An expression screen reveals modulators of class II histone deacetylase phosphorylation - PubMed (original) (raw)

An expression screen reveals modulators of class II histone deacetylase phosphorylation

Shurong Chang et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Class II histone deacetylases (HDACs) repress transcription by associating with a variety of transcription factors and corepressors. Phosphorylation of a set of conserved serine residues in the N-terminal extensions of class II HDACs creates binding sites for 14-3-3 chaperone proteins, which trigger nuclear export of these HDACs, thereby derepressing specific target genes in a signal-dependent manner. To identify intracellular signaling pathways that control phosphorylation of HDAC5, a class II HDAC, we designed a eukaryotic cDNA expression screen in which a GAL4-dependent luciferase reporter was expressed with the DNA-binding domain of GAL4 fused to the N-terminal extension of HDAC5 and the VP16 transcription activation domain fused to 14-3-3. The transfection of COS cells with cDNA expression libraries results in activation of luciferase expression by cDNAs encoding HDAC5 kinases or modulators of such kinases that enable phosphorylated GAL4-HDAC5 to recruit 14-3-3-VP16 with consequent reconstitution of a functional transcriptional complex. Our results reveal a remarkable variety of signaling pathways that converge on the signal-responsive phosphorylation sites in HDAC5, thereby enabling HDAC5 to connect extracellular signals to the genome.

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Figures

Fig. 1.

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 kinases or activators of kinases that phosphorylate the 14-3-3-binding sites in HDAC5, resulting in the recruitment of 14-3-3-VP16 and reconstitution of a transcriptional complex. (C) Results from a transfection assay in a representative 96-well plate are shown. Each well received a pool of ≈50–100 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.

Fig. 2.

Fig. 2.

Activation of UAS-luciferase by expression of cDNAs that promote the association of GAL4-HDAC5 and 14-3-3-VP16. COS cells were transfected with UAS-luciferase and expression plasmids encoding GAL4 fused to the wild-type HDAC5 N-terminal extension or mutants of this region in which serine 259 and/or 498 were mutated to alanines, as indicated, along with 14-3-3-VP16 and expression plasmids for individual activating cDNAs. Mutation of single serines severely impaired activation of UAS-luciferase and mutation of both serines abolished activation.

Fig. 3.

Fig. 3.

Phosphorylation of HDAC4 and HDAC5 by extracts from Mark2-transfected cells. COS cells were transiently transfected with an empty pcDNA3 expression plasmid (-) or a pcDNA3-Mark2 expression plasmid (+). Cell extracts were prepared and used for in vitro kinase assays with GST–HDAC fusion proteins and [γ-32P]ATP. WT, GST fusion proteins with the wild-type amino acid sequence; Mut, GST fusion proteins in which the signal-responsive serines were mutated to alanines.

Fig. 4.

Fig. 4.

Effects of activating cDNAs on nuclear/cytoplasmic distribution of class II HDACs. (A) COS cells were transiently transfected with expression plasmids encoding FLAG-tagged HDACs, as shown in each column, and activators of HDAC5 phosphorylation, as shown in each row. The subcellular distribution of HDACs was determined by immunostaining as described in Materials and Methods. Representative fields are shown. (B) The percentage of cells containing FLAG-tagged HDACs in the cytoplasm was determined by counting at least 100 transfected cells.

Fig. 5.

Fig. 5.

Signaling pathways directed at serines 259 and 498 of HDAC5. The ability of class II HDACs to inhibit activity of transcription factors is abolished by phosphorylation at signal-responsive serines, which creates 14-3-3 docking sites and results in the nuclear export of the complex. A variety of inducers of cardiac hypertrophy, including ET-1, sphingosine-1 phosphate, lysophosphatidic acid, serotonin (5-HT), and RhoA signaling, stimulate HDAC kinase activity and consequently derepress HDAC targeted transcription factors. We propose that class II HDACs serve as a nodal point that transmits extracellular and intracellular signals to the genome and controls gene expression.

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