Identification and Characterization of a Novel Nuclear Protein Complex Involved in Nuclear Hormone Receptor-mediated Gene Regulation

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 ϳ1.5 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-␣ 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.A number of crucial insights into the multilayered regulation of transcription have been uncovered through study of nuclear hormone receptor-mediated gene expression. Nuclear hormone receptors are hormone-and ligand-dependent transcription factors that control the coordinated expression of gene networks in numerous physiological, developmental, and metabolic processes (1). Dysfunction of nuclear receptor signaling leads to a number of proliferative, reproductive, and metabolic diseases such as cancer, infertility, obesity, and diabetes (2). The biological functions of nuclear hormone receptors rely on coactivators that represent a diverse group of proteins that enhance nuclear receptor-mediated transcription (3). Extensive studies on the in vivo functions of nuclear receptors have led to the identification and characterization of ϳ200 coactivators, all of which have been catalogued on-line at the Nuclear Receptor Signaling Atlas. Coactivators, in general, are known to act by: (i) bridging factors to recruit additional cofactors to DNA-bound nuclear receptors, e.g. p160/SRC proteins (4, 5); (ii) exhibiting various enzymatic activities such as methylation, acetylation, and others to modulate chromatin (6 -8); and (iii) interfacing between DNA-bound nuclear receptors and the basal transcriptional machinery, e.g. TRAP/DRIP complex (9).Nuclear receptor coregulator (NRC) 3 also referred to as ASC-2, TRBP...

A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex

“…Whether WRAD brings a histone methyltransferase activity to these complexes remains to be determined. Intriguingly, enzymatic assays with an immunoprecipitated NIF1 complex shows histone H3 methylation activity, albeit in a manner that is largely independent of H3K4 (65). It is therefore likely that the majority of enzymatic activity of the NIF1 complex is due to co-immunopurification of other histone lysine or arginine methyltransferases.…”

“…The apparent molecular mass of this complex determined by gel filtration is comparable with the theoretical molecular mass for the WDR5, RbBP5, and Ash2L complex (156 kDa) with 1:1:1 stoichiometry and is consistent with our sedimentation velocity analytical ultracentrifugation characterization of WRA(D). WRA(D) has also been identified in other complexes that appear to lack SET1 family enzymes, such as the CHD8 and NIF1 complexes (63)(64)(65). Whether WRAD brings a histone methyltransferase activity to these complexes remains to be determined.…”

DBC1 does not function as a negative regulator of SIRT1 in liver cancer

“…DBC1 induces p53-mediated apoptosis by negatively regulating silent mating type information regulation 2 homolog 1 (SIRT1) activity (6,7), and by directly interacting with estrogen receptor-α (ERα) promotes breast cancer cell survival (8). In addition, DBC1 regulates cancer cell growth by mediating the transcriptional activation of retinoic acid receptor α (RAR α) in breast cancer or androgen receptor (AR) in prostate cancer cells (9,10). DBC1 also regulates epigenetic mechanisms by inhibiting SUV39H1 methyltransferase and histone deacetylase 3 (HDAC3) (11,12).…”

WDR5, ASH2L, and RBBP5 control the efficiency of FOS transcript processing

“…Amplification and overexpression of the RBBP5 gene were found in patient-derived tumor samples [14]. Although biochemical purification and co-immunoprecipitation studies indicate the presence of the WAR subcomplex [7,12,13,35], compelling evidence for such an independently functioning complex under physiological conditions is still lacking as the experimental conditions may result in the loss of an integral partner of a protein complex. Overall, our understanding of IEG regulation is mainly based on FOS, and detailed descriptions of the regulatory mechanisms for other IEGs such as ZFP36 and EGR1 are less abundant [24].…”