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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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Primary chicken skeletal cells were co‐transfected with the DNA constructs containing wild‐type IRE, (pMLC2.1Luc) or IRE mutant (pMutIRELuc) along with Nished expression vector, pNished‐V5, or the control mammalian expression vector (pcDNA6V5/HisB). Data are plotted as percentage increase or decrease of promoter activity relative to that of pMLC2.1LucDNA. Experiments were performed in triplicate (n= 7). □ signifies differences with one sample t‐test with Bonferroni correction, P < 0.008.
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f4: Primary chicken skeletal cells were co‐transfected with the DNA constructs containing wild‐type IRE, (pMLC2.1Luc) or IRE mutant (pMutIRELuc) along with Nished expression vector, pNished‐V5, or the control mammalian expression vector (pcDNA6V5/HisB). Data are plotted as percentage increase or decrease of promoter activity relative to that of pMLC2.1LucDNA. Experiments were performed in triplicate (n= 7). □ signifies differences with one sample t‐test with Bonferroni correction, P < 0.008.

Mentions: We then examined the functional activity of Nished/IRE complex in transient cotransfection assay using the wild‐type promoter, pMLC2.1Luc, and mutant IRE‐ containing plasmid, pMutIRELuc, as reporters along with the Nished expression vector pNished‐V5 in primary cardiac cells (see Materials and Methods). We reasoned that in pMutIRE plasmid the presence of non‐functional IRE will simulate the transcriptional state of the gene in skeletal muscle cells where IRE is not accessible to DNA binding protein(s). We observed (Fig. 4) that the ectopic expression of Nished does not repress the activity of MLC2v promoter with wild‐type IRE, supporting our argument (see Fig. 1) that IRE/Nished interaction overrides the repressor activity of the CSS/Nished complex. Repression of promoter activity occurs when IRE is mutated. Apparently, the absence of wild‐type IRE and loss of IRE/Nished interaction would drive Nished to CSS to cause inhibition of transcription.


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)

Primary chicken skeletal cells were co‐transfected with the DNA constructs containing wild‐type IRE, (pMLC2.1Luc) or IRE mutant (pMutIRELuc) along with Nished expression vector, pNished‐V5, or the control mammalian expression vector (pcDNA6V5/HisB). Data are plotted as percentage increase or decrease of promoter activity relative to that of pMLC2.1LucDNA. Experiments were performed in triplicate (n= 7). □ signifies differences with one sample t‐test with Bonferroni correction, P < 0.008.
© Copyright Policy
Related In: Results  -  Collection

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

f4: Primary chicken skeletal cells were co‐transfected with the DNA constructs containing wild‐type IRE, (pMLC2.1Luc) or IRE mutant (pMutIRELuc) along with Nished expression vector, pNished‐V5, or the control mammalian expression vector (pcDNA6V5/HisB). Data are plotted as percentage increase or decrease of promoter activity relative to that of pMLC2.1LucDNA. Experiments were performed in triplicate (n= 7). □ signifies differences with one sample t‐test with Bonferroni correction, P < 0.008.
Mentions: We then examined the functional activity of Nished/IRE complex in transient cotransfection assay using the wild‐type promoter, pMLC2.1Luc, and mutant IRE‐ containing plasmid, pMutIRELuc, as reporters along with the Nished expression vector pNished‐V5 in primary cardiac cells (see Materials and Methods). We reasoned that in pMutIRE plasmid the presence of non‐functional IRE will simulate the transcriptional state of the gene in skeletal muscle cells where IRE is not accessible to DNA binding protein(s). We observed (Fig. 4) that the ectopic expression of Nished does not repress the activity of MLC2v promoter with wild‐type IRE, supporting our argument (see Fig. 1) that IRE/Nished interaction overrides the repressor activity of the CSS/Nished complex. Repression of promoter activity occurs when IRE is mutated. Apparently, the absence of wild‐type IRE and loss of IRE/Nished interaction would drive Nished to CSS to cause inhibition of transcription.

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