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An Expression Screen Reveals Modulators of Class II Histone Deacetylase Phosphorylation

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An Expression Screen Reveals Modulators of Class II Histone Deacetylase Phosphorylation An expression screen reveals modulators of class II histone deacetylase phosphorylation Shurong Chang, Svetlana Bezprozvannaya, Shijie Li, and Eric N. Olson* Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390 Contributed by Eric N. Olson, April 20, 2005 Class II histone deacetylases (HDACs) repress transcription by repress the responsiveness of skeletal muscle genes to motor associating with a variety of transcription factors and corepressors. innervation (18). The actions of these HDACs in cardiac and Phosphorylation of a set of conserved serine residues in the skeletal myocytes correlate with their ability to associate with and N-terminal extensions of class II HDACs creates binding sites for repress the activity of myocyte enhancer factor 2 (MEF2). HDAC4 14-3-3 chaperone proteins, which trigger nuclear export of these acts as a repressor of chondrocyte hypertrophy by means of its HDACs, thereby derepressing specific target genes in a signal- association with the Runx2 transcription factor, a master regulator dependent manner. To identify intracellular signaling pathways of chondrocyte hypertrophy; chondrocytes from HDAC4 knockout that control phosphorylation of HDAC5, a class II HDAC, we mice undergo precocious and ectopic hypertrophy, resulting in designed a eukaryotic cDNA expression screen in which a GAL4- lethal ossification of endochondral cartilage (19). HDAC7 has been dependent luciferase reporter was expressed with the DNA- implicated in negative selection and apoptosis of T cells as a result binding domain of GAL4 fused to the N-terminal extension of of its association with the orphan nuclear receptor Nur77 (11). HDAC5 and the VP16 transcription activation domain fused to In an effort to identify kinases and other signaling molecules 14-3-3. The transfection of COS cells with cDNA expression libraries capable of regulating the phosphorylation of HDAC5, we designed results in activation of luciferase expression by cDNAs encoding a eukaryotic cDNA expression screen for cDNAs whose products HDAC5 kinases or modulators of such kinases that enable phos- could induce the interaction of GAL4-HDAC5 and 14-3-3-VP16 phorylated GAL4–HDAC5 to recruit 14-3-3-VP16 with consequent chimeric proteins and consequent activation of a GAL4-dependent reconstitution of a functional transcriptional complex. Our results luciferase reporter in transfected COS cells. Here, we describe a reveal a remarkable variety of signaling pathways that converge collection of regulators of HDAC5 phosphorylation uncovered in on the signal-responsive phosphorylation sites in HDAC5, thereby this screen. Our results reveal a remarkable number of signaling enabling HDAC5 to connect extracellular signals to the genome. pathways that culminate with the phosphorylation of HDAC5 and suggest that HDAC5 integrates diverse signaling pathways and endothelial differentiation genes ͉ lysophosphatidic acid receptors ͉ transduces their effects to ‘‘downstream’’ target genes as a result of Rho signaling ͉ sphingosine-1 phosphate its signal-dependent nuclear export. hanges in histone acetylation represent a key mechanism for Materials and Methods Cthe modulation of gene transcription (1). Acetylation of nu- Constructs and cDNA Expression Library. HDAC5 derivatives in cleosomal histones by histone acetyltransferases enhances tran- which residues 2–664 were fused to the DNA-binding domain of scription by relaxing the condensed structure of the nucleosome, GAL4 (GAL4BD-HDAC5), and 14-3-3 was fused to the VP16 whereas deacetylation of histones by histone deacetylase (HDAC) transcription activation domain have been described (9). Human activity reverses this process and promotes chromatin condensation fetal heart and mouse embryonic day 10.5 cDNA expression and transcriptional repression. The recruitment of histone acetyl- libraries were purchased from Invitrogen. cDNA pools were pre- transferases and HDACs by specific transcription factors enables pared by using the PerfectPrep Plasmid 96 Vac Direct Bind kit these chromatin-modifying enzymes to regulate specific sets of (Eppendorf). Each cDNA pool used in the screening contained target genes. 50–100 single cDNA clones. Three different classes of HDACs can be distinguished by their structures, expression patterns, and catalytic mechanisms (2). The Transient Transfections and Luciferase Reporter Assays. In each well four class II HDACs (HDAC4, HDAC5, HDAC7, and HDAC9) of a 24-well plate, 5 ϫ 104 COS cells were cultured in DMEM with contain a bipartite structure with an N-terminal extension of Ϸ600 10% FBS. Cells were transfected with 250 ng of cDNA expression aa followed by a catalytic domain (3). The N-terminal regions of library together with 100 ng of upstream activating sequence class II HDACs interact with a variety of transcription factors and (UAS)-luciferase reporter plasmid, and 50 ng each of expression corepressors and contain a set of conserved phosphorylation sites plasmids encoding GAL4BD-HDAC5 and VP16-14-3-3 by using that control their distribution between the nucleus and the cyto- 1.4 ␮l of FuGENE 6 reagent (Roche Molecular Biochemicals). plasm (4–10). The signal-responsive serines in the N-terminal Transfection efficiency was normalized by cotransfection of 10 ng regions of class II HDACs are phosphorylated by calcium͞calmod- of pCMV-LacZ. At 48 h posttransfection, the cells were harvested ulin-dependent protein kinase (CaMK) and protein kinase D in 150 ␮l of passive lysate buffer (Promega), and 20 ␮l of cell lysate (PKD) (5–9, 11, 12). When phosphorylated, these sites recruit the was used for luciferase or ␤-galactosidase assays. For sib-selection, chaperone protein 14-3-3, which masks the nuclear localization positive pools of cDNA expression clones were transformed into sequence in class II HDACs with consequent export of the HDAC͞ Escherichia coli DH5␣ competent cells and plated on LB agar 14-3-3 complex from the nucleus to the cytoplasm and derepression dishes. For each positive pool, 96 single clones were picked from the of specific genes (4, 6, 13–16). Thus, phosphorylation of class II HDACs provides a mechanism for coupling external signals to the genome. Abbreviations: HDAC, histone deacetylase; CaMK, calcium͞calmodulin-dependent protein kinase; PKD, protein kinase D; MEF2, myocyte enhancer factor 2; ET-1, endothelin 1; Mark2, Class II HDACs have been implicated in a variety of develop- microtubule-associating regulatory kinase 2; EDG, endothelial differentiation gene; SENP2, mental and signal-dependent processes. HDAC5 and HDAC9 small ubiquitin-related modifier 1͞sentrin͞SMT3-specific protease 2; UAS, upstream acti- suppress hypertrophic growth of cardiomyocytes, such that knock- vating sequence. out mice lacking these HDACs develop grossly enlarged hearts in *To whom correspondence should be addressed: E-mail: [email protected]. response to cardiac stress (8, 17). HDAC9 also has been shown to © 2005 by The National Academy of Sciences of the USA 8120–8125 ͉ PNAS ͉ June 7, 2005 ͉ vol. 102 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503275102 Downloaded by guest on September 26, 2021 Fig. 1. Schematic diagram of HDAC5 and the cDNA expression screening strategy. (A) Schematic diagram of HDAC5. The positions of the two signal-responsive serines flanking the nuclear localization sequence (NLS) in the N-terminal extension of HDAC5 are shown. Amino acid positions are indicated. (B) The cDNA expression screen. The N-terminal extension of HDAC5 was fused to the DNA-binding domain of GAL4, and 14-3-3 was fused to the activation domain of VP16. A luciferase reporter controlled by the GAL4 DNA-binding site, referred to as the UAS, is expressed at a basal level in control COS cells. Transfection of COS cells with pools of cDNAs results in the activation of UAS-luciferase expression by pools containing ki- nases or activators of kinases that phosphorylate the 14-3-3-binding sites in HDAC5, resulting in the recruit- ment of 14-3-3-VP16 and reconstitution of a transcrip- tional complex. (C) Results from a transfection assay in a representative 96-well plate are shown. Each well received a pool of Ϸ50–100 cDNAs as described in B. The UAS-luciferase plasmid was specifically activated in well F2. Sib-selection from this pool identified ET-1 receptor A as the activating cDNA. dish and grown in LB liquid media. Twelve single clones were was fused to the VP16 transcription activation domain. In trans- combined as subpools, and plasmids were prepared and transfected fected COS cells, HDAC5 is not phosphorylated, so GAL4- for the reporter assay as described above. Single clones from the HDAC5 cannot interact with 14-3-3-VP16, and a GAL4-dependent positive subpools were prepared and tested for their ability to luciferase reporter (UAS-luciferase) cannot be activated. Expres- promote HDAC5 phosphorylation. sion plasmids encoding these fusion proteins, together with UAS- luciferase, were transfected into COS cells along with pools of Ϸ100 Immunocytochemistry. COS cells plated on glass coverslips were cDNA clones each from a fetal human heart or a mouse embryonic fixed and stained as described in ref. 7. Fluorescent images were day 10.5 cDNA expression library. Any cDNA pool containing a collected on a fluorescent microscope (Leica, Deerfield, IL) and kinase capable of phosphorylating the 14-3-3-binding sites in were processed with PHOTOSHOP (Adobe Systems, San Jose, CA). HDAC5 or an upstream activator of an endogenous kinase in COS Anti-FLAG (Sigma) and fluorescein-conjugated anti-mouse IgG cells for these sites will result in the creation of a phospho-14-3-3 (Vector Laboratories) were used at a dilution of 1:200. recognition motif, which will recruit 14-3-3-VP16 to activate the reporter. Individual clones were isolated from positive pools by HDAC Kinase Assay. Kinase assays were performed as described in sib-selection. Results obtained from a typical screen of cDNA pools ref. 8. Briefly, lysates were prepared from COS cells transfected in a 96-well plate and the luciferase expression in response to an with a microtubule-associating regulatory kinase 2 (Mark2) plas- activating pool containing a cDNA encoding the endothelin 1 mid.
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