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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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Chip assay with extracts from Chicken cardiac and skeletal muscle tissues. (A) Upper panel shows the ChIP assay performed in cardiac tissue for detection of active chromatin antiH3K4 (trimethylated) and inactive chromatin anti‐H3K9 (monomethylated) antibodies. We used primer flanking the IRE region in the cMLC2 gene, and as positive control the GAPDH primer, and as negative control primers that recognize the ApoB gene. (B) Lower panel shows a representative PCR using the same antibodies and target primers described in A but using skeletal muscle tissue.
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f3: Chip assay with extracts from Chicken cardiac and skeletal muscle tissues. (A) Upper panel shows the ChIP assay performed in cardiac tissue for detection of active chromatin antiH3K4 (trimethylated) and inactive chromatin anti‐H3K9 (monomethylated) antibodies. We used primer flanking the IRE region in the cMLC2 gene, and as positive control the GAPDH primer, and as negative control primers that recognize the ApoB gene. (B) Lower panel shows a representative PCR using the same antibodies and target primers described in A but using skeletal muscle tissue.

Mentions: In previous studies, Arnold and co‐workers [16] have noted the existence of a DNase‐I hypersensitive region (HR1) in the first intron of the cardiac MLC2v gene. Since IRE appeared to be the only functional sequence element in that region [15], we speculated that IRE is in an open conformation in cardiac cells, and perhaps not in skeletal muscle cells where the gene is repressed. To test this possibility, we performed the ChIP assay (see Materials and Methods) to read the histone code in the vicinity of IRE in cardiac and skeletal muscle cells. Euchromatin is characterized by the presence of histone modifications such as acetylated H3K9 and dimethylated H3K4. The heterochromatin is enriched by the presence of mono‐, di‐ and trimethylated H3K9. We used anti‐K4 methyl antibody to immunoprecipate chromatin from both heart and skeletal muscle tissues. Anti‐K4 methyl antibody was raised against the methylated lysine residue 4 and serves as an indicator of transcriptionally active DNA. PCR was performed with DNA primers flanking the IRE sequence. Results in Fig. 3 confirmed that the IRE containing DNA is in open conformation and hence accessible in cardiac cells, but under identical conditions it was not accessible in skeletal muscle cells. The use of monomethylated H3K9 antibody revealed the closed conformation in skeletal muscle. The expression of GAPDH gene was measured as a control reaction for evaluating the transcriptionally active selection of Anti‐K4 methyl antibody. Conversely, inactive chromatin activity was determined by evaluating the close chromatin status of the ApoB gene.


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)

Chip assay with extracts from Chicken cardiac and skeletal muscle tissues. (A) Upper panel shows the ChIP assay performed in cardiac tissue for detection of active chromatin antiH3K4 (trimethylated) and inactive chromatin anti‐H3K9 (monomethylated) antibodies. We used primer flanking the IRE region in the cMLC2 gene, and as positive control the GAPDH primer, and as negative control primers that recognize the ApoB gene. (B) Lower panel shows a representative PCR using the same antibodies and target primers described in A but using skeletal muscle tissue.
© Copyright Policy
Related In: Results  -  Collection

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

f3: Chip assay with extracts from Chicken cardiac and skeletal muscle tissues. (A) Upper panel shows the ChIP assay performed in cardiac tissue for detection of active chromatin antiH3K4 (trimethylated) and inactive chromatin anti‐H3K9 (monomethylated) antibodies. We used primer flanking the IRE region in the cMLC2 gene, and as positive control the GAPDH primer, and as negative control primers that recognize the ApoB gene. (B) Lower panel shows a representative PCR using the same antibodies and target primers described in A but using skeletal muscle tissue.
Mentions: In previous studies, Arnold and co‐workers [16] have noted the existence of a DNase‐I hypersensitive region (HR1) in the first intron of the cardiac MLC2v gene. Since IRE appeared to be the only functional sequence element in that region [15], we speculated that IRE is in an open conformation in cardiac cells, and perhaps not in skeletal muscle cells where the gene is repressed. To test this possibility, we performed the ChIP assay (see Materials and Methods) to read the histone code in the vicinity of IRE in cardiac and skeletal muscle cells. Euchromatin is characterized by the presence of histone modifications such as acetylated H3K9 and dimethylated H3K4. The heterochromatin is enriched by the presence of mono‐, di‐ and trimethylated H3K9. We used anti‐K4 methyl antibody to immunoprecipate chromatin from both heart and skeletal muscle tissues. Anti‐K4 methyl antibody was raised against the methylated lysine residue 4 and serves as an indicator of transcriptionally active DNA. PCR was performed with DNA primers flanking the IRE sequence. Results in Fig. 3 confirmed that the IRE containing DNA is in open conformation and hence accessible in cardiac cells, but under identical conditions it was not accessible in skeletal muscle cells. The use of monomethylated H3K9 antibody revealed the closed conformation in skeletal muscle. The expression of GAPDH gene was measured as a control reaction for evaluating the transcriptionally active selection of Anti‐K4 methyl antibody. Conversely, inactive chromatin activity was determined by evaluating the close chromatin status of the ApoB gene.

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