An HF-1a/HF-1b/MEF-2 combinatorial element confers cardiac ventricular specificity and established an anterior-posterior gradient of expression

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1799-1809 ◽  
Author(s):  
R.S. Ross ◽  
S. Navankasattusas ◽  
R.P. Harvey ◽  
K.R. Chien
Keyword(s):  
Gene Expression ◽  
Light Chain ◽  
Base Pair ◽  
Myosin Light Chain ◽  
Right Atrial ◽  
Specific Gene ◽  
Heart Tube ◽  
Endogenous Gene ◽  
Myosin Light Chain 2 ◽  
Anterior Posterior

The molecular determinants that direct gene expression to the ventricles of the heart are for the most part unknown. Additionally, little data is available on how the anterior/posterior axis of the heart tube is determined and whether the left and right atrial and ventricular chambers are assigned as part of this process. Utilizing myosin light chain-2 ventricular promoter/beta-galactosidase reporter transgenes, we have determined the minimal cis-acting sequences required for ventricular-specific gene expression. In multiple independent transgenic mouse lines, we found that both a 250 base pair myosin light chain-2 ventricular promoter fragment, as well as a dimerized 28 bp sub-element (HF-1) containing binding sites for HF1a and HF1b/MEF2 factors, directed ventricular-specific reporter expression from as early as the endogenous gene, at day 7.5-8.0 post coitum. While the endogenous gene is expressed uniformly throughout both ventricles, the transgenes were expressed in a right ventricular/conotruncal dominant fashion, suggesting that they contain only a subset of the elements which respond to positional information in the developing heart tube. Expression of the transgene was cell autonomous and its temporospatial characteristics not affected by mouse strain/methylation state of the genome. To determine whether ventricular-specific expression of the transgene was dependent upon regulatory genes required for correct ventricular differentiation, the 250 base pair transgene was bred into both retinoid X receptoralpha and Nkx2-5 null backgrounds. The transgene was expressed in both mutant backgrounds, despite the absence of endogenous myosin light chain-2 ventricular transcript in Nkx2-5 null embryos. Ventricular specification, as judged by transgene expression, appeared to occur normally in both mutants. Thus, the HF-1 element, directs chamber-specific transcription of a transgene reporter independently of retinoid X receptoralpha and Nkx2-5, and defines a minimal combinatorial pathway for ventricular chamber gene expression. The patterned expression of this transgene may provide a model system in which to investigate the cues that dictate anterior-posterior (right ventricle/left ventricle) gradients during mammalian heart development.

scholarly journals Expression of the rat myosin light-chain 2 gene in transgenic mice: stage specificity, developmental regulation, and interrelation with the endogenous gene.

1988 ◽  
Vol 8 (2) ◽  
pp. 1006-1009 ◽  
Author(s):  
M Shani ◽  
I Dekel ◽  
O Yoffe
Keyword(s):  
Transgenic Mice ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Developmental Regulation ◽  
Endogenous Gene ◽  
Temporal Regulation ◽  
Stage Specificity ◽  
Myosin Light Chain 2 ◽  
Skeletal Myosin ◽  
Transgenic Strains

The expression of the rat skeletal myosin light-chain 2 gene in two transgenic strains was tissue specific and stage specific. However, the temporal regulation during development of the transgene was different from that of the endogenous gene. Surprisingly, in one strain, the expression of the transgene was associated with a significant down-regulation of the endogenous gene. The possible mechanisms to account for the suppression of the endogenous gene and the potential implications of this suppression are discussed.

CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway

Development ◽  
1997 ◽  
Vol 124 (4) ◽  
pp. 793-804 ◽  
Author(s):  
Y. Zou ◽  
S. Evans ◽  
J. Chen ◽  
H.C. Kuo ◽  
R.P. Harvey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Myocytes ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Homeobox Gene ◽  
Ankyrin Repeat ◽  
Repeat Protein ◽  
Ankyrin Repeat Protein ◽  
Myosin Light Chain 2 ◽  
Cardiac Ankyrin Repeat Protein

To identify the molecular pathways that guide cardiac ventricular chamber specification, maturation and morphogenesis, we have sought to characterize factors that regulate the expression of the ventricular myosin light chain-2 gene, one of the earliest markers of ventricular regionalization during mammalian cardiogenesis. Previously, our laboratory identified a 28 bp HF-la/MEF-2 element in the MLC-2v promoter region, which confers cardiac ventricular chamber-specific gene expression during murine cardiogenesis, and showed that the ubiquitous transcription factor YB-1 binds to the HF-la site in conjunction with a co-factor. In a search for interacting co-factors, a nuclear ankyrin-like repeat protein CARP (cardiac ankyrin repeat protein) was isolated from a rat neonatal heart cDNA library by yeast two-hybrid screening, using YB-1 as the bait. Co-immunoprecipitation and GST-CARP pulldown studies reveal that CARP forms a physical complex with YB-1 in cardiac myocytes and immunostaining shows that endogenous CARP is localized in the cardiac myocyte nucleus. Co-transfection assays indicate that CARP can negatively regulate an HF-1-TK minimal promoter in an HF-1 sequence-dependent manner in cardiac myocytes, and CARP displays a transcriptional inhibitory activity when fused to a GAL4 DNA-binding domain in both cardiac and noncardiac cell context. Northern analysis revealed that carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expression was first clearly detected as early as E8.5 specifically in heart and is regulated temporally and spatially in the myocardium. Nkx2-5, the murine homologue of Drosophila gene tinman was previously shown to be required for heart tube looping morphogenesis and ventricular chamber-specific myosin light chain-2 expression during mammalian heart development. In Nkx2-5(−/−)embryos, carp expression was found to be significantly and selectively reduced as assessed by both whole-mount in situ hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network. Co-transfection assays using a dominant negative mutant Nkx2-5 construct with CARP promoter-luciferase reporter constructs in cardiac myocytes confirms that Nkx2-5 either directly or indirectly regulates carp at the transcriptional level. Finally, a carp promoter-lacZ transgene, which displays cardiac-specific expression in wild-type and Nkx2-5(+/−) background, was also significantly reduced in Nkx2-5(−/−) embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes. Thus, CARP is a YB-1 associated factor and represents the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway and may serve as a negative regulator of HF-1-dependent pathways for ventricular muscle gene expression.

scholarly journals The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression.

1991 ◽  
Vol 11 (7) ◽  
pp. 3735-3744 ◽  
Author(s):  
H Ernst ◽  
K Walsh ◽  
C A Harrison ◽  
N Rosenthal
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Light Chain ◽  
Promoter Activity ◽  
Myosin Light Chain ◽  
Specific Gene ◽  
Sequence Motif ◽  
Factor Binding ◽  
Actin Promoter ◽  
Alpha Actin

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.

The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression

1991 ◽  
Vol 11 (7) ◽  
pp. 3735-3744
Author(s):  
H Ernst ◽  
K Walsh ◽  
C A Harrison ◽  
N Rosenthal
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Light Chain ◽  
Promoter Activity ◽  
Myosin Light Chain ◽  
Specific Gene ◽  
Sequence Motif ◽  
Factor Binding ◽  
Actin Promoter ◽  
Alpha Actin

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.

scholarly journals Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element.

1991 ◽  
Vol 11 (3) ◽  
pp. 1676-1685 ◽  
Author(s):  
R A Shen ◽  
S K Goswami ◽  
E Mascareno ◽  
A Kumar ◽  
M A Siddiqui
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Tissue Specific ◽  
Myosin Light Chain 2 ◽  
Functional Components

Physiological expression of the cardiac muscle myosin light-chain 2 (MLC-2) gene in chickens is restricted to cardiac muscle tissue only, at least during the late embryonic to adult stages of development. The mechanism by which cardiac MLC-2 gene expression is repressed in differentiated noncardiac muscle tissues is unknown. Using sequential 5'-deletion mutants of the cardiac MLC-2 promoter introduced into primary skeletal muscle cells in culture, we have demonstrated that a 89-bp region, designated the cardiac-specific sequence (CSS), is essential for repression of cardiac MLC-2 expression in skeletal muscle. Removal of the CSS sequence alone allows transcription in skeletal muscle cells without affecting the transcriptional activity of the promoter in cardiac muscle cells. DNase I footprinting and gel shift assays indicate that protein binding to sequences in the CSS domain occurs readily in nuclear extracts obtained from skeletal muscle but not in extracts isolated under identical conditions from cardiac muscle. Thus, it appears that a negative regulatory mechanism accounts for the lack of expression of the cardiac MLC-2 gene in skeletal muscle and that the CSS element and its binding proteins are important functional components of the regulatory apparatus which ensures the developmental program for cardiac tissue-specific gene expression.

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