Abstract
NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of approximately 1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-alpha was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.
Highlights
A number of crucial insights into the multilayered regulation of transcription have been uncovered through study of nuclear hormone receptor-mediated gene expression
The NRC interacting factor 1 (NIF-1) complex contains Ash2L, Retinoblastoma binding protein 5 (RbBP5), and WD-40 repeat protein 5 (WDR5), suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4
To understand how NIF-1 enhances the activity of nuclear receptors and other transcription factors, we developed methodology to purify NIF-1 complexes from cells and identify their components by mass spectrometry
Summary
Cell Culture and Plasmids—The 293T packaging cell line (A) and the HeLa S3 suspension cell line were a kind gift from Dr Jeffrey Ye. NIF-1 and other associated proteins were eluted off the beads using a FLAG peptide, and the eluates were immunoprecipitated with either control IgG or with antibodies against Ash2L, DBC-1, or EMSY. Whole cell lysates were prepared in lysis buffer (50 mM KCl, pH 7.4, 250 mM KCl, 1 mM dithiothreitol, 0.25% Nonidet P-40, 7ϫ protease inhibitor tablets (Roche Applied Science)), and equal amounts of protein were analyzed by SDS-PAGE followed by Western blotting with an anti-Sox antibody. The Vector control samples or the NIF-1-purified complex(es) were incubated at 30 °C for 1 h in buffer containing 50 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 4 mM dithiothreitol, 1 M 3H-labeled S-adenosylmethionine (Amersham Biosciences). A twohybrid assay using X-gal intensity on plates was carried out as described earlier [28]
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