binding DNA either alone or as a heterodimer with tissue specific HLH transcription factors such as the muscle determination gene, MyoD1 (Benezra et al., 1990: Cell 61:49-59). Since Id has been shown to be down-regulated during induced differentiation in several cell lines, it has been postulated that Id plays a general inhibitory role in cell differentiation (Benezra et al., 1990). In situ analysis of Id mRNA expression in the mouse embryo was performed in order to determine whether the pattern of Id expression is consistent with this postulate. A detailed study throughout the entirety of mouse postimplantation development reveals that Id is expressed upon gastrulation at very high levels in almost all regions of the mouse embryo and expression declines as embryogenesis proceeds. In skeletal muscle, in which the inhibitory action of Id has been established in tissue culture models (Benezra et al., 1990), Id and the HLH myogenic factors are expressed in a mutually exclusive manner suggesting that myogenic precursors do not express both types of HLH gene products. In addition, Id colocalizes both spatially and temporally with Hox-7.1, a murine homeobox gene which is associated with regions of high cell proliferation and positional fate assignment.

Animals↗
PubMed1992

The gene for the helix-loop-helix protein, Id, is specifically expressed in neural precursors.

While mammalian neurogenesis has been characterized extensively, the molecules involved in regulating neural cell determination and differentiation remain ill-defined. There is accruing evidence that various members of the basic helix-loop-helix (bHLH) protein family critically regulate these biological processes in a number of tissues. Id, a negative regulator of bHLH proteins, was found to exhibit peak gene expression during mouse embryogenesis with a striking pattern in the central nervous system. Id transcripts were specifically localized to undifferentiated neural precursors of the ventricular zone and were not present in their differentiated derivatives. In addition, in the peripheral nervous system, dorsal root ganglia sensory precursors, known to be undifferentiated while dividing, also expressed Id mRNA. However, in the sympathetic nervous system and adrenal medulla, where differentiation and division occur simultaneously in precursors, Id was not expressed. Since Id transcript abundance inversely correlated with differentiation, this protein, similar to its Drosophila homolog, extramacrochaetae, may play a negative regulatory role in neural differentiation.

Animals↗
PubMed1992

Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins.

The helix-loop-helix (HLH) protein Id lacks the basic DNA-binding domain common to this class of proteins. In vitro experiments suggested that Id could associate tightly with two other HLH proteins encoded by the E2A gene, E12 and E47 (referred to here collectively as E proteins) and prevent their binding to a sequence present in the muscle creatine kinase (MCK) enhancer either as homo-oligomers or hetero-oligomers with MyoD. In this report we present evidence for the in vivo roles of Id and E proteins: (1) Id and E proteins co-fractionate and co-immunoprecipitate in whole-cell extracts prepared from myoblasts; (2) the loss of Id protein observed during the conversion of proliferating myoblasts into mature myotubes correlates with the formation of MyoD/E hetero-oligomeric complexes in whole-cell extracts (these complexes do not form when purified Id protein is added to the extracts); and (3) stable overexpression of Id mRNA and protein in the C2C12 muscle cell line inhibits differentiation in these cells 16 hr post-induction. The myotubes that do eventually form 48 hr post-induction have no detectable Id protein in the nucleus despite the persistence of exogenous Id mRNA. These data support a model in which Id can inhibit muscle cell differentiation by associating with E proteins and preventing them from forming active hetero-oligomeric complexes with the muscle determination gene products.

Animals↗
PubMed1991

The myoD gene family: nodal point during specification of the muscle cell lineage.

The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription; this may stabilize commitment to myogenesis.

Animals↗
PubMed1991

Repression of immunoglobulin enhancers by the helix-loop-helix protein Id: implications for B-lymphoid-cell development.

It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.

3T3 Cells↗
PubMed1990

The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.

We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.

Amino Acid Sequence↗
PubMed1986

Nucleosomes are phased along the mouse beta-major globin gene in erythroid and nonerythroid cells.

We have used the chemical cleavage reagent methidiumpropyl-EDTA-Fe(II) to determine the location of the nucleosomes along the mouse beta-major globin gene in erythroid and nonerythroid cells. In mouse L cells, in which the globin gene is inactive, the nucleosomes are precisely positioned with respect to the underlying DNA sequence from positions -3000 to +1500 relative to the cap site. In uninduced and induced murine erythroleukemia cells, the same phasing persists but is interrupted from positions -200 to +500. This gap in the phased distribution of nucleosomes appears to be protected from MPE-Fe(II) digestion, and is bounded on both sides by hypersensitive sites. These results define at least two structural states for the globin gene: an inactive state in which the gene is covered with a continuous array of phased nucleosomes and an active state in which this array is disrupted over the 5' half of the structural gene.

Animals↗
PubMed1984

Human B-like lymphoblastoid cell lines obtained by long-term culture of normal spleen leukocytes.

B-like lymphoblastoid cell lines were obtained by long-term culture of human spleen leukocytes in RPMI 1640 medium containing human plasma fraction instead of whole foetal calf serum. These cell lines, which did not form E-rosettes had membrane immunoglobulins, and expressed Epstein-Barr virus antigens. Most synthesized intracytoplasmic immunoglobulins and were shown to be diploid and to remain so after subcultures. All produced interferon upon induction with Sendai virus.

B-Lymphocytes↗
PubMed1981

Immunological and organizational heterogeneity of histone H2a variants within chromatin of cells at different stages of Friend leukemia.

We have used antibodies directed against two histone H2a variants, H2a.1 and H2a.2, to probe chromatin structure in Friend erythroleukemia cells. Each molecule has at least one unique antigenic determinant, as well as determinants shared by both variants. Some Friend leukemia cell types contained H2a molecules which showed altered immunologic reactivity with the two antisera. The accessibility of the H2a variants in chromatin to anti-H2a antibody was different as measured by the use of whole chromatin as an immunoabsorbent and by binding of antibody to nucleosomes in a solid phase radioimmunoassay. While anti-H2a.1 IgG bound to chromatin, anti-H2a.2 IgG did not. Moreover, anti-H2a.1 IgG binding to chromatin from different Friend cell types reflected, in general, the relative amounts of H2a.1 in total chromatin. The different reactivity of the two antisera with chromatin was also observed with isolated nucleosomes: anti-H2a.1 IgG bound but anti-H2a.2 did not. Furthermore, the binding of anti-H2a.1 Ig with subfractions of nucleosomes varied; H1-depleted, high mobility group-enriched nucleosomes reacted better than H1-containing, high mobility group-depleted nucleosomes. These findings demonstrated a heterogeneity in the organization of H2a variants in chromatin within nucleosomal subfractions of chromatin and among chromatin of different Friend leukemia cell types. Moreover, most of the antigenic determinants common to both H2a variants were shown to be buried within the nucleosome core; only H2a.1-unique determinants were accessible to an anti-H2a.1 IgG molecule.

Animals↗