scholarly journals A single transcription factor binds to two divergent sequence elements with a common function in cardiac myosin light chain-2 promoter.

1992 ◽  
Vol 12 (3) ◽  
pp. 1107-1116 ◽  
Author(s):  
P Qasba ◽  
E Lin ◽  
M D Zhou ◽  
A Kumar ◽  
M A Siddiqui
Keyword(s):  
Transcription Factor ◽  
Gene Transcription ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Cardiac Myosin ◽  
Divergent Sequence ◽  
Specific Element ◽  
Myosin Light Chain 2 ◽  
Sequence Elements ◽  
Gel Shift

The cardiac myosin light chain-2 (MLC-2) gene promoter contains several positive and negative cis-acting sequences that are involved in the regulation of its expression. We describe here the properties of two activator sequences, elements A and P, and their DNA-binding factors (ABFs). Element A (CCAAAAGTGG), located at -61, has homology with the evolutionarily conserved sequence CC(A/T)6GG, present in the genes of many contractile proteins. Element P (TAACCTTGAAAGC), located 114 bp upstream of element A, is conserved in both chicken and rat cardiac MLC-2 gene promoters. Deletion mutagenesis demonstrated that these two elements are involved in the positive regulation of MLC-2 gene transcription. At least two sequence-specific element A-binding proteins, ABF-1 and ABF-2, were identified by gel shift analysis of the fractionated cardiac nuclear proteins. ABF-1 binds to element A with strict dependence on the internal element A sequence AAAAGT. In contrast, ABF-2 exhibits a relaxed sequence requirement, as it recognizes the consensus CArG and CCAAT box sequences as well. ABF-2 also recognizes the distal element P despite the fact that the sequences of elements A and P are divergent. DNase I footprinting, methylation interference, and gel shift analyses demonstrated unequivocally that the element A-DNA affinity-purified protein ABF-2 binds to element P with sequence specificity. Since both elements A and P play a positive regulatory role in MLC-2 gene transcription and bind to a single protein (ABF-2), it would appear that ABF-2 is a key transcription factor with the ability to recognize divergent sequence elements involved in a common regulatory pathway during myogenesis.

A single transcription factor binds to two divergent sequence elements with a common function in cardiac myosin light chain-2 promoter

1992 ◽  
Vol 12 (3) ◽  
pp. 1107-1116
Author(s):  
P Qasba ◽  
E Lin ◽  
M D Zhou ◽  
A Kumar ◽  
M A Siddiqui
Keyword(s):  
Transcription Factor ◽  
Gene Transcription ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Cardiac Myosin ◽  
Divergent Sequence ◽  
Specific Element ◽  
Myosin Light Chain 2 ◽  
Sequence Elements ◽  
Gel Shift

The cardiac myosin light chain-2 (MLC-2) gene promoter contains several positive and negative cis-acting sequences that are involved in the regulation of its expression. We describe here the properties of two activator sequences, elements A and P, and their DNA-binding factors (ABFs). Element A (CCAAAAGTGG), located at -61, has homology with the evolutionarily conserved sequence CC(A/T)6GG, present in the genes of many contractile proteins. Element P (TAACCTTGAAAGC), located 114 bp upstream of element A, is conserved in both chicken and rat cardiac MLC-2 gene promoters. Deletion mutagenesis demonstrated that these two elements are involved in the positive regulation of MLC-2 gene transcription. At least two sequence-specific element A-binding proteins, ABF-1 and ABF-2, were identified by gel shift analysis of the fractionated cardiac nuclear proteins. ABF-1 binds to element A with strict dependence on the internal element A sequence AAAAGT. In contrast, ABF-2 exhibits a relaxed sequence requirement, as it recognizes the consensus CArG and CCAAT box sequences as well. ABF-2 also recognizes the distal element P despite the fact that the sequences of elements A and P are divergent. DNase I footprinting, methylation interference, and gel shift analyses demonstrated unequivocally that the element A-DNA affinity-purified protein ABF-2 binds to element P with sequence specificity. Since both elements A and P play a positive regulatory role in MLC-2 gene transcription and bind to a single protein (ABF-2), it would appear that ABF-2 is a key transcription factor with the ability to recognize divergent sequence elements involved in a common regulatory pathway during myogenesis.

Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription

1989 ◽  
Vol 9 (6) ◽  
pp. 2513-2525
Author(s):  
T Braun ◽  
E Tannich ◽  
G Buschhausen-Denker ◽  
H H Arnold
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Mutational Analysis ◽  
Nuclear Proteins ◽  
Chain Gene ◽  
Cardiac Myosin ◽  
Mobility Shift ◽  
Light Chain Gene ◽  
Sequence Elements

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

scholarly journals Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.

1989 ◽  
Vol 9 (6) ◽  
pp. 2513-2525 ◽  
Author(s):  
T Braun ◽  
E Tannich ◽  
G Buschhausen-Denker ◽  
H H Arnold
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Mutational Analysis ◽  
Nuclear Proteins ◽  
Chain Gene ◽  
Cardiac Myosin ◽  
Mobility Shift ◽  
Light Chain Gene ◽  
Sequence Elements

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

A conserved 28-base-pair element (HF-1) in the rat cardiac myosin light-chain-2 gene confers cardiac-specific and alpha-adrenergic-inducible expression in cultured neonatal rat myocardial cells

1991 ◽  
Vol 11 (4) ◽  
pp. 2273-2281
Author(s):  
H Zhu ◽  
A V Garcia ◽  
R S Ross ◽  
S M Evans ◽  
K R Chien
Keyword(s):  
Transient Expression ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myocardial Cell ◽  
Inducible Expression ◽  
Neonatal Rat ◽  
Cardiac Myosin ◽  
Myocardial Cells ◽  
Myosin Light Chain 2 ◽  
Regulated Expression

To study the transcriptional regulatory mechanisms which mediate cardiac-specific and inducible expression during myocardial cell hypertrophy, we have extensively characterized the rat cardiac myosin light-chain-2 (MLC-2) gene as a model system. The MLC-2 gene encodes a relatively abundant contractile protein in slow skeletal and cardiac muscle and is upregulated during in vivo cardiac hypertrophy and alpha-adrenergic-mediated hypertrophy of neonatal rat myocardial cells. In transient expression assays employing a series of MLC-2-luciferase constructs, recent studies have identified a 250-bp fragment which is sufficient for both cardiac-specific and alpha-adrenergic-inducible expression. Within this 250-bp fragment lie three regions (HF-1, HF-2, and HF-3), each greater than 10 bp in length, which are conserved between the chicken and rat cardiac MLC-2 genes, suggesting their potential role in the regulated expression of this contractile protein gene. As assessed by substitution mutations within each of the conserved regions, the present study demonstrates that HF-1 and HF-2 are important in both cardiac-specific and inducible expression, while HF-3 has no detectable role in the regulated expression of the MLC-2 gene in transient expression assays. HF-1 sequences confer both cardiac-specific and inducible expression to a neutral promoter-luciferase construct but have no significant effect in the skeletal muscle or nonmuscle cell contexts. Thus, these studies have identified a new cardiac-specific regulatory element (HF-1) which plays a role in both cardiac-specific and inducible expression during myocardial cell hypertrophy.

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