Ligand-induced transrepressive function of VDR requires a chromatin remodeling complex, WINAC

We have previously shown that the novel ATP-dependent chromatin remodeling complex WINAC is required for the ligand-bound Vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D₃ 1-hydroxylase [1(OH)ase] gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated histones, that appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1(OH)ase gene expression.     

Ligand-induced transrepression by VDR through association of WSTF with acetylated histones

We have previously shown that the novel ATP-dependent chromatin-remodeling complex WINAC is required for the ligand-bound vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D3 1alpha-hydroxylase (1alpha(OH)ase) gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1alpha(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated his tones, which appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1alpha(OH)ase gene expression.     

Cloning and developmental expression of WSTF during Xenopus laevis embryogenesis

The gene WSTF is deleted in the autosomal dominant hereditary disorder Williams-Beuren syndrome. This disorder is caused by a 1.3 megabase deletion in human chromosome 7, encompassing at least 17 genes. The WSTF protein contains a bromodomain, found predominantly in chromatin-associated proteins. Reported association of WSTF with chromatin remodeling factors and functional data support a role for WSTF during chromatin remodeling. Here, we report the cloning and developmental expression pattern of Xenopus laevis WSTF. Xenopus laevis WSTF is a protein with a predicted amino acid sequence of 1441 amino acids. Three discrete domains can be identified in the Xenopus laevis WSTF protein, a PHD finger, a DDT domain and a bromodomain. Alignment of Xenopus WSTF with the corresponding orthologues from Homo sapiens, Gallus gallus, Mus musculus and Danio rerio demonstrates an evolutionary conservation of WSTF amino acid sequence and domain organization. In situ hybridization reveals a dynamic expression profile during embryonic development. WSTF is expressed differentially in neural tissue, especially during neurulae stages in the eye, in neural crest cells and the brain.     

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.     

The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes

The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4's ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4-PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF-SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection.