PubMed Health⌕ Search

Biomedical subjects

M Nemer

Publications and source records attributed to M Nemer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Combinatorial interactions regulating cardiac transcription.

In vertebrates, heart development is a multistep process that starts with formation and patterning of the primitive heart tube and is followed by complex morphological events to give rise to the mature four-chambered heart. These various stages are characterized by distinct patterns of gene expression. Although chamber specificity and developmental regulation can be demonstrated in transgenic mice using short promoter fragments, the mechanism underlying spatial and temporal specificity within the heart remains largely unclear. Combinatorial interaction between a limited number of cardiac-specific and ubiquitous transcription factors may account for the diverse genetic inputs required to generate the complex transcriptional patterns that characterize the developing myocardium. We have used the cardiac atrial natriuretic peptide (ANP) promoter to test this hypothesis. The ANP gene is transcribed in a spatial- and temporal-specific manner in the heart, and a 500 bp promoter fragment is sufficient to recapitulate both chamber and developmental specificity. This promoter is composed of three modules, a "basal" cardiac promoter that is essential for transcription in embryonic and postnatal atrial and ventricular myocytes and two other independent modules that behave as chamber-specific enhancers. The basal cardiac promoter is the target of two cardiac-specific transcription factors, the zinc finger GATA-4 protein and the Nkx2-5 homeodomain, which bind to contiguous elements within this region. At low concentrations--a situation that likely occurs during the very first stages of cardiac cell fate determination--the two proteins synergistically activate transcription from the ANP promoter. This functional synergy requires physical interaction between the GATA-4 protein and an extended C-terminal homeodomain on Nkx2-5. This interaction, which unmasks an activation domain present just N-terminal of the homeodomain, is specific for GATA-4 and-5, but is not observed with the other cardiac GATA factor, GATA-6. Optimal synergy requires binding of both proteins to their cognate sites, although modest synergy also could be observed on heterologous promoters containing only multimerized Nkx binding sites, suggesting that Nkx2-5 is able to recruit GATA-4 into a transcriptionally active complex. The GATA/Nkx interaction, which appears to have been evolutionary conserved in nematode, fly, and mammals, provides a paradigm for analyzing transcription factor interaction during organogenesis. The data are also discussed in the context of our present knowledge of the roles of GATA and NK2 proteins in cardiac development.

Animals↗
PubMed1998

Histone deacetylase mRNA temporally and spatially regulated in its expression in sea urchin embryos.

SpHDAC1, a cDNA homolog of the yeast Rpd3 and higher eukaryotic histone deacetylases (HDAC), was cloned from the sea urchin Strongylocentrotus purpuratus. Its predicted polypeptide and the Rpd3 homologs were highly identical in two-thirds of their lengths, but diverged in their carboxyl-terminal regions in both length and sequence. SpHDAC1 transcripts, which reached maximal concentration at the blastula stages, and diminished thereafter, were neither ubiquitously expressed nor restricted to particular cell lineages, but appeared successively in distinct embryonic regions. In the blastula, transcripts were concentrated in a ring within the vegetal plate, comprising primordial endoderm, and, at the outset of gastrulation, in primordial hindgut endoderm. However, in early to mid-gastrula transcripts, they also appeared in oral ectoderm. In the late-stage gastrula, expression developed in the foregut. These shifts in spatial expression, together with an observed developmental blockage prior to sea urchin gastrulation by the histone deacetylase inhibitor trichostatin A, suggest a stepwise involvement of SpHDAC1 gene expression or SpHDAC1 functionality in the events of normal gastrulation.

Amino Acid Sequence↗
PubMed1998

GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression.

The cardiogenic homeodomain factor Nkx-2.5 and serum response factor (SRF) provide strong transcriptional coactivation of the cardiac alpha-actin (alphaCA) promoter in fibroblasts (C. Y. Chen and R. J. Schwartz, Mol. Cell. Biol. 16:6372-6384, 1996). We demonstrate here that Nkx-2.5 also cooperates with GATA-4, a dual C-4 zinc finger transcription factor expressed in early cardiac progenitor cells, to activate the alphaCA promoter and a minimal promoter, containing only multimerized Nkx-2.5 DNA binding sites (NKEs), in heterologous CV-1 fibroblasts. Transcriptional activity requires the N-terminal activation domain of Nkx-2.5 and Nkx-2.5 binding activity through its homeodomain but does not require GATA-4's activation domain. The minimal interactive regions were mapped to the homeodomain of Nkx-2.5 and the second zinc finger of GATA-4. Removal of Nkx-2.5's C-terminal inhibitory domain stimulated robust transcriptional activity, comparable to the effects of GATA-4 on wild-type Nkx-2.5, which in part facilitated Nkx-2.5 DNA binding activity. We postulate the following simple model: GATA-4 induces a conformational change in Nkx-2.5 that displaces the C-terminal inhibitory domain, thus eliciting transcriptional activation of promoters containing Nkx-2.5 DNA binding targets. Therefore, alphaCa promoter activity appears to be regulated through the combinatorial interactions of at least three cardiac tissue-enriched transcription factors, Nkx-2.5, GATA-4, and SRF.

Animals↗
PubMed1998

The C-terminal domain of c-fos is required for activation of an AP-1 site specific for jun-fos heterodimers.

The proto-oncogenes jun and fos are members of the AP-1 family of transcription factors, which activate transcription of target genes via the tetradecanoyl phorbol acetate response element (TRE). Both jun and fos contain activation domains, but their relative contributions to transcriptional activation of different TREs remain unclear. It is not apparent whether the cellular availability of specific AP-1 members is the major determinant for regulation of TREs or whether other factors including the TRE sequence itself contribute to selectivity. We have identified in the promoter of the rat atrial natriuretic factor (ANF) a novel AP-1 site which is unresponsive to jun homodimers and is inducible only in the presence of c-fos. This activation is potentiated by mitogen-activated protein (MAP) kinase. The jun proteins appear to be required solely to tether c-fos to the promoter, and c-fos mutants lacking putative activation domains abrogate transactivation. Unexpectedly, the oncogenic form of c-fos which diverges most significantly in the carboxy-terminal 50 amino acids is unable to mediate transactivation at this specialized AP-1 site. Mutations within the C terminus of c-fos at serine residues that are phosphorylation targets for growth factors and MAP kinase completely abrogate transactivation and block potentiation by MAP kinase. Using GAL4 fusions, we show that the 90-amino-acid C terminus of c-fos contains autonomous activation domains and that the serine residues are essential for full activity. These results suggest that phosphorylation of the C terminus of c-fos affects its transactivation properties and provide evidence for novel regulatory mechanisms that may contribute to biologic specificities of the AP-1 transcription complex.

Animals↗
PubMed1998

Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter.

Mammalian gonadal development and sexual differentiation are complex processes that require the coordinated expression of a specific set of genes in a strict spatiotemporal manner. Although some of these genes have been identified, the molecular pathways, including transcription factors, that are critical for the early events of lineage commitment and sexual dimorphism, remain poorly understood. GATA-4, a member of the GATA family of transcription factors, is present in the gonads and may be a regulator of gonadal gene expression. We have analyzed the ontogeny of gonadal GATA-4 expression by immunohistochemistry. GATA-4 protein was detected as early as embryonic day 11.5 in the primitive gonads of both XX and XY mouse embryos. In both sexes, GATA-4 specifically marked the developing somatic cell lineages (Sertoli in testis and granulosa in ovary) but not primordial germ cells. Interestingly, abundant GATA-4 expression was maintained in Sertoli cells throughout embryonic development but was markedly down-regulated shortly after the histological differentiation of the ovary on embryonic day 13.5. This pattern of expression suggested that GATA-4 might be involved in early gonadal development and possibly sexual dimorphism. Consistent with this hypothesis, we found that the Müllerian inhibiting substance promoter which harbors a conserved GATA element is a downstream target for GATA-4. Thus, transcription factor GATA-4 may be a new factor in the cascade of regulators that control gonadal development and sex differentiation in mammals.

Animals↗
PubMed1998

Regulation of gene expression in the endocrine heart.

Cardiac growth and contractility is profoundly altered in response to various hormones and neurotransmitters as well as by changes in electrolyte balance, blood volume, and blood pressure. In turn, the endocrine heart contributes to body homeostasis by secreting a number of biologically active peptide hormones that act on several target tissues. Atrial natriuretic peptide (ANP) and B-type natiuretic peptide (BNP) are the major secretory products of the endocrine myocardium. These peptide hormones act on many target organs via guanylate cyclase-linked membrane receptors to produce natriuresis, diuresis, vasodilatation, and hypotension. ANP and BNP receptors are found on most organs involved in cardiovascular homeostasis (e.g., kidney, adrenal, vasculature, brain). They are also present in gonads and in pituitary, where they alter steroidogenesis and pituitary hormone secretion. Not surprisingly, changes in blood pressure, volume, or hormone status influence ANP and BNP expression, which is also altered in almost all diseases that affect cardiac function. Thus, studies of ANP and BNP gene expression are relevant for many clinical settings. Moreover, transcription of the ANP and BNP genes characterizes cardiac cells at very early stages of development and is tightly linked to cardiac growth--be it proliferation, as in the fetal heart, or trophic growth, as in postnatal ventricular hypertrophy. Thus, analysis of the molecular circuitry that controls ANP and BNP expression might shed important insight into the complex regulatory pathways that underlie normal and pathologic heart development. Indeed, over the past few years, we have analysed transcriptional control of the ANP and BNP genes in embryonic and postnatal cardiomyocytes and in cardiomyocytes treated with hormones and neurotransmitters that cause cardiomyocyte hypertrophy. These studies have lead to the identification of distinct cis-acting DNA elements that modulate basal and hormone-stimulated transcription. Most importantly, the work also resulted in the isolation and characterization of cardiac-specific transcription factors that play critical roles for cardiac cell differentiation and survival.

Animals↗
PubMed1997

The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors.

The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.

Animals↗
PubMed1997

Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor.

GATA-4 is a cardiac-specific member of the GATA family of zinc finger transcription factors. During embryogenesis, GATA-4 expression is detected very early in the cardiogenic area and persists later in the developing heart. Studies have shown that GATA-4 is a potent transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program. Consistent with a role for GATA-4 in cardiomyocyte formation, inhibition of GATA-4 expression by antisense transcripts interferes with expression of cardiac muscle genes and blocks development of beating cardiomyocytes in P19 embryonic stem cells. In order to better define the function of GATA-4 in cardiogenesis, we have carried out molecular analysis of early stages of cardiomyocyte differentiation in GATA-4-deficient P19 cell lines and in P19 cells stably overexpressing GATA-4. The results indicate that GATA-4 is not required for either endodermal or mesodermal commitment or for initiation of the cardiac pathway. However, in the absence of GATA-4, differentiation is blocked at the precardiac (cardioblasts) stage and cells are lost through extensive apoptosis. In contrast, ectopic expression of GATA-4 in P19 cells accelerates cardiogenesis and markedly increases (over 10-fold) the number of terminally differentiated beating cardiomyocytes following cell aggregation. Together, these findings suggest that, in addition to its role in activation of the cardiac genetic program, GATA-4 may be the nuclear target of inductive and/or survival factors for precardiac cells.

Animals↗
PubMed1996

WEE1-like CDK tyrosine kinase mRNA level is regulated temporally and spatially in sea urchin embryos.

A cDNA from the sea urchin Strongylocentrotus purpuratus encodes a 624 amino acid polypeptide (WEE1S.purp) with a high degree of similarity to the Mik1 and Wee1 protein tyrosine kinases. These kinases act as negative regulators of mitosis by inactivating cyclin-dependent kinases (CDK). Wee1 activity varies during the cell-cycle, and is generated only when required. The pattern of WEE1S.purp mRNA expression was examined temporally and spatially in sea urchin embryos. Only a trace amount of WEE1S.purp mRNA is present in the egg and through the fifth cell cycle post-fertilization. During the next three cycles to the mid-blastula stage, its concentration rises transiently to 2.5 x 10(4) transcripts per embryo. Its developmental profile during this early period is the inverse of that reported for cyclin mRNAs, which are at a high level in the egg and through the fifth cell cycle, then decline upon further development. WEE1S.purp mRNA in the gastrula and pluteus stages becomes restricted to cells engaged in DNA replication, including the endoderm (gut), oral ectoderm, and arm rudiments. It is absent from the aboral ectoderm, which lacks cycling cells. In the pluteus larva of the species Lytechinus pictus, WEE1 mRNA was detected in the arm rudiments during cellular proliferation and arm elongation, but not after the completion of the arms. Putative regulatory motifs in the sea urchin Wee1-like cDNA suggest a capacity for rapid turnover of both its mRNA and protein: The WEE1S.purp mRNA 3' UTR contains 13 AUUUA pentamers, which have been characterized as determinants of mRNA lability; and the N-terminal domain of the predicted WEE1S.purp polypeptide is enriched in S/TP-containing, potential kinase-target sites, as well as high-value "PEST' sequences, associated with protein lability. The developmental appearance of WEE1S.purp mRNA may coincide with the introduction of a gap phase in the cell cycle. Its spatial pattern during embryogenesis appears to reflect distinct programs of regulated cell cycling in differentiating tissues.

Age Factors↗
PubMed1996

The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium.

The recently described NK2 family of homeodomain proteins are key developmental regulators. In Drosophila melanogaster, two members of this family, bagpipe and tinman, are required for visceral and cardiac mesoderm formation, respectively. In vertebrates, tinman appears to represent a family of closely related NK2 genes, including Nkx-2.5, that are expressed at an early stage in precardiac cells. Consistent with a role for Nkx-2.5 in heart development, inactivation of the Nkx-2.5 gene in mice causes severe cardiac malformations and embryonic lethality. However, little is known about the molecular action of Nkx-2.5 and its targets in cardiac muscle. In this paper, we report the identification and characterization of a functional and highly conserved Nkx-2.5 response element, termed the NKE, in the proximal region of the cardiac atrial natriuretic factor (ANF) promoter. The NKE is composed of two near-consensus NK2 binding sites that are each able to bind purified Nkx-2.5. The NKE is sufficient to confer cardiac cell-specific activity to a minimal TATA-containing promoter and is required for Nkx-2.5 activation of the ANF promoter in heterologous cells. Interestingly, in primary cardiocyte cultures, the NKE contributes to ANF promoter activity in a chamber- and developmental stage-specific manner, suggesting that Nkx-2.5 and/or other related cardiac proteins may play a role in chamber specification. This work provides the identification of a direct target for NK2 homeoproteins in the heart and lays the foundation for further molecular analyses of the role of Nkx-2.5 and other NK2 proteins in cardiac development.

Animals↗
PubMed1995

Localization of the human B-type natriuretic peptide precursor (NPPB) gene to chromosome 1p36.

Cardiac myocytes synthesize and secrete a family of peptide hormones with potent natriuretic, diuretic, and vasodilatory properties. These peptides are derived from precursor molecules that are encoded by two different genes, the atrial natriuretic peptide precursor A (NPPA) and the B-type natriuretic peptide or natriuretic peptide precursor B (NPPB). A human genomic clone for the NPPB gene was used to determine the chromosomal location of the NPPB gene. Analysis of Southern blot hybridization to DNAs from various somatic cell hybrids and fluorescence in situ hybridization allowed assignment of the NPPB locus to human chromosome 1p36. This location coincided with that of the NPPA locus; pulsed-field gel electrophoresis placed NPPA and NPPB within 50 kb of each other. This close chromosomal linkage, together with the conserved primary sequences and structural organization of the two natriuretic peptide precursor genes, suggests that the natriuretic peptide loci may have evolved from a common ancestor gene.

Animals↗
PubMed1995

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1.

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5'-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.

Actins↗
PubMed1995

Inhibition of transcription factor GATA-4 expression blocks in vitro cardiac muscle differentiation.

Commitment of mesodermal cells to the cardiac lineage is a very early event that occurs during gastrulation, and differentiation of cardiac muscle cells begins in the presomite stage prior to formation of the beating heart tube. However, the molecular events, including gene products that are required for differentiation of cardiac muscle cells, remain essentially unknown. GATA-4 is a recently characterized cardiac muscle-restricted transcription factor whose properties suggest an important regulatory role in heart development. We tested the role of GATA-4 in cardiac differentiation, using the pluripotent P19 embryonal carcinoma cells, which can be differentiated into beating cardiac muscle cells. In this system, GATA-4 transcripts and protein are restricted to cells committed to the cardiac lineage, and induction of GATA-4 precedes expression of cardiac marker genes and appearance of beating cells. Inhibition of GATA-4 expression by antisense transcripts blocks development of beating cardiac muscle cells and interferes with expression of cardiac muscle markers. These data indicate that GATA-4 is necessary for development of cardiac muscle cells and identify for the first time a tissue-specific transcription factor that may be crucial for early steps of mammalian cardiogenesis.

Antisense Elements (Genetics)↗
PubMed1994

Modulation of gene expression by calreticulin binding to the glucocorticoid receptor.

Calreticulin is a multifunctional protein that acts as a major Ca(2+)-binding (storage) protein in the lumen of the endoplasmic reticulum. It is also found in the nucleus, suggesting that it may have a role in transcription regulation. Calreticulin has been reported to bind to the synthetic peptide KLGFFKR, which is almost identical to an amino-acid sequence in the DNA-binding domain of the superfamily of nuclear receptors. Could calreticulin interact with the DNA-binding domain of these receptors and affect their function? Here we report that the amino terminus of calreticulin interacts with the DNA-binding domain of the glucocorticoid receptor and prevents the receptor from binding to its specific glucocorticoid response element. Overexpression of calreticulin in mouse L fibroblasts inhibits glucocorticoid-response-mediated transcriptional activation of a glucocorticoid-sensitive reporter gene and of the endogenous, glucocorticoid-sensitive gene encoding cytochrome P450. Together these results indicate that calreticulin may be important in gene transcription, regulating the glucocorticoid receptor and perhaps other members of the super-family of nuclear receptors.

Amino Acid Sequence↗
PubMed1994

Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells.

Cardiac myocytes undergo a major genetic switch within the first week of postnatal development, when cell division ceases terminally and many cardiac genes are either activated or silenced. We have developed stage-specific cardiocyte cultures to analyze transcriptional control of the rat atrial natriuretic factor (ANF) gene to identify the mechanisms underlying tissue-specific and developmental regulation of this gene in the heart. The first 700 bp of ANF flanking sequences was sufficient for cardiac muscle- and stage-specific expression in both atrial and ventricular myocytes, and a cardiac muscle-specific enhancer was localized between -136 and -700 bp. Deletion of this enhancer markedly reduced promoter activity in cardiac myocytes and derepressed ANF promoter activity in nonexpressing cells. Two distinct domains of the enhancer appeared to contribute differentially to cardiac specificity depending on the differentiation stage of the myocytes. DNase I footprinting of the enhancer domain active in differentiated cells revealed four putative regulatory elements including an A+T-rich region and a CArG element. Deletion mutagenesis and promoter reconstitution assays revealed an important role for the CArG-containing element exclusively in cardiac cells, where its activity was switched on in differentiated myocytes. Transcriptional activity of the ANF-CArG box correlated with the presence of a cardiac- and stage-specific DNA-binding complex which was not recognized by the c-fos serum response element. Thus, the use of this in vitro model system representing stage-specific cardiac development unraveled the presence of different regulatory mechanisms for transcription of the ANF gene during cardiac differentiation and may be useful for studying the regulatory pathways of other genes that undergo switching during cardiac myogenesis.

Animals↗