Phosphorylation of thyroid hormone receptor-associated nuclear receptor corepressor holocomplex by the DNA-dependent protein kinase enhances its histone deacetylase activity.
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.
Jeyakumar
M
M
Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801.
Liu
Xue-Feng
XF
Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801.
Erdjument-Bromage
Hediye
H
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Tempst
Paul
P
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Bagchi
Milan K
MK
Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801. Electronic address: mbagchi@life.uiuc.edu.
D051747:DNA-Activated Protein Kinase / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D056971:Nuclear Receptor Co-Repressor 1; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D047488:Retinoid X Receptors / Q000378:metabolism; D000099186:Histone Deacetylase 3
D051747:DNA-Activated Protein Kinase; D006655:Histone Deacetylases; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D011988:Receptors, Thyroid Hormone; D012097:Repressor Proteins; D047488:Retinoid X Receptors; D000099186:Histone Deacetylase 3; C539127:NCOR1 protein, human