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(PGC-1 ) and Mitochondrial Function by MEF2 and HDAC5

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(PGC-1 ) and Mitochondrial Function by MEF2 and HDAC5 Regulation of peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) and mitochondrial function by MEF2 and HDAC5 Michael P. Czubryt*, John McAnally*, Glenn I. Fishman†, and Eric N. Olson*‡ *Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148; and †Division of Cardiology, New York University School of Medicine, 550 First Avenue, OBV-A615, New York, NY 10016 Contributed by Eric N. Olson, December 13, 2002 The myocyte enhancer factor-2 (MEF2) transcription factor regu- To further investigate the importance of HDAC5 in cardiac lates muscle development and calcium-dependent gene expres- function, we generated transgenic mice harboring a doxycycline sion. MEF2 activity is repressed by class II histone deacetylases (DOX)-inducible cardiac-specific transgene encoding the (HDACs), which dissociate from MEF2 when phosphorylated on HDAC5S͞A signal-resistant mutant. Because this mutant is able two serine residues in response to calcium signaling. To explore the to efficiently inhibit MEF2 transactivation (4), we hypothesized potential importance of MEF2͞HDAC interactions in the heart, we that it would allow us to more clearly identify MEF2͞HDAC generated transgenic mice expressing a signal-resistant form of targets free from complications of unknown kinases and phos- HDAC5 under cardiac-specific and doxycycline-inducible regula- phatases that may alter the activity of endogenous HDAC5. tion. Transgene expression resulted in sudden death in male mice Expression of HDAC5S͞A in the adult heart resulted in sudden accompanied by loss and morphologic changes of cardiac mito- death of male mice accompanied by gross aberrations in mito- chondria and down-regulation of mitochondrial enzymes. The chondrial architecture and down-regulation of mitochondrial ␣ transcriptional coactivator PGC-1 , a master regulator of mito- enzymes and the transcriptional coactivator peroxisome prolif- chondrial biogenesis and fatty acid oxidation, was also down- erator-activated receptor (PPAR) ␥ coactivator 1␣ (PGC-1␣). regulated in response to HDAC5 expression. Examination of the PGC-1␣ coactivates numerous transcription factors involved in CELL BIOLOGY ␣ PGC-1 promoter revealed two MEF2-binding sites that mediate metabolism, including MEF2, and cardiac-specific overexpres- transcriptional activation by MEF2 and repression by HDAC5. These ␣ ␣ ͞ sion of PGC-1 is sufficient to induce mitochondrial biogenesis findings identify PGC-1 as a key target of the MEF2 HDAC (9–11). We show that two MEF2-binding sites in the PGC-1␣ regulatory pathway and demonstrate this pathway’s importance upstream region mediate transcriptional responsiveness to in maintenance of cardiac mitochondrial function. MEF2 and repression by HDAC5, identifying the MEF2͞HDAC pathway as a key regulator of the PGC-1␣ gene and mitochon- cetylation of nucleosomal histones represents a central drial biogenesis in the heart. Amechanism for regulation of gene expression. Histone acetylation relaxes chromatin, allowing access of the transcrip- Methods tional machinery to specific regions of DNA. Control of this Generation of Transgenic Animals. We cloned the cDNA for human process is tightly regulated and can affect individual genes in a HDAC5 containing serine-to-alanine mutations at amino acids specific and directed fashion (1). Acetylation of histones by 259 and 498 into the tetracycline (tet)-responsive pUHG-10 histone acetyltransferases is associated with activation of gene vector to generate the tetHDAC5S͞A vector (4). This vector was expression. Conversely, gene repression is achieved by histone injected into B6C3F1 mouse oocytes and implanted into surro- deacetylases (HDACs) (2). gate female ICR mice. Transgenic offspring were bred to HDACs in mammalian cells comprise three distinct classes on ␣-myosin heavy chain (MHC)-tet transactivator transgenic mice the basis of protein structure and homology to yeast HDACs. while receiving 200 ␮g͞ml DOX in water (12). Animals were Class I and III HDACs are ubiquitously expressed, whereas class maintained on DOX as needed. All animal protocols were II HDACs are highly enriched in striated muscles and brain (2). approved by the Institutional Animal Care and Use Committee Class II HDACs (HDAC4, 5, 7, and 9) repress transcriptional of the University of Texas Southwestern Medical Center. activity of myocyte enhancer factor-2 (MEF2) transcription factors, which are also highly expressed in muscle and brain (3). Histology and Microscopy. For light microscopy, hearts were fixed This repression is relieved by phosphorylation of HDACs on two overnight in 10% formalin, then embedded in paraffin, sectioned conserved serine residues, creating docking sites for the 14–3-3 at 5 ␮m, and stained with hematoxylin͞eosin. Alternatively, chaperone protein, which mediates translocation of class II sections were immunostained with anti-FLAG primary antibody HDACs out of the nucleus (4). Mutation of these serine residues ␮ ͞ to alanines in HDAC5 prevents 14–3-3 binding and export to the (20 g ml; Sigma–Aldrich) and goat anti-mouse fluorescein cytosol (4–6). This signal-resistant HDAC mutant, which we isothiocyanate conjugated secondary antibody (1:1,000; Vector refer to here as HDAC5S͞A, acts as a dominant negative Laboratories, Burlingame, CA). For transmission electron mi- regulator of MEF2 signaling, because it binds and represses croscopy, hearts were fixed overnight in 3% glutaraldehyde in MEF2 factors but cannot be exported from the nucleus. Because PBS at 4°C, then postfixed in 1% OsO4 and dehydrated in an histone acetyltransferases (HATs) and HDACs compete for the ethanol series. Samples were then embedded in Spurr resin (Ted same binding site on MEF2, class II HDACs may also inhibit Pella, Redding, CA), stained with uranyl acetate and lead citrate, binding of HATs to MEF2 and subsequent histone acetylation and sectioned at 80 nm. (7). Expression of HDAC5S͞A in neonatal rat cardiomyocytes prevents agonist-induced hypertrophy and activation of the fetal Abbreviations: HDAC, histone deacetylase; MEF2, myocyte enhancer factor-2; HDAC5S͞A, cardiac gene program (8). The notion that class II HDACs may HDAC5 double serine-to-alanine mutant; PPAR, peroxisome proliferator-activated recep- counteract hypertrophic signals is supported by the recent tor; PGC-1␣, PPAR␥ coactivator 1␣; DOX, doxycycline, ChIP, chromatin immunoprecipita- demonstration that HDAC9 mutant mice show an exaggerated tion; MHC, myosin heavy chain; tet, tetracycline. response to hypertrophic stimuli (8). ‡To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0337639100 PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 1711–1716 Downloaded by guest on October 1, 2021 Western Blot Analysis. Protein was isolated from hearts by ho- mogenization in lysis buffer (PBS, 0.1% Triton X-100͞1mM EDTA, pH 7.4) containing Complete protease inhibitors (Roche Diagnostics). After electrophoresis and blotting onto Sequi-blot poly(vinylidene difluoride) membranes (Bio-Rad), blots were probed with anti-FLAG antibodies and horseradish peroxidase- conjugated secondary antibodies (Amersham Pharmacia Bio- sciences). Signal was detected with Western blotting Luminol Reagent (Santa Cruz Biotechnology), followed by exposure of blots to BioMax film (Kodak). Fig. 1. Characterization of HDAC5S͞A expression in DOX-inducible ͞ Cell Culture and Luciferase Assays. COS cells were maintained in HDAC5S A mice. (A) Western blotting of equal amounts of total cardiac protein of ␣-MHC-tTA͞tetHDAC5S͞A double transgenic mice before or after 10% FBS in DMEM. Fugene 6 (Roche Diagnostics) was used for DOX withdrawal. (B) Immunohistochemical staining for FLAG-HDAC5S͞Aof all transfections. The first 3.1 kb of genomic sequence upstream cardiac sections from double transgenic mice before or after DOX withdrawal. of the ATG translational start codon for mouse PGC-1␣ was amplified from BAC RPCI23–260A10 (CHORI-BacPac, Oak- land, CA; www.chori.org) by high-fidelity PCR and TA cloned Results into the pCRII-TOPO vector (upstream primer: 5Ј-GGGG- ͞ Ј Generation of Transgenic HDAC5S A Mice. Our initial goal was to TACCCCATTTGGGAATCCTCTATACAAAGTTG-3 ; express the signal-resistant HDAC5 protein, HDAC5S͞A, in the downstream primer: 5Ј-GAAGATCTTCCCAGCTCCCGAAT- Ј heart and to determine the consequences on cardiac gene GACG-3 ), then subcloned into the pGL3 basic luciferase expression. Attempts to generate mice transgenic for reporter vector (Promega). A QuikChange kit (Stratagene) was HDAC5S͞A under control of the ␣-MHC promoter were un- used for site-directed mutagenesis of this reporter with primers successful, suggesting that constitutive high-level expression of to introduce several A͞TtoC͞G mutations in the two putative Ј this HDAC5 mutant caused lethality (T. A. McKinsey and MEF2-binding sites (MEF2 site 1-distal: 5 -AAGACA- E.N.O., unpublished results). We therefore incorporated a tet- GAGAGAAAATTAACCATGGAAACTGCCTGGGGAG- off inducible system in which transgene expression is inhibited in 3Ј; MEF2 site 2-proximal: 5Ј-ATGGTGCTTTATAAATTA- the presence of the tet analog, DOX, but is induced upon GGTCTAGATGCATAGGGACTTT-3Ј; mutations are withdrawal of the drug (12). FLAG-tagged HDAC5S͞A was underlined). Luciferase assays were performed on COS cell cloned into a tet-responsive expression vector to generate a lysates by using a kit (Promega). Cardiomyocytes were isolated tetHDAC5 mouse line. This line was crossed with transgenic
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