Biomedical subjects

H Weintraub

Publications and source records attributed to H Weintraub.

At least 19 recordsLinked to original sources

PubMed1996

I-mf, a novel myogenic repressor, interacts with members of the MyoD family.

During embryogenesis, cells from the ventral and dorsal parts of the somites give rise to sclerotome and dermomyotome, respectively. Dermomyotome contains skeletal muscle precursors that are determined by the MyoD family of myogenic factors. We have isolated a novel myogenic repressor, I-mf (Inhibitor of MyoD family), which is highly expressed in the sclerotome. In contrast, MyoD family members are concentrated in the dermomyotome. We demonstrate that I-mf inhibits the transactivation activity of the MyoD family and represses myogenesis. I-mf associates with MyoD family members and retains them in the cytoplasm by masking their nuclear localization signals. I-mf can also interfere with the DNA binding activity of MyoD family members. We postulate that I-mf plays an important role in the patterning of the somite early in development.

3T3 Cells↗

PubMed1996

Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain.

Previous studies imply that the intracellular domain of Notch1 must translocate to the nucleus for its activity. In this study, we demonstrate that a mNotch1 mutant protein that lacks its extracellular domain but retains its membrane-spanning region becomes proteolytically processed on its intracellular surface and, as a result, the activated intracellular domain (mNotchIC) is released and can move to the nucleus. Proteolytic cleavage at an intracellular site is blocked by protease inhibitors. Intracellular cleavage is not seen in cells transfected with an inactive variant, which includes the extracellular lin-Notch-glp repeats. Collectively, the studies presented here support the model that mNotch1 is proteolytically processed and the cleavage product is translocated to the nucleus for mNotch1 signal transduction.

3T3 Cells↗

PubMed1996

Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro.

The epiblast layer of the chick embryo gives rise to all embryonic tissues. In vitro analyses were carried out to determine whether epiblast cells could form skeletal muscle prior to entry into the primitive streak. Epiblasts were separated from the mesoderm, hypoblast, and primitive streak, dissociated to produce a single cell suspension, and plated at high density. Myogenesis began on the first day in culture, and by the fifth day most cells had differentiated into skeletal muscle. Some cells differentiated without replicating. MyoD messenger RNA was present in epiblast tissue and translated in practically all cells in culture. Cells from regions of the epiblast which do not form muscle later in the embryo did so in vitro. Epiblasts cultured for 2 days as an intact epithelium, or in the presence of the mesoderm and hypoblast, did not undergo myogenesis. These findings demonstrate that myogenic potential is wide-spread within the primitive streak stage epiblast, and that muscle differentiation, which occurs relatively autonomously in culture, can be prevented by cell and tissue interactions.

Animals↗

PubMed1996

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Animals↗

PubMed1996

B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB.

B-lymphocyte development requires the basic helix-loop-helix proteins encoded by the E2A gene. In this study, the control mechanism of E2A was further explored by disruption of the E2A-related genes, E2-2 and HEB. In contrast to E2A, E2-2 and HEB are not essential for the establishment of the B-cell lineage. However, both E2-2 and HEB are required for the generation of the normal numbers of pro-B cells in mouse embryos. Breeding tests among mice carrying different mutations revealed that E2-2 and HEB interact with E2A in many developmental processes including generation of B cells. Specifically, mice transheterozygous for any two mutations of these three genes produced fewer pro-B cells than the singly heterozygous littermates. This study indicates that B-cell development is dependent not only on an essential function provided by the E2A gene but also on a combined dosage set by E2A, E2-2, and HEB.

Animals↗

PubMed1996

Differences between MyoD DNA binding and activation site requirements revealed by functional random sequence selection.

A method has been developed for selecting functional enhancer/promoter sites from random DNA sequences in higher eukaryotic cells. Of sequences that were thus selected for transcriptional activation by the muscle-specific basic helix-loop-helix protein MyoD, only a subset are similar to the preferred in vitro binding consensus, and in the same promoter context an optimal in vitro binding site was inactive. Other sequences with full transcriptional activity instead exhibit sequence preferences that, remarkably, are generally either identical or very similar to those found in naturally occurring muscle-specific promoters. This first systematic examination of the relation between DNA binding and transcriptional activation by basic helix-loop-helix proteins indicates that binding per se is necessary but not sufficient for transcriptional activation by MyoD and implies a requirement for other DNA sequence-dependent interactions or conformations at its binding site.

3T3 Cells↗

PubMed1995

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

DNA-Binding Proteins↗

PubMed1995

Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and S100a (alpha alpha) proteins.

MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.

Amino Acid Sequence↗

PubMed1995

Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein.

Basic helix-loop-helix (bHLH) proteins are instrumental in determining cell type during development. A bHLH protein, termed NeuroD, for neurogenic differentiation, has now been identified as a differentiation factor for neurogenesis because (i) it is expressed transiently in a subset of neurons in the central and peripheral nervous systems at the time of their terminal differentiation into mature neurons and (ii) ectopic expression of neuroD in Xenopus embryos causes premature differentiation of neuronal precursors. Furthermore, neuroD can convert presumptive epidermal cells into neurons and also act as a neuronal determination gene. However, unlike another previously identified proneural gene (XASH-3), neuroD seems competent to bypass the normal inhibitory influences that usually prevent neurogenesis in ventral and lateral ectoderm and is capable of converting most of the embryonic ectoderm into neurons. The data suggest that neuroD may participate in the terminal differentiation step during vertebrate neuronal development.

Amino Acid Sequence↗

PubMed1995

Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.

Amino Acid Sequence↗

PubMed1995

Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage- and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.

Amino Acid Sequence↗

PubMed1994

The helix-loop-helix gene E2A is required for B cell formation.

Heterodimers between tissue-specific basic-helix-loop-helix proteins and the gene products of E2A play major roles in determining tissue-specific cell fate. To understand the broad role of E2A in development, we have generated E2A mutant mice following homologous recombination in embryonic stem cells. Homozygous mutant mice develop to full term without apparent abnormalities, but then display a high rate of postnatal death. The surviving mice show retarded postnatal growth. Detailed examination of hematopoiesis reveals that the homozygous mutant mice contain no B cells while other lineages including T cell, granulocyte, macrophage, and erythroid are intact. The block to B cell differentiation occurs prior to immunoglobulin gene DH-JH rearrangement and the expression of the B lineage-specific marker B220. Surprisingly, heterozygous embryos contain, on average, about half as many B cells as wild-type embryos, suggesting the existence of a counting mechanism that translates levels of E2A into numbers of B cells.

Animals↗

PubMed1994

Formation of a monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements.

Maternally expressed Skn-1 protein is required for the correct specification of certain blastomere fates in early Caenorhabditis elegans embryos. Skn-1 contains a basic region similar to those of basic leucine zipper (bZIP) proteins but, paradoxically, it lacks a leucine zipper dimerization segment. Random sequence selection methods were used to show that Skn-1 binds to specific DNA sequences as a monomer. The Skn-1 basic region lies at the carboxyl terminus of an 85-amino acid domain that binds preferentially to a bZIP half-site and also recognizes adjacent 5' AT-rich sequences in the minor groove, apparently with an amino (NH2)-terminal "arm" related to those of homeodomain proteins. The intervening residues appear to stabilize interactions of these two subdomains with DNA. The Skn-1 DNA binding domain thus represents an alternative strategy for promoting binding of a basic region segment recognition helix to its cognate half-site. The results point to an underlying modularity in subdomains within established DNA binding domains.

Amino Acid Sequence↗

PubMed1994

Tissue-specific gene activation by MyoD: determination of specificity by cis-acting repression elements.

MyoD is a muscle-specific transcriptional activator; E12 is a B-cell activator. An IgH enhancer is activated almost 100-fold by E12 but not at all by Myo; an MCK enhancer is activated almost 1000-fold by MyoD and not at all by E12. MyoD and E12 are both basic helix-loop-helix proteins that bind to similar E-box sequences (CANNTG); the IgH enhancer contains the same E boxes as the MCK enhancer, yet each retains exclusive specificity for either E12 or MyoD, respectively. We show that the IgH enhancer contains a cis-acting negative element that is directed at MyoD, but not at E12. This repression requires the mu E5 E box within the IgH enhancer; however, the specificity for repression, as opposed to activation, is associated with 2 bp flanking each side of the mu E5 E box. The target for repression of MyoD in the IgH enhancer is the bHLH region of MyoD. Our results suggest that MyoD only activates myogenic genes because nonmuscle enhancers that contain E boxes also contain negative elements that prevent MyoD activity.

3T3 Cells↗

PubMed1994

Positive control mutations in the MyoD basic region fail to show cooperative DNA binding and transcriptional activation in vitro.

An in vitro transcription system from HeLa cells has been established in which MyoD and E47 proteins activate transcription both as homodimers and heterodimers. However, heterodimers activate transcription more efficiently than homodimers, and function synergistically from multiple binding sites. Positive control mutants in the basic region of MyoD that have previously been shown to be defective in initiating the myogenic program, can bind DNA but have lost their ability to function as transcriptional activators in vitro. Additionally, positive control mutants, unlike wild-type MyoD, fail to bind cooperatively to DNA. We propose that binding of MyoD complexes to high affinity MyoD binding sites induces conformational changes that facilitate cooperative binding to multiple sites and promote transcriptional activation.

Amino Acid Sequence↗

PubMed1994

Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.

In Drosophila, the proneural genes of the achaete-scute complex encode transcriptional activators that can commit cells to a neural fate. We have isolated cDNAs for two Xenopus achaete-scute homologs, ASH3a and ASH3b, which are expressed in a subset of central nervous system (CNS) neuroblasts during early neurogenesis. After expressing either ASH3 protein in developing Xenopus embryos, we find enlargement of the CNS at the expense of adjacent non-neural ectoderm. Analysis of molecular markers for neural, epidermal, and neural crest cells indicates that CNS expansion occurs as early as neural plate formation. ASH3-dependent CNS enlargement appears to require neural induction, as it does not occur in animal cap explants. Inhibition of DNA synthesis shows that additional CNS tissue does not depend on cell division--rather it reflects conversion of prospective neural crest and epidermal cells to a neural fate. The differentiation of the early forming primary neurons also seems to be prevented by ASH3 expression. This may be secondary to the observed activation of Xotch transcription by ASH3.

Amino Acid Sequence↗

PubMed1994

Xenopus embryos regulate the nuclear localization of XMyoD.

Injection of Xenopus myoD mRNA into Xenopus embryos leads to only a modest activation of myogenic markers. In contrast, we show that injected mouse myoD mRNA leads to a potent activation. We postulate that XMyoD is under negative control in frog embryos, but because of slight sequence differences, mouse MyoD fails to see the negative signal. Whereas mMyoD is constitutively nuclear, XMyoD is largely cytoplasmic except in a region of the embryo that includes the location where mesoderm induction occurs; there, it is nuclear. At MBT, endogenous XmyoD mRNA is expressed ubiquitously in the frog embryo. Our results suggest that this expression would lead to cytoplasmic XMyoD protein. Among other events, muscle induction might remove this negative regulation, allow MyoD to enter the nucleus, and establish an autoregulatory loop that could commit cells to myogenesis.

Animals↗

PubMed1994

Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation.

The crystal structure of a MyoD basic-helix-loop-helix (bHLH) domain-DNA complex has been solved and refined at 2.8 A resolution. This structure proves that bHLH and bHLH-leucine zipper (bHLH-ZIP) proteins are remarkably similar; it helps us understand subtle differences in binding preferences for these proteins; and it has surprising implications for our understanding of transcription. Specifically, Ala-114 and Thr-115, which are required for positive control in the myogenic proteins, are buried at the protein-DNA interface. These residues are not available for direct protein-protein contacts, but they may determine the conformation of Arg-111. Comparisons with Max suggest that the conformation of this arginine, which is different in the two structures, may play an important role in myogenic transcription.

Amino Acid Sequence↗