Nuclear Coactivator-62 kDa/Ski-interacting Protein Is a Nuclear Matrix-associated Coactivator That May Couple Vitamin D Receptor-mediated Transcription and RNA Splicing

Abstract

Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDRactivated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D 3 -dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D 3 -induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing. The vitamin D receptor (VDR)1 is a nuclear receptor (NR) family member that mediates the biological actions of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the active hormone of the vitamin D endocrine system. VDR forms a heterodimer with retinoid X receptor (RXR) and binds to specific vitamin Dresponsive elements (VDREs) in the promoter region of target genes to regulate transcription by RNA polymerase II (1, 2). Transcriptional activation through 1,25-(OH) 2 D 3 and VDR is enhanced by nuclear receptor coactivator proteins such as steroid receptor coactivators (SRCs) (3-5) and proteins of the vitamin D receptor-interacting protein (DRIP) complex (6, 7). The SRCs and DRIPs utilize leucine-rich LXXLL motifs (8, 9) to interact in a 1,25-(OH) 2 D 3 -dependent manner with a complementary hydrophobic cleft on the surface of the VDR ligand binding domain. This hydrophobic surface is composed of helices H3, H4, H5, and H12, and it is the ligand-dependent folding of H12 that creates the interaction cleft for SRC binding to NRs (10 -15). The H12 helix of the NRs contains the critical ligand-dependent activation function-2 domain that is essential for ligand activated transcription (16, 17) and ligand-dependent S...

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