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Repression of the cardiac myosin light chain-2 gene in skeletal muscle requires site-specific association of antithetic regulator, Nished, and HDACs.

Mathew S, Galatioto J, Mascareno E, Siddiqui MA - J. Cell. Mol. Med. (2009)

Bottom Line: We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively.Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor.Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Center for Cardiovascular and Muscle Research and Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA.

ABSTRACT
The transcriptional activation mechanisms that regulate tissue-specific expression of cardiac muscle genes have been extensively investigated, but little is known of the regulatory events involved in repression of cardiac-specific genes in non-cardiac cells. We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively. Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor. Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner. Co-transfection studies in primary muscle cells in culture and in Nished expressing stable skeletal muscle cell line demonstrate that Nished down-regulates the cardiac MLC2 gene expression when its association is restricted to CSS alone. Chromatin immunoprecipitation data suggest that the CSS-mediated repression of cardiac MLC2v gene in skeletal muscle cells excludes the participation of the positive element IRE despite the presence of an identical Nished binding site. Taken together, it appears that the negative control of MLC2v transcription is based on a dual mode of regulations, one that affords inaccessibility of IRE to Nished and second that promotes the formation of the transcription repression complex at the inhibitory CSS site to silence the cardiac gene in skeletal muscle cell.

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(A) Schematic representation of the chicken cardiac MLC2 promoter constructs with either the wild‐type (pMLC2.1Luc), IRE mutant, (pMutIRELuc) or mutations in both CSS and IRE, (pMutCSSIRELuc). (B) Transient transfection and MLC2 promoter activity. Chicken primary skeletal muscle cells in culture were transfected with plasmids pMLC2.1Luc, pMutIRELuc or pMutCSSIRELuc and activity of luciferase was measured as described in ‘Material and Methods’. Data are shown as percentage of wild‐type promoter activity. (n= 4 in triplicate; □ one sample t‐test with Bonferroni correction, P < 0.0167).
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f1: (A) Schematic representation of the chicken cardiac MLC2 promoter constructs with either the wild‐type (pMLC2.1Luc), IRE mutant, (pMutIRELuc) or mutations in both CSS and IRE, (pMutCSSIRELuc). (B) Transient transfection and MLC2 promoter activity. Chicken primary skeletal muscle cells in culture were transfected with plasmids pMLC2.1Luc, pMutIRELuc or pMutCSSIRELuc and activity of luciferase was measured as described in ‘Material and Methods’. Data are shown as percentage of wild‐type promoter activity. (n= 4 in triplicate; □ one sample t‐test with Bonferroni correction, P < 0.0167).

Mentions: We have previously identified a negative regulatory cis‐element (CSS) in the MLC2v promoter that contains a palindrome sequence 5′GAAGCTTC3′[10]. We observed subsequently that an identical sequence motif resides in the downstream activator element IRE located in the first intron of the MLC2v gene [15]. To examine the possibility of functional interaction between CSS and IRE in transcription regulation of the cardiac MLC2v gene, transient transfection was done with gene constructs that contain mutations in either IRE or in both IRE and CSS sequences. Specifically, the GAAGCTTC palindrome in IRE was mutated in the 2.1 kb SmaI‐StuI genomic fragment that extends from −1293 to +815 bp and encompasses the promoter and the first intron of the gene. The DNA fragment was linked to the luciferase reporter (pMutIRLuc) (see Materials and Methods). The same domain was also mutated in CSS as above to generate a double mutant pMutCSS+pMutIRELuc (Fig. 1A). The activity of these constructs was examined in transient transfection assay. In the wild‐type construct, the IRE positive activity apparently overrides the inhibitory activity of CSS. Mutation in IRE in pMutIRELuc resulted in a significant loss (70%) of the promoter function. However, when the same mutation was introduced in CSS as well as in IRE in the double mutant pMutIRE+MutCSSLuc, there was a loss of inhibition (Fig. 1B), suggesting that CSS and IRE interact with common DNA binding protein(s) that recognizes the conserved GAAG/CTTC motif present in both regulatory elements. Indeed, we have previously identified a transcription factor, Nished, that binds with sequence specificity to the palindrome in CSS [14] as well as in IRE where the IRE/Nished complex mediates the activation function of MLC2v gene promoter in cardiac cells [15].


Repression of the cardiac myosin light chain-2 gene in skeletal muscle requires site-specific association of antithetic regulator, Nished, and HDACs.

Mathew S, Galatioto J, Mascareno E, Siddiqui MA - J. Cell. Mol. Med. (2009)

(A) Schematic representation of the chicken cardiac MLC2 promoter constructs with either the wild‐type (pMLC2.1Luc), IRE mutant, (pMutIRELuc) or mutations in both CSS and IRE, (pMutCSSIRELuc). (B) Transient transfection and MLC2 promoter activity. Chicken primary skeletal muscle cells in culture were transfected with plasmids pMLC2.1Luc, pMutIRELuc or pMutCSSIRELuc and activity of luciferase was measured as described in ‘Material and Methods’. Data are shown as percentage of wild‐type promoter activity. (n= 4 in triplicate; □ one sample t‐test with Bonferroni correction, P < 0.0167).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940774&req=5

f1: (A) Schematic representation of the chicken cardiac MLC2 promoter constructs with either the wild‐type (pMLC2.1Luc), IRE mutant, (pMutIRELuc) or mutations in both CSS and IRE, (pMutCSSIRELuc). (B) Transient transfection and MLC2 promoter activity. Chicken primary skeletal muscle cells in culture were transfected with plasmids pMLC2.1Luc, pMutIRELuc or pMutCSSIRELuc and activity of luciferase was measured as described in ‘Material and Methods’. Data are shown as percentage of wild‐type promoter activity. (n= 4 in triplicate; □ one sample t‐test with Bonferroni correction, P < 0.0167).
Mentions: We have previously identified a negative regulatory cis‐element (CSS) in the MLC2v promoter that contains a palindrome sequence 5′GAAGCTTC3′[10]. We observed subsequently that an identical sequence motif resides in the downstream activator element IRE located in the first intron of the MLC2v gene [15]. To examine the possibility of functional interaction between CSS and IRE in transcription regulation of the cardiac MLC2v gene, transient transfection was done with gene constructs that contain mutations in either IRE or in both IRE and CSS sequences. Specifically, the GAAGCTTC palindrome in IRE was mutated in the 2.1 kb SmaI‐StuI genomic fragment that extends from −1293 to +815 bp and encompasses the promoter and the first intron of the gene. The DNA fragment was linked to the luciferase reporter (pMutIRLuc) (see Materials and Methods). The same domain was also mutated in CSS as above to generate a double mutant pMutCSS+pMutIRELuc (Fig. 1A). The activity of these constructs was examined in transient transfection assay. In the wild‐type construct, the IRE positive activity apparently overrides the inhibitory activity of CSS. Mutation in IRE in pMutIRELuc resulted in a significant loss (70%) of the promoter function. However, when the same mutation was introduced in CSS as well as in IRE in the double mutant pMutIRE+MutCSSLuc, there was a loss of inhibition (Fig. 1B), suggesting that CSS and IRE interact with common DNA binding protein(s) that recognizes the conserved GAAG/CTTC motif present in both regulatory elements. Indeed, we have previously identified a transcription factor, Nished, that binds with sequence specificity to the palindrome in CSS [14] as well as in IRE where the IRE/Nished complex mediates the activation function of MLC2v gene promoter in cardiac cells [15].

Bottom Line: We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively.Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor.Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Center for Cardiovascular and Muscle Research and Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA.

ABSTRACT
The transcriptional activation mechanisms that regulate tissue-specific expression of cardiac muscle genes have been extensively investigated, but little is known of the regulatory events involved in repression of cardiac-specific genes in non-cardiac cells. We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively. Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor. Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner. Co-transfection studies in primary muscle cells in culture and in Nished expressing stable skeletal muscle cell line demonstrate that Nished down-regulates the cardiac MLC2 gene expression when its association is restricted to CSS alone. Chromatin immunoprecipitation data suggest that the CSS-mediated repression of cardiac MLC2v gene in skeletal muscle cells excludes the participation of the positive element IRE despite the presence of an identical Nished binding site. Taken together, it appears that the negative control of MLC2v transcription is based on a dual mode of regulations, one that affords inaccessibility of IRE to Nished and second that promotes the formation of the transcription repression complex at the inhibitory CSS site to silence the cardiac gene in skeletal muscle cell.

Show MeSH
Related in: MedlinePlus