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 VDR-activated 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 D3-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 D3-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.
Highlights
Lated to other vitamin D receptor (VDR) coactivators such as those in the responsive elements (VDREs) in the promoter region of target steroid receptor coactivator (SRC) family
Chromatin immunoprecipitation studies revealed that endogenous Nuclear coactivator-62 kDa (NCoA62)/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells
NCoA62/SKIP lacks LXXLL type motifs, it interacts with VDR in an activation function-2-independent manner [18], and it interacts with VDR through domains that are distinct from the H3–5/H12 surface cleft [20]
Summary
Lated to other VDR coactivators such as those in the responsive elements (VDREs) in the promoter region of target steroid receptor coactivator (SRC) family. We show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-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, suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing. Important differences exist between NCoA62/SKIP and SRCs. For example, NCoA62/SKIP lacks LXXLL type motifs, it interacts with VDR in an activation function-2-independent manner [18], and it interacts with VDR through domains that are distinct from the H3–5/H12 surface cleft [20]. § To whom all correspondence should be addressed: Dept. of Phar- NR, nuclear receptor; SRC, steroid receptor coactivator; GST, glutathimacology, Case Western Reserve University, 10900 Euclid Ave., one S-transferase; ChIP, chromatin immunoprecipitation; DRIP, vita-
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