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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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Gel mobility shift assay. Nuclear extracts of Chicken skeletal muscle cells pre‐incubated with anti‐Nished antibody (Nished) or pre‐immune serum (PI) were incubated with radiolabelled CSS oligonucleotide and subjected to gel mobility shift assay as described in ‘Materials and Methods’. □ denotes the DNA‐protein complexes disrupted by anti‐Nished antibody.
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f2: Gel mobility shift assay. Nuclear extracts of Chicken skeletal muscle cells pre‐incubated with anti‐Nished antibody (Nished) or pre‐immune serum (PI) were incubated with radiolabelled CSS oligonucleotide and subjected to gel mobility shift assay as described in ‘Materials and Methods’. □ denotes the DNA‐protein complexes disrupted by anti‐Nished antibody.

Mentions: In order to test whether the CSS/Nished complex is formed in skeletal muscle cells, we performed the gel shift assay with CSS DNA as probe using the skeletal muscle nuclear extract (Fig. 2). We observed that one major (CSSBPA) and two minor (CSSBP1 & 2) complexes are formed and that preincubation of the extracts with anti‐Nished antibody disrupts the major complex, CSSBPA, and one of the minor complexes, CSSBP1. Thus, Nished appears to be the common CSS and IRE DNA binding protein that is likely to be involved in both activation and repression mechanism of transcription of cardiac MLC2v gene in the distinct cellular environments, i.e. cardiac and skeletal muscle.


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)

Gel mobility shift assay. Nuclear extracts of Chicken skeletal muscle cells pre‐incubated with anti‐Nished antibody (Nished) or pre‐immune serum (PI) were incubated with radiolabelled CSS oligonucleotide and subjected to gel mobility shift assay as described in ‘Materials and Methods’. □ denotes the DNA‐protein complexes disrupted by anti‐Nished antibody.
© Copyright Policy
Related In: Results  -  Collection

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

f2: Gel mobility shift assay. Nuclear extracts of Chicken skeletal muscle cells pre‐incubated with anti‐Nished antibody (Nished) or pre‐immune serum (PI) were incubated with radiolabelled CSS oligonucleotide and subjected to gel mobility shift assay as described in ‘Materials and Methods’. □ denotes the DNA‐protein complexes disrupted by anti‐Nished antibody.
Mentions: In order to test whether the CSS/Nished complex is formed in skeletal muscle cells, we performed the gel shift assay with CSS DNA as probe using the skeletal muscle nuclear extract (Fig. 2). We observed that one major (CSSBPA) and two minor (CSSBP1 & 2) complexes are formed and that preincubation of the extracts with anti‐Nished antibody disrupts the major complex, CSSBPA, and one of the minor complexes, CSSBP1. Thus, Nished appears to be the common CSS and IRE DNA binding protein that is likely to be involved in both activation and repression mechanism of transcription of cardiac MLC2v gene in the distinct cellular environments, i.e. cardiac and skeletal muscle.

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