Abstract
It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR.retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR.RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR.RXR.corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR.RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR.RXR.corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex.
Highlights
Several laboratories have characterized the corepressors that interact with thyroid hormone receptor (TR) [8, 13]
About fifteen additional polypeptides with relative molecular masses ranging from 50 to 400 kDa were detected in the fraction eluted from the GST-TR1⁄7RXR column but were absent in the fraction eluted from the control beads
The present study is aimed at the biochemical identification and functional characterization of transcriptional coregulatory proteins recruited by the repressive form of the TR1⁄7RXR heterodimer
Summary
Reagents—Glutathione-Sepharose-4B and reduced glutathione were purchased from Amersham Biosciences. For analyses of the TR1⁄7RXR holocomplex by glycerol density gradient fractionation or immunoprecipitation, the bead-bound complex is eluted under native conditions using 20 mM GSH. Following repeated washings with wash buffer, the bound proteins were eluted with SDS sample buffer and analyzed by Western blotting using antibodies against NCoR/SMRT and DNA-PK. Phosphorylation of TR1⁄7RXR Holocomplex and HDAC3—The TR1⁄7RXR heterodimer-bound protein complexes were isolated from HeLa nuclear extract as detailed above, except that phosphatase inhibitor cocktails (I and II) were used during receptor immobilization and incubation with nuclear extract. For immunoprecipitation of phosphorylated proteins, 32Plabeled TR1⁄7RXR holocomplex was suspended in radioimmune precipitation assay buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% IGEPAL, 0.5% deoxycholate, 0.1% SDS) and incubated with protein-A-Sepharose beads bound to an antibody against GST or NCoR or DNA-PKcs or HDAC3 or control IgG (10 g) for 2 h at 4 °C. The TRE-driven firefly luciferase and the internal control Renilla luciferase activities were measured by the instruction provided with the dual luciferase assay system (Promega)
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