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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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Tricostatin A releases CSS mediates repression of MLC2v promoter in C2C12 cells. (A) C2C12 cells were transfected with MLC2v promoter containing mutated IRE (pMutIRELuc). After 24 hrs, cells were treated with Tricostation A (TSA), an inhibitor of Class I and II HDACs, and 25 μM each of Sirtinol and M15, inhibitors of Class III HDACs. The HDACs inhibitors were dissolved in DMSO. Luciferase assays were performed 18 hrs after treatment. Nished repression of MLC2v promoter was relieved by TSA in a dose‐dependent manner, whereas Sirtionol and M15 had no effect. (B) Similar transient transfection experiment was performed in chicken primary cardiac cells, however, TSA treatment failed to modify the MLC2v promoter activity (n= 4 in triplicate, □ one sample t‐test, P < 0.05).
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f7: Tricostatin A releases CSS mediates repression of MLC2v promoter in C2C12 cells. (A) C2C12 cells were transfected with MLC2v promoter containing mutated IRE (pMutIRELuc). After 24 hrs, cells were treated with Tricostation A (TSA), an inhibitor of Class I and II HDACs, and 25 μM each of Sirtinol and M15, inhibitors of Class III HDACs. The HDACs inhibitors were dissolved in DMSO. Luciferase assays were performed 18 hrs after treatment. Nished repression of MLC2v promoter was relieved by TSA in a dose‐dependent manner, whereas Sirtionol and M15 had no effect. (B) Similar transient transfection experiment was performed in chicken primary cardiac cells, however, TSA treatment failed to modify the MLC2v promoter activity (n= 4 in triplicate, □ one sample t‐test, P < 0.05).

Mentions: To evaluate the mechanism by which Nished inhibits MLC2v gene transcription in non‐cardiac cells, we examined the involvement of HDAC activity in MLC‐2v gene repression. Upon exposure to Trichostatin A (TSA), a Class I‐II HDAC inhibitor, the promoter activity of the pMutIRELuc reporter in skeletal muscle cells was activated in a dose‐dependent manner (5, 10, 25 nM), to nearly 2.5 fold (Fig. 7A), whereas the pMutIRELuc was not activated in primary cardiac cells exposed to TSA (Fig. 7B). The differential response of MLC‐2v gene to TSA in cardiac and skeletal muscle cells indicates the specific requirement of HDAC activity for active repression in skeletal muscle cells. The selective requirement of classes I and II HDACs was demonstrated by lack of release of inhibition by the HDAC class III inhibitors, sirtinol [17] and M15 [18]. To identify the HDAC, which interacts with Nished, tissue extracts of heart and skeletal muscle were immunoprecipitated with anti‐Nished antibody followed by Western blotting with antibodies against HDAC3, HDAC5 and Nished. We observed interaction of Nished with HDAC 3 and HDAC 5 in extracts from skeletal muscle only (Fig. 8A), indicating that Nished’s association with HDAC3 and 5 might be important in repression of cardiac MLC2v gene in skeletal muscle. In an attempt to identify the putative HDAC3/5 binding domain(s) in Nished, we tested the deletion mutants of Nished for Nished/HDAC3/5 binding assay as above. Figure 8B shows that NΔ2 does not bind with HDAC3 and 5, whereas NΔ1 binds with HDAC 3 but not with HDAC5, suggesting that the optimal binding activity, at least for HDAC3, resides in CΔ2, and involves amino acids 37–64 in Nished.


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)

Tricostatin A releases CSS mediates repression of MLC2v promoter in C2C12 cells. (A) C2C12 cells were transfected with MLC2v promoter containing mutated IRE (pMutIRELuc). After 24 hrs, cells were treated with Tricostation A (TSA), an inhibitor of Class I and II HDACs, and 25 μM each of Sirtinol and M15, inhibitors of Class III HDACs. The HDACs inhibitors were dissolved in DMSO. Luciferase assays were performed 18 hrs after treatment. Nished repression of MLC2v promoter was relieved by TSA in a dose‐dependent manner, whereas Sirtionol and M15 had no effect. (B) Similar transient transfection experiment was performed in chicken primary cardiac cells, however, TSA treatment failed to modify the MLC2v promoter activity (n= 4 in triplicate, □ one sample t‐test, P < 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940774&req=5

f7: Tricostatin A releases CSS mediates repression of MLC2v promoter in C2C12 cells. (A) C2C12 cells were transfected with MLC2v promoter containing mutated IRE (pMutIRELuc). After 24 hrs, cells were treated with Tricostation A (TSA), an inhibitor of Class I and II HDACs, and 25 μM each of Sirtinol and M15, inhibitors of Class III HDACs. The HDACs inhibitors were dissolved in DMSO. Luciferase assays were performed 18 hrs after treatment. Nished repression of MLC2v promoter was relieved by TSA in a dose‐dependent manner, whereas Sirtionol and M15 had no effect. (B) Similar transient transfection experiment was performed in chicken primary cardiac cells, however, TSA treatment failed to modify the MLC2v promoter activity (n= 4 in triplicate, □ one sample t‐test, P < 0.05).
Mentions: To evaluate the mechanism by which Nished inhibits MLC2v gene transcription in non‐cardiac cells, we examined the involvement of HDAC activity in MLC‐2v gene repression. Upon exposure to Trichostatin A (TSA), a Class I‐II HDAC inhibitor, the promoter activity of the pMutIRELuc reporter in skeletal muscle cells was activated in a dose‐dependent manner (5, 10, 25 nM), to nearly 2.5 fold (Fig. 7A), whereas the pMutIRELuc was not activated in primary cardiac cells exposed to TSA (Fig. 7B). The differential response of MLC‐2v gene to TSA in cardiac and skeletal muscle cells indicates the specific requirement of HDAC activity for active repression in skeletal muscle cells. The selective requirement of classes I and II HDACs was demonstrated by lack of release of inhibition by the HDAC class III inhibitors, sirtinol [17] and M15 [18]. To identify the HDAC, which interacts with Nished, tissue extracts of heart and skeletal muscle were immunoprecipitated with anti‐Nished antibody followed by Western blotting with antibodies against HDAC3, HDAC5 and Nished. We observed interaction of Nished with HDAC 3 and HDAC 5 in extracts from skeletal muscle only (Fig. 8A), indicating that Nished’s association with HDAC3 and 5 might be important in repression of cardiac MLC2v gene in skeletal muscle. In an attempt to identify the putative HDAC3/5 binding domain(s) in Nished, we tested the deletion mutants of Nished for Nished/HDAC3/5 binding assay as above. Figure 8B shows that NΔ2 does not bind with HDAC3 and 5, whereas NΔ1 binds with HDAC 3 but not with HDAC5, suggesting that the optimal binding activity, at least for HDAC3, resides in CΔ2, and involves amino acids 37–64 in Nished.

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