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HDAC4 regulates muscle fiber type-specific gene expression programs.

Cohen TJ, Choi MC, Kapur M, Lira VA, Yan Z, Yao TP - Mol. Cells (2015)

Bottom Line: The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers.Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers.Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA.

ABSTRACT
Fiber type-specific programs controlled by the transcription factor MEF2 dictate muscle functionality. Here, we show that HDAC4, a potent MEF2 inhibitor, is predominantly localized to the nuclei in fast/glycolytic fibers in contrast to the sarcoplasm in slow/oxidative fibers. The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers. Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers. Indeed, HDAC4 represses the MEF2-dependent, PGC-1α-mediated oxidative metabolic gene program. Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

No MeSH data available.


HDAC4 inhibits MEF2-dependent expression of PGC-1α and related oxidative genes. (A) Tibialis muscles (TA) were electroporated with expression plasmids containing control, wild type HDAC4, or the nuclear-localized HDAC4-3SA mutant and oxidative gene expression was evaluated by RNA analysis of isolated TA muscle lysates by real-time RT-PCR. Oxidative gene expression was determined using gene-specific primers detecting PGC-1α, and myoglobin, all of which are up-regulated in slow-fibers. Values represent fold repression relative to actin levels. Columns, mean; bars, SEM (n = 3). *P < 0.05. (B) RNA analysis similar to panel A was performed to monitor oxidative gene expression after HDAC4 siRNA knockdown in TA muscles. Columns, mean; bars, SEM (n = 3). *P < 0.05. (C) PGC-1α promoter luciferase activities were evaluated in transfected myotubes using wild-type PGC-1α or a mutant promoter containing deletion of AT-rich elements harboring MEF2-binding sites (deltaMEF2). HDAC4 suppressed PGC-1α promoter activity in a MEF2-dependent manner. (D) MEF2 promoter activity was examined in transfected myotubes using wild-type HDAC4, HDAC4-3SA mutant, or HDAC5.
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f4-molce-38-4-343: HDAC4 inhibits MEF2-dependent expression of PGC-1α and related oxidative genes. (A) Tibialis muscles (TA) were electroporated with expression plasmids containing control, wild type HDAC4, or the nuclear-localized HDAC4-3SA mutant and oxidative gene expression was evaluated by RNA analysis of isolated TA muscle lysates by real-time RT-PCR. Oxidative gene expression was determined using gene-specific primers detecting PGC-1α, and myoglobin, all of which are up-regulated in slow-fibers. Values represent fold repression relative to actin levels. Columns, mean; bars, SEM (n = 3). *P < 0.05. (B) RNA analysis similar to panel A was performed to monitor oxidative gene expression after HDAC4 siRNA knockdown in TA muscles. Columns, mean; bars, SEM (n = 3). *P < 0.05. (C) PGC-1α promoter luciferase activities were evaluated in transfected myotubes using wild-type PGC-1α or a mutant promoter containing deletion of AT-rich elements harboring MEF2-binding sites (deltaMEF2). HDAC4 suppressed PGC-1α promoter activity in a MEF2-dependent manner. (D) MEF2 promoter activity was examined in transfected myotubes using wild-type HDAC4, HDAC4-3SA mutant, or HDAC5.

Mentions: Since MEF2 and PGC-1α expression positively regulates oxidative gene expression (Lin et al., 2002; Potthoff et al., 2007b), we next examined whether HDAC4 regulates PGC-1α gene expression. To this end, we electroporated wild type and constitutively nuclear HDAC4-3SA mutant (Zhao et al., 2001) into TA muscles. As shown in Fig. 4A, PGC-1α expression was modestly repressed by wild-type HDAC4, while more prominent repression was observed by the HDAC4-3SA mutant. The relatively modest effect of ectopic wild-type HDAC4 could be due to endogenous HDAC4 enrichment in the nuclei of TA muscle. Supporting a repressive activity of HDAC4 on PGC-1α-dependent oxidative programming, knockdown of HDAC4 by a siRNA in TA muscle led to elevated expression of PGC-1α, myoglobin and Cytochrome C (Fig. 4B). Furthermore, HDAC4 repressed PGC-1α promoter activity in a MEF2-dependent manner, as deletion of AT-rich MEF2-binding elements prevented HDAC4-mediated repression of PGC-1α (Fig. 4C). Confirming that HDAC4-mediated transcriptional repression occurs via a nuclear mechanism, both wild-type HDAC4 and most robustly, the nuclear-targeted HDAC4-3SA mutant, strongly suppressed MEF2 promoter activity (Fig. 4D). Collectively, these results indicate that HDAC4 establishes fast fiber gene expression via repression of MEF2-dependent PGC-1α and related oxidative genes.


HDAC4 regulates muscle fiber type-specific gene expression programs.

Cohen TJ, Choi MC, Kapur M, Lira VA, Yan Z, Yao TP - Mol. Cells (2015)

HDAC4 inhibits MEF2-dependent expression of PGC-1α and related oxidative genes. (A) Tibialis muscles (TA) were electroporated with expression plasmids containing control, wild type HDAC4, or the nuclear-localized HDAC4-3SA mutant and oxidative gene expression was evaluated by RNA analysis of isolated TA muscle lysates by real-time RT-PCR. Oxidative gene expression was determined using gene-specific primers detecting PGC-1α, and myoglobin, all of which are up-regulated in slow-fibers. Values represent fold repression relative to actin levels. Columns, mean; bars, SEM (n = 3). *P < 0.05. (B) RNA analysis similar to panel A was performed to monitor oxidative gene expression after HDAC4 siRNA knockdown in TA muscles. Columns, mean; bars, SEM (n = 3). *P < 0.05. (C) PGC-1α promoter luciferase activities were evaluated in transfected myotubes using wild-type PGC-1α or a mutant promoter containing deletion of AT-rich elements harboring MEF2-binding sites (deltaMEF2). HDAC4 suppressed PGC-1α promoter activity in a MEF2-dependent manner. (D) MEF2 promoter activity was examined in transfected myotubes using wild-type HDAC4, HDAC4-3SA mutant, or HDAC5.
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f4-molce-38-4-343: HDAC4 inhibits MEF2-dependent expression of PGC-1α and related oxidative genes. (A) Tibialis muscles (TA) were electroporated with expression plasmids containing control, wild type HDAC4, or the nuclear-localized HDAC4-3SA mutant and oxidative gene expression was evaluated by RNA analysis of isolated TA muscle lysates by real-time RT-PCR. Oxidative gene expression was determined using gene-specific primers detecting PGC-1α, and myoglobin, all of which are up-regulated in slow-fibers. Values represent fold repression relative to actin levels. Columns, mean; bars, SEM (n = 3). *P < 0.05. (B) RNA analysis similar to panel A was performed to monitor oxidative gene expression after HDAC4 siRNA knockdown in TA muscles. Columns, mean; bars, SEM (n = 3). *P < 0.05. (C) PGC-1α promoter luciferase activities were evaluated in transfected myotubes using wild-type PGC-1α or a mutant promoter containing deletion of AT-rich elements harboring MEF2-binding sites (deltaMEF2). HDAC4 suppressed PGC-1α promoter activity in a MEF2-dependent manner. (D) MEF2 promoter activity was examined in transfected myotubes using wild-type HDAC4, HDAC4-3SA mutant, or HDAC5.
Mentions: Since MEF2 and PGC-1α expression positively regulates oxidative gene expression (Lin et al., 2002; Potthoff et al., 2007b), we next examined whether HDAC4 regulates PGC-1α gene expression. To this end, we electroporated wild type and constitutively nuclear HDAC4-3SA mutant (Zhao et al., 2001) into TA muscles. As shown in Fig. 4A, PGC-1α expression was modestly repressed by wild-type HDAC4, while more prominent repression was observed by the HDAC4-3SA mutant. The relatively modest effect of ectopic wild-type HDAC4 could be due to endogenous HDAC4 enrichment in the nuclei of TA muscle. Supporting a repressive activity of HDAC4 on PGC-1α-dependent oxidative programming, knockdown of HDAC4 by a siRNA in TA muscle led to elevated expression of PGC-1α, myoglobin and Cytochrome C (Fig. 4B). Furthermore, HDAC4 repressed PGC-1α promoter activity in a MEF2-dependent manner, as deletion of AT-rich MEF2-binding elements prevented HDAC4-mediated repression of PGC-1α (Fig. 4C). Confirming that HDAC4-mediated transcriptional repression occurs via a nuclear mechanism, both wild-type HDAC4 and most robustly, the nuclear-targeted HDAC4-3SA mutant, strongly suppressed MEF2 promoter activity (Fig. 4D). Collectively, these results indicate that HDAC4 establishes fast fiber gene expression via repression of MEF2-dependent PGC-1α and related oxidative genes.

Bottom Line: The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers.Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers.Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA.

ABSTRACT
Fiber type-specific programs controlled by the transcription factor MEF2 dictate muscle functionality. Here, we show that HDAC4, a potent MEF2 inhibitor, is predominantly localized to the nuclei in fast/glycolytic fibers in contrast to the sarcoplasm in slow/oxidative fibers. The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers. Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers. Indeed, HDAC4 represses the MEF2-dependent, PGC-1α-mediated oxidative metabolic gene program. Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

No MeSH data available.