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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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Schematic representation of Nished and its C‐and N‐terminal deletion mutants. (A) The deletions were made and cloned in the mammalian expression vector, pcDNA6V5/HisB, as described in ‘Material and Methods’. Numbers denote the amino acids in the constructs. (B) microaffinity isolation of S35 methionine labelled Nished and its N and C‐terminal deletion proteins generated by coupled transcription‐translation reaction with biotinylated CSS oligonucleotide as described in ‘Material and Methods’. (C) One tenth of reaction mixture volume of S35 methionine labelled Nished and its N‐and C‐terminal deletions proteins were electrophoresed on an 18% SDS gel. (D) Functional analysis of Nished deletion mutants. Primary skeletal muscle cells were co‐transfected with the IRE mutant (pMutIRELuc and Nished or the N and C terminal deletions mutants). The luciferase activity was normalized against the internal control, Renilla luciferase activity. (n= 7 in triplicate, □ one sample t‐test with Bonferroni correction, P < 0.05). Nished full length protein; NΔ1 (34–137 a.a); NΔ2 (90137 a.a.); CΔ1 (1–64 a.a); CΔ2 (1–115 a.a); V, Vector alone (negative control); Nished 10X, competition with lysate contouring vector alone.
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f6: Schematic representation of Nished and its C‐and N‐terminal deletion mutants. (A) The deletions were made and cloned in the mammalian expression vector, pcDNA6V5/HisB, as described in ‘Material and Methods’. Numbers denote the amino acids in the constructs. (B) microaffinity isolation of S35 methionine labelled Nished and its N and C‐terminal deletion proteins generated by coupled transcription‐translation reaction with biotinylated CSS oligonucleotide as described in ‘Material and Methods’. (C) One tenth of reaction mixture volume of S35 methionine labelled Nished and its N‐and C‐terminal deletions proteins were electrophoresed on an 18% SDS gel. (D) Functional analysis of Nished deletion mutants. Primary skeletal muscle cells were co‐transfected with the IRE mutant (pMutIRELuc and Nished or the N and C terminal deletions mutants). The luciferase activity was normalized against the internal control, Renilla luciferase activity. (n= 7 in triplicate, □ one sample t‐test with Bonferroni correction, P < 0.05). Nished full length protein; NΔ1 (34–137 a.a); NΔ2 (90137 a.a.); CΔ1 (1–64 a.a); CΔ2 (1–115 a.a); V, Vector alone (negative control); Nished 10X, competition with lysate contouring vector alone.

Mentions: In an attempt to localize the DNA binding domain in Nished, we created two N‐terminal, NΔ1 (34–137) and NΔ2 (90–137) and two C‐terminal CΔ1 (1–64) and CΔ2 (1–115) mutants (Fig. 6A) (see Materials & Methods). The wild‐type Nished and the mutants in lanes NΔ1, CΔ1, NΔ2 and CΔ2 were tested by micro‐affinity isolation assay (see Materials & Methods) where the in vitro translated and S35‐labelled products were subjected to binding to the biotinylated oligonucleotide containing CSS. Results in Fig. 6B show that mutants CΔ1 (1–64) and NΔ2 (90–137) that lack the segment do not bind CSS suggesting that the DNA binding activity resides within this region. The weak signals might be due to the proteolysis activity during transcription/translation assay. The results were nonetheless reproducible. The input of the reaction mixtures used in the assay, where the oligonucleotide was omitted, is shown in Fig. 6C. Specificity of the binding reaction was demonstrated by competition (Fig. 6B) with 10‐fold (10×) excess of non‐labelled in vitro translated wild‐type protein pNishedV5 product. Then, each mutant was evaluated for their repression activity on the pMutIRELuc, and we observed that NΔ1 lacking the first 34 aa although binds to the CSS motifs, yet fails to repress the promoter activity suggesting that the trans‐repression domain might be located within the first 34 aa region of the protein (Fig. 6D).


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)

Schematic representation of Nished and its C‐and N‐terminal deletion mutants. (A) The deletions were made and cloned in the mammalian expression vector, pcDNA6V5/HisB, as described in ‘Material and Methods’. Numbers denote the amino acids in the constructs. (B) microaffinity isolation of S35 methionine labelled Nished and its N and C‐terminal deletion proteins generated by coupled transcription‐translation reaction with biotinylated CSS oligonucleotide as described in ‘Material and Methods’. (C) One tenth of reaction mixture volume of S35 methionine labelled Nished and its N‐and C‐terminal deletions proteins were electrophoresed on an 18% SDS gel. (D) Functional analysis of Nished deletion mutants. Primary skeletal muscle cells were co‐transfected with the IRE mutant (pMutIRELuc and Nished or the N and C terminal deletions mutants). The luciferase activity was normalized against the internal control, Renilla luciferase activity. (n= 7 in triplicate, □ one sample t‐test with Bonferroni correction, P < 0.05). Nished full length protein; NΔ1 (34–137 a.a); NΔ2 (90137 a.a.); CΔ1 (1–64 a.a); CΔ2 (1–115 a.a); V, Vector alone (negative control); Nished 10X, competition with lysate contouring vector alone.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940774&req=5

f6: Schematic representation of Nished and its C‐and N‐terminal deletion mutants. (A) The deletions were made and cloned in the mammalian expression vector, pcDNA6V5/HisB, as described in ‘Material and Methods’. Numbers denote the amino acids in the constructs. (B) microaffinity isolation of S35 methionine labelled Nished and its N and C‐terminal deletion proteins generated by coupled transcription‐translation reaction with biotinylated CSS oligonucleotide as described in ‘Material and Methods’. (C) One tenth of reaction mixture volume of S35 methionine labelled Nished and its N‐and C‐terminal deletions proteins were electrophoresed on an 18% SDS gel. (D) Functional analysis of Nished deletion mutants. Primary skeletal muscle cells were co‐transfected with the IRE mutant (pMutIRELuc and Nished or the N and C terminal deletions mutants). The luciferase activity was normalized against the internal control, Renilla luciferase activity. (n= 7 in triplicate, □ one sample t‐test with Bonferroni correction, P < 0.05). Nished full length protein; NΔ1 (34–137 a.a); NΔ2 (90137 a.a.); CΔ1 (1–64 a.a); CΔ2 (1–115 a.a); V, Vector alone (negative control); Nished 10X, competition with lysate contouring vector alone.
Mentions: In an attempt to localize the DNA binding domain in Nished, we created two N‐terminal, NΔ1 (34–137) and NΔ2 (90–137) and two C‐terminal CΔ1 (1–64) and CΔ2 (1–115) mutants (Fig. 6A) (see Materials & Methods). The wild‐type Nished and the mutants in lanes NΔ1, CΔ1, NΔ2 and CΔ2 were tested by micro‐affinity isolation assay (see Materials & Methods) where the in vitro translated and S35‐labelled products were subjected to binding to the biotinylated oligonucleotide containing CSS. Results in Fig. 6B show that mutants CΔ1 (1–64) and NΔ2 (90–137) that lack the segment do not bind CSS suggesting that the DNA binding activity resides within this region. The weak signals might be due to the proteolysis activity during transcription/translation assay. The results were nonetheless reproducible. The input of the reaction mixtures used in the assay, where the oligonucleotide was omitted, is shown in Fig. 6C. Specificity of the binding reaction was demonstrated by competition (Fig. 6B) with 10‐fold (10×) excess of non‐labelled in vitro translated wild‐type protein pNishedV5 product. Then, each mutant was evaluated for their repression activity on the pMutIRELuc, and we observed that NΔ1 lacking the first 34 aa although binds to the CSS motifs, yet fails to repress the promoter activity suggesting that the trans‐repression domain might be located within the first 34 aa region of the protein (Fig. 6D).

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