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1.
Cell Death Differ ; 21(12): 1950-60, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25146928

ABSTRACT

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass, which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos, as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor, small interfering RNAs, or a dominant-negative approach suppresses Nanog expression, and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1, aka Smarca4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4)), a key regulator of ESC self-renewal and pluripotency, in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene, providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.


Subject(s)
Blastocyst/metabolism , Embryonic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Blastocyst/cytology , Cell Differentiation , DNA Helicases/metabolism , Female , Gene Expression , Gene Expression Regulation, Developmental , HEK293 Cells , Homeodomain Proteins/genetics , Humans , Male , Mice, Inbred C57BL , Nanog Homeobox Protein , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolism
2.
Dev Dyn ; 222(4): 667-80, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11748835

ABSTRACT

The proximal-distal patterning of lung epithelium involves a complex series of signaling and transcriptional events resulting in the programmed differentiation of highly specialized cells for gas exchange and surfactant protein expression essential for postnatal lung function. The BMP signaling pathway has been shown to regulate cellular differentiation in the lung as well as other tissues. In this report, we show that the can family of related BMP antagonists, including gremlin, cer-1, PRDC, and Dan are expressed in the lung during embryonic development with gremlin expression observed in the proximal airway epithelium. The role of gremlin in lung development was explored by overexpressing it in the distal lung epithelium of transgenic mice using the human SP-C promoter. SP-C/gremlin transgenic mice exhibited a disruption of the proximal-distal patterning found in the airways of the mammalian lung. Expanded expression of the proximal epithelial cell markers CC10 and HFH-4 (Foxj1) was observed in the distal regions of transgenic lungs. Furthermore, smooth muscle alpha-actin expression was observed surrounding the distal airways of SP-C/gremlin mice, indicating a proximalization of distal lung tubules. These data suggest that gremlin plays an important role in lung morphogenesis by regulating the proximal-distal patterning of the lung during development.


Subject(s)
Body Patterning/physiology , Bone Morphogenetic Proteins/antagonists & inhibitors , Intercellular Signaling Peptides and Proteins , Lung/embryology , Proteins/physiology , Animals , Cytokines , Embryo, Mammalian/metabolism , Embryo, Mammalian/physiology , Embryonic and Fetal Development , Humans , Mice/embryology , Mice, Transgenic/genetics , Muscle, Smooth/embryology , Proteins/genetics , Proteins/metabolism , Proteolipids/genetics , Pulmonary Surfactants/genetics , Pulmonary Surfactants/metabolism , Transcription, Genetic/physiology
3.
Mech Dev ; 107(1-2): 69-82, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11520664

ABSTRACT

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. We show that expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, we find that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis.


Subject(s)
Hair Follicle/metabolism , Nerve Tissue Proteins , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Animals , Embryonic Induction , Gene Expression Profiling , Gene Expression Regulation, Developmental , Hair Follicle/cytology , Hair Follicle/embryology , Hair Follicle/growth & development , Hedgehog Proteins , In Situ Hybridization , Mice , Morphogenesis , Mutation , Proto-Oncogene Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Skin/embryology , Skin/metabolism , Trans-Activators/metabolism , Up-Regulation , Wnt Proteins , Wnt-5a Protein
4.
J Biol Chem ; 276(29): 27488-97, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11358962

ABSTRACT

Epithelial gene expression in the lung is thought to be regulated by the coordinate activity of several different families of transcription factors including the Fox family of winged-helix/forkhead DNA-binding proteins. In this report, we have identified and characterized two members of this Fox gene family, Foxp1 and Foxp2, and show that they comprise a new subfamily of Fox genes expressed in the lung. Foxp1 and Foxp2 are expressed at high levels in the lung as early as E12.5 of mouse development with Foxp2 expression restricted to the airway epithelium. In addition, Foxp1 and Foxp2 are expressed at lower levels in neural, intestinal, and cardiovascular tissues during development. Upon differentiation of the airway epithelium along the proximal-distal axis, Foxp2 expression becomes restricted to the distal alveolar epithelium whereas Foxp1 expression is observed in the distal epithelium and mesenchyme. Foxp1 and Foxp2 can regulate epithelial lung gene transcription as was demonstrated by their ability to dramatically repress the mouse CC10 promoter and, to a lesser extent, the human surfactant protein C promoter. In addition, GAL4 fusion proteins encoding subdomains of Foxp1 and Foxp2 demonstrate that an independent and homologous transcriptional repression domain lies within the N-terminal end of the proteins. Together, these studies suggest that Foxp1 and Foxp2 are important regulators of lung epithelial gene transcription.


Subject(s)
Gene Expression Regulation, Developmental , Lung/metabolism , Repressor Proteins/genetics , Repressor Proteins/physiology , Transcription Factors , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Epithelium/metabolism , Forkhead Transcription Factors , Lung/embryology , Mice , Molecular Sequence Data , Repressor Proteins/chemistry , Repressor Proteins/pharmacology , Sequence Homology, Amino Acid
6.
J Biol Chem ; 275(26): 19949-54, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10779506

ABSTRACT

Gene targeting studies have demonstrated that the zinc finger transcription factor GATA-6 lies upstream in a transcriptional cascade that controls differentiation of the visceral endoderm. To understand the function of GATA-6 in the visceral endoderm and to identify genes regulated by GATA-6 in this tissue, subtractive hybridization was performed using template cDNAs derived from differentiated wild-type embryonic stem (ES) cells and GATA-6(-/-) ES cells, respectively. These analyses revealed that the gene encoding Dab2, a mitogen-responsive phosphoprotein, is differentially expressed in wild-type and GATA-6-deficient ES cells. Consistent with these findings, Dab2 is expressed in the visceral endoderm of wild-type embryos but not in the visceral endoderm of GATA-6-deficient embryos. Cotransfection experiments demonstrate that the human Dab2 promoter can be transactivated by forced expression of GATA-6 in NIH-3T3 cells. In contrast, forced expression of GATA-4 does not transactivate the human Dab2 promoter and Dab2 is expressed in the visceral endoderm of GATA-4 null embryos. Surprisingly, the specificity of GATA-6-induced transactivation of the Dab2 promoter is not mediated through its zinc finger DNA-binding domain. Taken together, these data demonstrate that the mitogen-responsive phosphoprotein Dab2 is a downstream target of GATA-6 in the visceral endoderm. Moreover, these data demonstrate that molecular mechanisms have evolved that direct, and distinguish, the functional specificity of GATA family members when they are developmentally coexpressed.


Subject(s)
Adaptor Proteins, Vesicular Transport , DNA-Binding Proteins/metabolism , Endoderm/metabolism , Gene Expression Regulation, Developmental , Phosphoproteins/metabolism , Proteins , Transcription Factors/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing , Animals , Apoptosis Regulatory Proteins , Base Sequence , Cell Differentiation , DNA, Complementary/metabolism , DNA-Binding Proteins/physiology , GATA4 Transcription Factor , GATA6 Transcription Factor , Genes, Tumor Suppressor , Humans , In Situ Hybridization , Mice , Molecular Sequence Data , Phosphoproteins/genetics , Plasmids , Promoter Regions, Genetic , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Stem Cells/cytology , Transcription Factors/physiology , Transcription, Genetic , Transcriptional Activation , Tumor Suppressor Proteins
7.
J Biol Chem ; 275(2): 1043-9, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10625644

ABSTRACT

Surfactant protein A (SP-A) is a member of the collectin family of innate host defense molecules expressed primarily in respiratory epithelial cells of the lung. SP-A concentrations are influenced by both cell-specific and ubiquitous nuclear proteins that regulate SP-A gene transcription in a cell-selective and temporally regulated manner. In this work, a consensus GATA-binding site (GBS) was identified at positions -69 to -64 of the mouse SP-A gene. The transcriptional activity of wild-type SP-A reporter constructs in HeLa cells was increased 5-10-fold when cotransfected with a GATA-6 expression plasmid. Deletion of the GBS completely blocked transactivation by GATA-6. Transfection of a construct expressing GATA-6-engrailed fusion protein inhibited basal expression of the SP-A/chloramphenicol acetyltransferase construct in MLE-15 cells. Nuclear extract proteins from MLE-15 cells bound to the GBS in the mouse SP-A gene, and a supershifted band was detected with a GATA-6-specific antibody. Transactivation of the wild-type SP-A constructs by GATA-6 increased transcriptional activity 7-10-fold, whereas thyroid transcription factor-1 (TTF-1) increased the activity of these constructs 12-18-fold. The effects of cotransactivating with both GATA-6 and TTF-1 expression constructs were additive. However, mutation of the TTF-1-binding sites alone or in combination decreased GATA-6 transactivation. Likewise, mutation of the GBS blocked TTF-1 activation of the SP-A promoter. In situ hybridization demonstrated GATA-6 mRNA in the peripheral epithelial cells of fetal mouse lung, consistent with the sites of SP-A expression. GATA-6 is expressed in respiratory epithelial cells and binds to a cis-acting element in the SP-A gene promoter, activating the transcriptional activity of the gene.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Proteolipids/genetics , Pulmonary Surfactants/genetics , Transcription Factors/metabolism , Transcription, Genetic , Animals , Base Sequence , Cell Line , Cell Nucleus/metabolism , Chloramphenicol O-Acetyltransferase/genetics , Conserved Sequence , GATA6 Transcription Factor , Glycoproteins/genetics , HeLa Cells , Humans , Lung/embryology , Lung/metabolism , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins/metabolism , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Proteins , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Respiratory Mucosa/metabolism , Sequence Alignment , Sequence Homology, Nucleic Acid , Thyroid Nuclear Factor 1 , Transcriptional Activation , Transfection , Zinc Fingers
8.
Genes Dev ; 12(22): 3579-90, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9832509

ABSTRACT

GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient (GATA6(-/-)) ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6(-/-) ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, alpha-fetoprotein (AFP), and HNF3beta. Homozygous GATA6(-/-) mice died between embryonic day (E) 6.5 and E7. 5 and exhibited a specific defect in endoderm differentiation including severely down-regulated expression of GATA4 and absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activated the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development, GATA6(-/-)-C57BL/6 chimeric mice were generated. lacZ-tagged GATA6(-/-) ES cells contributed to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium.


Subject(s)
Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Embryonic and Fetal Development/genetics , Endoderm/cytology , Gene Expression Regulation, Developmental/genetics , Phosphoproteins/genetics , Transcription Factors/genetics , Viscera/growth & development , 3T3 Cells , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Embryo, Mammalian/pathology , GATA6 Transcription Factor , Gene Targeting , Genotype , Hepatocyte Nuclear Factor 4 , Histocytochemistry , In Situ Hybridization , Lung/embryology , Mice , Mice, Knockout , Microscopy, Electron , RNA, Messenger/genetics , Transcriptional Activation/genetics
9.
Genes Dev ; 11(8): 1048-60, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9136932

ABSTRACT

Previous studies have suggested that the GATA4 transcription factor plays an important role in regulating mammalian cardiac development. In the studies described in this report we have used gene targeting to produce GATA4-deficient mice. Homozygous GATA4-deficient (GATA4-/-) mice died between 8.5 and 10.5 days post coitum (dpc). GATA4-/- embryos displayed severe defects in both rostral-to-caudal and lateral-to-ventral folding, which were reflected in a generalized disruption of the ventral body pattern. This resulted in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube. Analysis of cardiac development in the GATA4-/- mice demonstrated that these embryos developed splanchnic mesoderm, which differentiated into primitive cardiac myocytes that expressed contractile proteins. However, consistent with the observed defect in ventral morphogenesis, these GATA4-/- procardiomyocytes failed to migrate to the ventral midline to form a linear heart tube and instead formed aberrant cardiac structures in the anterior and dorsolateral regions of the embryo. The defect in ventral migration of the GATA4-/- procardiomyocytes was not cell intrinsic because GATA4-/- cardiac myocytes and endocardial cells populated the hearts of GATA4-/- -C57BL/6 chimeric mice. Taken together, these results demonstrated that GATA4 is not essential for the specification of the cardiac cell lineages. However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis.


Subject(s)
Body Patterning , DNA-Binding Proteins/physiology , Heart/embryology , Transcription Factors/physiology , Animals , Cell Differentiation , Chimera , Crosses, Genetic , DNA-Binding Proteins/genetics , Digestive System/embryology , Embryonic and Fetal Development , Female , GATA4 Transcription Factor , GATA6 Transcription Factor , Gene Expression Regulation, Developmental , Male , Mesoderm , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Morphogenesis , Myocardium/chemistry , Myocardium/cytology , RNA, Messenger/analysis , Transcription Factors/genetics , Viscera/chemistry , Viscera/embryology , Yolk Sac/chemistry , Yolk Sac/embryology
10.
Dev Biol ; 183(1): 21-36, 1997 Mar 01.
Article in English | MEDLINE | ID: mdl-9119112

ABSTRACT

Members of the GATA family of zinc finger transcription factors regulate critical steps of cellular differentiation during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the murine GATA-5 cDNA and protein and defined the temporal and spatial pattern of GATA-5 gene expression during mammalian development. The amino terminus of the mouse GATA-5 protein shares high level amino acid sequence identity with the murine GATA-4 and -6 proteins, but not with other members of the GATA family. GATA-5 binds to the functionally important CEF-1 nuclear protein binding site in the cardiac-specific slow/cardiac troponin C (cTnC) transcriptional enhancer and overexpression of GATA-5 transactivates the cTnC enhancer in noncardiac muscle cell lines. During embryonic and postnatal development, the pattern of GATA-5 gene expression differs significantly from that of other GATA family members. In the primitive streak embryo, GATA-5 mRNA is detectable in the precardiac mesoderm. Within the embryonic heart, the GATA-5 gene is expressed within the atrial and ventricular chambers (ED 9.5), becomes restricted to the atrial endocardium (ED 12.5), and is subsequently not expressed in the heart during late fetal and postnatal development. Moreover, coincident with the earliest steps in lung development, only the GATA-5 gene is expressed within the pulmonary mesenchyme. Finally, the GATA-5 gene is expressed in tissue-restricted subsets of smooth muscle cells (SMCs), including bronchial SMCs and SMCs in the bladder wall. These data are consistent with a model in which GATA-5 performs a unique temporally and spatially restricted function in the embryonic heart and lung. Moreover, these data suggest that GATA-5 may play an important role in the transcriptional program(s) that underlies smooth muscle cell diversity.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental/physiology , Trans-Activators/genetics , Transcription Factors/genetics , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cloning, Molecular , DNA, Complementary , Embryonic and Fetal Development , GATA5 Transcription Factor , Gastrula/chemistry , Heart/embryology , Lung/embryology , Mesoderm/chemistry , Mice , Molecular Sequence Data , Muscle, Smooth/chemistry , Muscle, Smooth/cytology , Organ Specificity , RNA, Messenger/analysis , Sequence Homology, Amino Acid
11.
J Biol Chem ; 272(13): 8515-24, 1997 Mar 28.
Article in English | MEDLINE | ID: mdl-9079680

ABSTRACT

GATA-4 is one of the earliest developmental markers of the precardiac mesoderm, heart, and gut and has been shown to activate regulatory elements controlling transcription of genes encoding cardiac-specific proteins. To elucidate the molecular mechanisms underlying the transcriptional activity of the GATA-4 protein, structure-function analyses were performed. These analyses revealed that the C-terminal zinc finger and adjacent basic domain of GATA-4 is bifunctional, modulating both DNA-binding and nuclear localization activities. The N terminus of the protein encodes two independent transcriptional Activation Domains (amino acids 1-74 and amino acids 130-177). Amino acid residues were identified within each domain that are required for transcriptional activation. Finally, we have shown that regions of Xenopus GATA-5 and -6 corresponding to Activation Domains I and II, respectively, function as potent transcriptional activators. The identification and functional characterization of two evolutionarily conserved transcriptional Activation Domains within the GATA-4/5/6 subfamily suggests that each of these domains modulates critical functions in the transcriptional regulatory program(s) encoded by GATA-4, -5, and -6 during vertebrate development. As such these data provide novel insights into the molecular mechanisms that control development of the heart.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic , Zinc Fingers , 3T3 Cells , Amino Acid Sequence , Animals , COS Cells , Conserved Sequence , DNA/metabolism , GATA4 Transcription Factor , GATA5 Transcription Factor , GATA6 Transcription Factor , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Alignment , Sequence Deletion , Structure-Activity Relationship , Troponin C/genetics
12.
Dev Biol ; 177(1): 309-22, 1996 Jul 10.
Article in English | MEDLINE | ID: mdl-8660897

ABSTRACT

Members of the GATA family of zinc finger transcription factors play important roles in the development of several mesodermally derived cell lineages. In the studies described in this report, we have isolated and functionally characterized the murine GATA-6 cDNA and protein and defined the temporal and spatial patterns of GATA-6 gene expression during mammalian development. The GATA-6 and -4 proteins share high-level amino acid sequence identity over a proline-rich region at the amino terminus of the protein that is not conserved in other GATA family members. GATA-6 binds to a functionally important nuclear protein binding site within the cardiac-specific cardiac troponin C (cTnC) transcriptional enhancer. Moreover, the cTnC promoter enhancer can be transactivated by overexpression of GATA-6 in noncardiac muscle cells. During early murine embryonic development, the patterns of GATA-6 and -4 gene expression are similar, with expression of GATA-6 restricted to the precardiac mesoderm, the embryonic heart tube, and the primitive gut. However, coincident with the onset of vasculogenesis and development of the respiratory and urogenital tracts, only the GATA-6 gene is expressed in arterial smooth muscle cells, the developing bronchi, and the urogenital ridge and bladder. These data are consistent with a model in which GATA-6 functions in concert with GATA-4 to direct tissue-specific gene expression during formation of the mammalian heart and gastrointestinal tract, but performs a unique function in programming lineage-restricted gene expression in the arterial system, the bladder, and the embryonic lung.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Mesoderm/physiology , Transcription Factors/genetics , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cardiovascular System/embryology , Cell Lineage/genetics , Clone Cells , DNA, Complementary/isolation & purification , Digestive System/embryology , GATA6 Transcription Factor , Lung/embryology , Mice , Molecular Sequence Data , Muscle, Skeletal/embryology , Muscle, Smooth, Vascular/embryology , RNA/analysis , Rats , Troponin/genetics , Troponin C , Urogenital System/embryology
13.
J Biol Chem ; 271(1): 395-403, 1996 Jan 05.
Article in English | MEDLINE | ID: mdl-8550594

ABSTRACT

Calponin-h1 is a 34-kDa myofibrillar thin filament, actin-binding protein that is expressed exclusively in smooth muscle cells (SMCs) in adult animals. To examine the molecular mechanisms that regulate SMC-specific gene expression, we have examined the temporal, spatial, and cell cycle-regulated patterns of expression of calponin-h1 gene expression and isolated and structurally characterized the murine calponin-h1 gene. Calponin-h1 mRNA is expressed exclusively in SMC-containing tissues in adult animals. During murine embryonic development, calponin-h1 gene expression is (i) detectable in E9.5 embryos in the dorsal aorta, cardiac outflow tract, and tubular heart, (ii) sequentially up-regulated in SMC-containing tissues, and (iii) down-regulated to non-detectable levels in the heart during late fetal development. In addition, the gene is expressed in resting rat aortic SMCs, but its expression is rapidly down-regulated when growth-arrested cells reenter phase G1 of the cell cycle and proliferate. Calponin-h1 is encoded by a 10.7-kilobase single copy gene composed of seven exons, which is part of a multigene family. Transient transfection analyses demonstrated that 1.5 kilobases of calponin-h1 5'-flanking sequence is sufficient to program high level transcription of a luciferase reporter gene in cultured primary rat aortic SMCs and the smooth muscle cell line, A7r5. Taken together, these data suggest that the calponin-h1 gene will serve as an excellent model system with which to examine the molecular mechanisms that regulate SMC lineage specification, differentiation, and phenotypic modulation.


Subject(s)
Calcium-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Muscle, Smooth, Vascular/metabolism , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Calcium-Binding Proteins/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary , Humans , Mice , Microfilament Proteins , Molecular Sequence Data , Multigene Family , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/physiology , Phenotype , Promoter Regions, Genetic , Rats , Sequence Homology, Amino Acid , Transcription, Genetic , Calponins
14.
J Biol Chem ; 270(22): 13460-9, 1995 Jun 02.
Article in English | MEDLINE | ID: mdl-7768949

ABSTRACT

SM22 alpha is expressed exclusively in smooth muscle-containing tissues of adult animals and is one of the earliest markers of differentiated smooth muscle cells (SMCs). To examine the molecular mechanisms that regulate SMC-specific gene expression, we have isolated and structurally characterized the murine SM22 alpha gene. SM22 alpha is a 6.2-kilobase single copy gene composed of five exons. SM22 alpha mRNA is expressed at high levels in the aorta, uterus, lung, and intestine, and in primary cultures of rat aortic SMCs, and the SMC line, A7r5. In contrast to genes encoding SMC contractile proteins, SM22 alpha gene expression is not decreased in proliferating SMCs. Transient transfection experiments demonstrated that 441 base pairs of SM22 alpha 5'-flanking sequence was necessary and sufficient to program high level transcription of a luciferase reporter gene in both primary rat aortic SMCs and A7r5 cells. DNA sequence analyses revealed that the 441-base pair promoter contains two CArG/SRF boxes, a CACC box, and one potential MEF-2 binding site, cis-acting elements which are each important regulators of striated muscle transcription. Taken together, these studies have identified the murine SM22 alpha promoter as an excellent model system for studies of developmentally regulated, lineage-specific gene expression in SMCs.


Subject(s)
Microfilament Proteins , Muscle Proteins/genetics , Muscle, Smooth, Vascular/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Cycle/genetics , Cells, Cultured , Cloning, Molecular , DNA, Complementary , Mice , Molecular Sequence Data , Muscle, Smooth, Vascular/cytology , Promoter Regions, Genetic , Rats
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