Limits...
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.

Show MeSH

Related in: MedlinePlus

Total cell lystate of mouse cardiac and skeletal muscle tissue was co‐immunoprecipicated with anti‐Nished antibodies and immunoblotted with anti‐HDAC3, anti‐HDAC5 and anti‐Nished antibody (loading control). (B) In vitro transcription/translation pull‐down assay was done using pcNishedV5, CΔ1, CΔ2, NΔ1, NΔ2, pcHDAC3′ flag, pcHDAC5′ flag as described in ‘Material and Methods’. Nished proteins were labelled with S35 methionine and combined with HDAC3 or HDAC5. Proteins were pulled down using M2 anti‐flag agarose, and run on a 18% polyacrylamide gel. Panel 1 (INPUT) shows translated Nished proteins. Panel 2 shows HDAC3 binding to Nished mutants, no binding is seen with NΔ2; panel 3 shows HDAC5 binding to Nished mutant proteins (no binding is seen with NΔ2).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4940774&req=5

f8: Total cell lystate of mouse cardiac and skeletal muscle tissue was co‐immunoprecipicated with anti‐Nished antibodies and immunoblotted with anti‐HDAC3, anti‐HDAC5 and anti‐Nished antibody (loading control). (B) In vitro transcription/translation pull‐down assay was done using pcNishedV5, CΔ1, CΔ2, NΔ1, NΔ2, pcHDAC3′ flag, pcHDAC5′ flag as described in ‘Material and Methods’. Nished proteins were labelled with S35 methionine and combined with HDAC3 or HDAC5. Proteins were pulled down using M2 anti‐flag agarose, and run on a 18% polyacrylamide gel. Panel 1 (INPUT) shows translated Nished proteins. Panel 2 shows HDAC3 binding to Nished mutants, no binding is seen with NΔ2; panel 3 shows HDAC5 binding to Nished mutant proteins (no binding is seen with NΔ2).

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)

Total cell lystate of mouse cardiac and skeletal muscle tissue was co‐immunoprecipicated with anti‐Nished antibodies and immunoblotted with anti‐HDAC3, anti‐HDAC5 and anti‐Nished antibody (loading control). (B) In vitro transcription/translation pull‐down assay was done using pcNishedV5, CΔ1, CΔ2, NΔ1, NΔ2, pcHDAC3′ flag, pcHDAC5′ flag as described in ‘Material and Methods’. Nished proteins were labelled with S35 methionine and combined with HDAC3 or HDAC5. Proteins were pulled down using M2 anti‐flag agarose, and run on a 18% polyacrylamide gel. Panel 1 (INPUT) shows translated Nished proteins. Panel 2 shows HDAC3 binding to Nished mutants, no binding is seen with NΔ2; panel 3 shows HDAC5 binding to Nished mutant proteins (no binding is seen with NΔ2).
© Copyright Policy
Related In: Results  -  Collection

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

f8: Total cell lystate of mouse cardiac and skeletal muscle tissue was co‐immunoprecipicated with anti‐Nished antibodies and immunoblotted with anti‐HDAC3, anti‐HDAC5 and anti‐Nished antibody (loading control). (B) In vitro transcription/translation pull‐down assay was done using pcNishedV5, CΔ1, CΔ2, NΔ1, NΔ2, pcHDAC3′ flag, pcHDAC5′ flag as described in ‘Material and Methods’. Nished proteins were labelled with S35 methionine and combined with HDAC3 or HDAC5. Proteins were pulled down using M2 anti‐flag agarose, and run on a 18% polyacrylamide gel. Panel 1 (INPUT) shows translated Nished proteins. Panel 2 shows HDAC3 binding to Nished mutants, no binding is seen with NΔ2; panel 3 shows HDAC5 binding to Nished mutant proteins (no binding is seen with NΔ2).
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