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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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Western blot of total cell lysates from C2C12 cells stably transfected with pcDNA6 vector (V6) or with plasmid expressing Nished (N1). (A) cell lysates were immunoprecipitated and blotted with anti‐Nished antibody or pre‐immune serum. As loading control we used equal amount of proteins from each cell line, and by Western blot determined the GAPDH levels. (B) Transient transfection of plasmid pMutIRELuc in C2C12 cells stably transfected with pNished (N1) or vector alone (V5). Data are expressed as percentage activity of pMutIRELuc transfected in wild‐type C2C12 cells. (n= 4 in triplicate; □, one sample t‐test; P < 0.05).
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f5: Western blot of total cell lysates from C2C12 cells stably transfected with pcDNA6 vector (V6) or with plasmid expressing Nished (N1). (A) cell lysates were immunoprecipitated and blotted with anti‐Nished antibody or pre‐immune serum. As loading control we used equal amount of proteins from each cell line, and by Western blot determined the GAPDH levels. (B) Transient transfection of plasmid pMutIRELuc in C2C12 cells stably transfected with pNished (N1) or vector alone (V5). Data are expressed as percentage activity of pMutIRELuc transfected in wild‐type C2C12 cells. (n= 4 in triplicate; □, one sample t‐test; P < 0.05).

Mentions: To further investigate the role of Nished as repressor, we used the mouse skeletal muscle cell line C2C12 to produce Nished expressing stable transfectants, N1, (see ‘Materials and Methods’). Immunoprecipitation and Western blotting of cell extracts with anti‐Nished antibody showed an enhanced level of Nished expression in N1 cells relative to the parent cells C2C12. GHAPDH levels were detected as loading control (Fig. 5A). Upon transient transfection of mutant MLC2 promoter (pMutIRELuc) in N1 cells, there was repression of the reporter plasmid compared to the level of expression in the parent cell line C2C12 (Fig. 5B). Taken together, these results suggest that Nished effectively down‐regulates the MLC2v promoter activity when its binding is restricted to CSS alone, an environment mimicking that of the skeletal muscle cell.


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)

Western blot of total cell lysates from C2C12 cells stably transfected with pcDNA6 vector (V6) or with plasmid expressing Nished (N1). (A) cell lysates were immunoprecipitated and blotted with anti‐Nished antibody or pre‐immune serum. As loading control we used equal amount of proteins from each cell line, and by Western blot determined the GAPDH levels. (B) Transient transfection of plasmid pMutIRELuc in C2C12 cells stably transfected with pNished (N1) or vector alone (V5). Data are expressed as percentage activity of pMutIRELuc transfected in wild‐type C2C12 cells. (n= 4 in triplicate; □, one sample t‐test; P < 0.05).
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Related In: Results  -  Collection

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

f5: Western blot of total cell lysates from C2C12 cells stably transfected with pcDNA6 vector (V6) or with plasmid expressing Nished (N1). (A) cell lysates were immunoprecipitated and blotted with anti‐Nished antibody or pre‐immune serum. As loading control we used equal amount of proteins from each cell line, and by Western blot determined the GAPDH levels. (B) Transient transfection of plasmid pMutIRELuc in C2C12 cells stably transfected with pNished (N1) or vector alone (V5). Data are expressed as percentage activity of pMutIRELuc transfected in wild‐type C2C12 cells. (n= 4 in triplicate; □, one sample t‐test; P < 0.05).
Mentions: To further investigate the role of Nished as repressor, we used the mouse skeletal muscle cell line C2C12 to produce Nished expressing stable transfectants, N1, (see ‘Materials and Methods’). Immunoprecipitation and Western blotting of cell extracts with anti‐Nished antibody showed an enhanced level of Nished expression in N1 cells relative to the parent cells C2C12. GHAPDH levels were detected as loading control (Fig. 5A). Upon transient transfection of mutant MLC2 promoter (pMutIRELuc) in N1 cells, there was repression of the reporter plasmid compared to the level of expression in the parent cell line C2C12 (Fig. 5B). Taken together, these results suggest that Nished effectively down‐regulates the MLC2v promoter activity when its binding is restricted to CSS alone, an environment mimicking that of the skeletal muscle cell.

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