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H Weintraub

Publications and source records attributed to H Weintraub.

At least 55 records · Page 3Linked to original sources
PubMed1992

Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12.

The basic helix-loop-helix (bHLH) protein MyoD is a transcription factor that is important for the induction of the myogenic phenotype. The DNA binding basic region (13 amino acids) is necessary for recognition of the consensus MyoD binding site, for transcriptional activation, and for conversion of fibroblasts to muscle. In contrast, the non-tissue-specific bHLH protein E12 can bind to the MyoD binding site but does not induce myogenesis. Here, it is shown that only two amino acids in the MyoD basic region and a single amino acid from the junction, which separates the basic region and helix 1, are sufficient for myogenic specificity when substituted into the corresponding region of E12. These findings suggest that the recognition of particular determinants in the basic region is required for conversion of fibroblasts to muscle.

Amino Acid Sequence↗
PubMed1992

Body-wall muscle formation in Caenorhabditis elegans embryos that lack the MyoD homolog hlh-1.

The myoD family of DNA binding proteins has been implicated in the control of myogenesis in a variety of organisms. Searches for homologs in the nematode Caenorhabditis elegans yielded only one gene, designated hlh-1, expressed in body-wall muscle cells and their precursors. To assess the role of hlh-1 in C. elegans myogenesis, genetic deficiencies spanning the hlh-1 locus were isolated after gamma irradiation. Embryos homozygous for these deficiencies exhibited extensive body-wall muscle differentiation, including expression of several characteristic myofilament proteins and weak contracile behavior. Thus, zygotic hlh-1 expression was not required for body-wall muscle precursors to adopt muscle cell fates.

Amino Acid Sequence↗
PubMed1992

Functional antagonism between c-Jun and MyoD proteins: a direct physical association.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.

Animals↗
PubMed1992

Storage of glycoprotein in NCTR-Balb/C mouse. Lectin histochemistry, and biochemical studies.

A strain of Balb/C mice carrying a lysosomal storage disorder exhibits metabolic and phenotypic abnormalities similar to patients with sphingomyelin-cholesterol lipidoses type II (i.e., Niemann-Pick C and D). Their foamy cells, which belong to the reticuloendothelial system, stained intensely by periodate-Schiff (PAS) reagent and were resistant to predigestion with diastase. To identify the chemical nature of the PAS-positive storage material, we applied lectin histochemistry and biochemical methods. Paraffin embedded sections, and delipidated frozen tissue sections, were treated with biotinylated lectins and localized with avidin-biotin-peroxidase complex. Araldite-embedded semithin sections were incubated with biotinylated lectins followed by avidin-gold and were enhanced with silver. By both histochemical methods the affected foamy cells stained positively as follows: Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Griffonia simplicifolia-I, Lens culinaris agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, wheat germ agglutinin (WGA), and succinylated-WGA. Biochemical analysis of liver extracts complemented the histochemical data and demonstrated accumulation of glycoproteins containing polylactosaminoglycans in affected mice. Our findings indicate that the storage material in NCTR-Balb/C mice is heterogeneous. The lipids that are extracted by organic solvents during the histologic preparations mask the occurrence of polylactosaminoglycan containing glycoproteins in native frozen sections.

Animals↗
PubMed1992

Low density lipoprotein-receptors in primary cultures of rat glial cells.

Newborn rat glial cells in primary culture contain an active cholesterol side chain cleavage cytochrome P450. Cholesterol can be supplied either by biosynthesis or derive from low density lipoproteins (LDL), which bind apolipoprotein Band E (apoB,E) (LDL)-receptors and undergo receptor-mediated endocytosis. Using antibodies to purified human plasma LDL and antibodies to bovine adreno-cortical LDL-receptor, the presence of LDL-receptors was demonstrated on rat glial cells after 3-4 weeks of primary culture, by ligand blotting, immunoblotting, and indirect immunofluorescence staining. The latter approach indicated that oligodendrocytes express higher levels of LDL-receptors than astrocytes present in the same culture. The immunofluorescence staining was observed not only at the cell surface, but also within the cytoplasm, suggesting that the LDL-receptor complexes had been internalized. Western blotting of LDL-receptors extracted from glial cells indicated a band of approximately 130 kDa, the size expected for intact receptors. Their functionality was shown by the conversion of [3H]cholesterol linoleate, incorporated into reconstituted LDL and added to the cell cultures, to [3H]pregnenolone and/or its 20 alpha-hydroxy-metabolite. This is the first characterization of functional LDL-receptors on isolated, well characterized, normal brain cells.

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↗
PubMed1992

A novel myoblast enhancer element mediates MyoD transcription.

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Animals↗
PubMed1992

Morphometric studies of pancreatic acinar granule formation in NCTR-Balb/c mice.

NCTR-Balb/c mice are afflicted with a cholesterol lysosomal storage disorder stemming from a defect in intracellular cholesterol processing. The clinical and biochemical abnormalities expressed in the mice resemble Niemann-Pick type C and D disorders in humans. One of the proposed mechanisms to explain the pathophysiology of the disorder implies a defect in the process of membrane transport that normally takes place in the vesicular movement of cholesterol to specific target sites in the cell. Secretory granule formation in pancreatic acinar cells is one of the biological processes known to involve massive membrane flow. Thus, we have undertaken a morphometric study of the regranulation mechanism in the pancreatic acinar cells of the mutant mice, as a way of studying cellular membrane movement. Electron micrographs of pancreatic acinar cells from mutant and normal mice were taken at several time points after extensive degranulation induced by pilocarpine injection. Two hours after stimulation the pancreatic cells demonstrated a complete loss of granules, and at later time points newly formed granules appeared. Identical unit granule volumes were observed in both groups, indicating that the progranules were of normal size. However, the rate of granule formation and maturation was reduced in the mutant mice, which might be the result of a defect in membrane function.

Animals↗
PubMed1992

Functional conservation of nematode and vertebrate myogenic regulatory factors.

The Caenorhabditis elegans protein, CeMyoD, is related to the vertebrate myogenic regulatory factors MyoD, myogenin, MRF-4 and Myf-5. Like its vertebrate counterparts, CeMyoD accumulates in the nucleus of striated muscle cells prior to the onset of terminal differentiation. CeMyoD also shares functional similarities with the vertebrate myogenic regulatory factors. Viral LTR driven expression of CeMyoD in mouse 10T1/2 cells can convert this cell line into myoblasts as well as efficiently trans-activate mouse muscle-specific promoters. Furthermore, mouse MyoD expression can activate a CeMyoD-beta-galactosidase reporter construct in a 10T1/2 co-transfection assay.

Amino Acid Sequence↗
PubMed1991

Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.

Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located in the viral long terminal repeat and the 5' end of all viral mRNAs. Sequences in TAR RNA can fold into a specific stem-loop structure, and certain features of the stem-loop are essential for Tat-mediated transactivation. In Xenopus oocytes, TAR sequences can inhibit the translation of 3' cis-linked mRNAs. However, coinjection of Tat and the TAR-containing RNA into oocyte nuclei relieves this translational inhibition [Braddock, M., Chambers, A., Wilson, W., Esnout, M. A., Adams, S.E. & Kingsman, S.M. (1989) Cell 58, 269-279]. We report here that the intramolecular TAR stem-loop structure is a substrate for the double-stranded RNA (dsRNA)-modifying activity, which converts adenosines to inosines. This activity is located in the nuclei of Xenopus oocytes. The specificity and extent of modification of adenosines in TAR is dependent on Tat. We propose that the dsRNA-modifying activity may be one of the cellular proteins that interacts with TAR in the nucleus. The possible role of TAR RNA modification in the expression of HIV-1 is discussed.

Adenosine↗
PubMed1991

Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.

In this report we provide four lines of evidence indicating that E12/E47-like proteins interact in vivo with the myogenic HLH proteins MyoD and myogenin. First, cotransfection of MyoD and E47 in COS cells indicates that these factors synergistically enhance transcription of a reporter gene containing an oligomerized MyoD-binding site. Second, mobility-shift assays of muscle cell nuclear extracts, "double shifted" with specific antisera, have identified complexes binding to the MEF1 site that contain either MyoD or myogenin in association with E12/E47-like proteins. Third, association with E47 alters the phosphorylation state of MyoD. Fourth, C3H10T1/2 cells expressing antisense E2A transcripts contain low levels of E2A gene products and display less terminal muscle differentiation when infected with retroviral MyoD or when challenged to differentiate with 5-azacytidine treatment. In addition we demonstrate that MyoD, in conjunction with E12/E47-like proteins, is functioning as a regulatory nodal point for activation of several other downstream muscle regulators.

Adenovirus Early Proteins↗
PubMed1991

Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X. laevis.

We have used a quantitative reverse transcription-polymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm.

Animals↗
PubMed1991

The MCK enhancer contains a p53 responsive element.

p53 is an antioncogene that is defective or absent in a large number of human tumors. Its function in normal cells is not known. We show that co-transfection of mouse p53 with muscle-specific creatine kinase-chloramphenicol acetyltransferase reporter gene, containing 3.3 kilobase of upstream control sequence for the muscle-specific creatine kinase gene, results in a 10- to 80-fold activation. The p53 responsive element maps to a region distinguished from the known MyoD binding region. Identification of a p53 responsive element should allow a more focused analysis of the effects of p53 in controlling gene activity.

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

Muscle-specific transcriptional activation by MyoD.

We focus on the mechanism by which MyoD activates transcription. Previous experiments showed that when the 13-amino-acid basic region of E12 replaced the corresponding basic region of MyoD, the resulting MyoD-E12Basic chimeric protein could bind specifically to muscle-specific enhancers in vitro and form dimers with E12, but could not activate a cotransfected reporter gene or convert 10T1/2 cells to muscle. Here we show that back mutation of this chimeric protein (with the corresponding residues in MyoD) re-establishes activation, and we identify a specific alanine involved in increasing DNA binding and a specific threonine required for activation. Using a reporter gene containing MyoD-binding sites located downstream from the transcription start site, we show that MyoD-E12Basic can bind in vivo and thereby inhibit expression of the reporter. In vivo binding is also supported by the fact that the addition of the "constitutive" VP16 activation domain to MyoD-E12Basic restores full trans-activation potential. The normal MyoD-activation domain maps within the amino-terminal 53 residues and can be functionally replaced by the activation domain of VP16. The activity of the MyoD-activation domain is dramatically elevated when deletions are made almost anywhere in the rest of the MyoD molecule, suggesting that the activation domain in MyoD is usually masked. Surprisingly, MyoD-E12Basic can activate transcription in CV1 and B78 cells (but not in 10T1/2 or 3T3 cells), suggesting that the activation function of the basic domain requires a specific factor present in CV1 and B78 cells. We propose that to function, the masked MyoD-activation domain requires the participation of a second factor that recognizes the basic region. We refer to such a factor as a recognition factor.

Amino Acid Sequence↗