Transcriptional silencing by many transcription factors is mediated by the nuclear receptor corepressor (N-CoR). The mechanism by which N-CoR represses basal transcription involves the direct or indirect recruitment of histone deacetylases (HDACs). We have isolated two multiprotein N-CoR complexes, designated N-CoR-1 and N-CoR-2, which possess histone deacetylase activity that is mediated by distinct HDACs. Based on Western blotting using antibodies against known subunits, the only HDAC found in the N-CoR-1 complex was HDAC3. In contrast, N-CoR-2 contained predominantly HDAC1 and HDAC2 as well as several other subunits that are found in the Sin3A⅐HDAC complex. Using mass spectrometry and Western blotting, we have identified several novel components of the N-CoR-1 complex including the SWI/SNF-related proteins BRG1, BAF 170, BAF 155, BAF 47/INI1, and the corepressor KAP-1 that is involved in silencing heterochromatin. Indirect immunofluorescence has revealed that both KAP-1 and N-CoR colocalize throughout the nucleus. These results suggest that N-CoR is found in distinct multiprotein complexes, which are involved in multiple pathways of transcriptional repression.Cellular proliferation and differentiation is critically dependent on the ability of specific DNA-binding transcription factors to activate or repress the transcription of target genes in a coordinated fashion. This is accomplished through transcription factor-mediated recruitment of coactivators and corepressors, which can regulate transcription through the modification of the local chromatin environment and by specifically interacting with components of the core transcriptional machinery (1, 2). The nuclear hormone receptor superfamily provides a unique model to study the mechanism of transcriptional activation and repression and the role of reversible chromatin modification in the control of gene expression. In the absence of ligand, nuclear hormone receptors such as the thyroid hormone (TR) 1 and retinoic acid receptors (RAR) function as potent repressors by interacting with specific corepressor proteins (3, 4). Ligand binding induces a conformational change in these receptors that results in corepressor release and the recruitment of coactivator proteins (2, 5). The nuclear receptor corepressor (N-CoR), and its related family member silencing mediator for retinoid and thyroid hormone receptor (SMRT), were initially identified by yeast two hybrid screening using unliganded TR or RAR as bait, respectively (3, 4). Several lines of evidence suggest that both N-CoR and SMRT mediate the repressive effects of nuclear hormone receptors. First, both N-CoR and SMRT contain two nuclear receptor interaction domains (IDs) in the carboxyl terminus. Molecular characterization of the IDs reveals the presence of a signature CoR box motif that is necessary and sufficient for receptor interaction and ligand-induced release of N-CoR or SMRT (6 -8). More recently, a strong correlation between repression mediated by the TR and recruitment of N-CoR or SMRT have also been ...

Corticosteroid-binding globulin (CBG) is a serine proteinase inhibitor (serpin) family member that transports glucocorticoids in blood and regulates their access to target cells. The 1.9 Å crystal structure of rat CBG shows that its steroid-binding site resembles the thyroxin-binding site in the related serpin, thyroxin-binding globulin, and mutagenesis studies have confirmed the contributions of key residues that constitute the steroid-binding pocket. Unlike thyroxin-bound thyroxin-binding globulin, the cortisol-bound CBG displays an "active" serpin conformation with the proteinase-sensitive, reactive center loop (RCL) fully expelled from the regulatory ␤-sheet A. Moreover, the CBG structure allows us to predict that complete insertion of the proteolytically cleaved RCL into the serpin fold occurs in concert with a displacement and unwinding of helix D that would disrupt the steroid-binding site. This allosteric coupling between RCL positioning and occupancy of the CBG steroidbinding site, which resembles the ligand (glycosamino-glycan)-dependent activation of the thrombin inhibitory serpins heparin cofactor II and anti-thrombin RCLs, ensures both optimal recognition of CBG by target proteinases and efficient release of steroid to sites of action.Glucocorticoid hormones (cortisol and corticosterone) and progesterone are transported in the blood by a glycoprotein known as corticosteroid-binding globulin (CBG) 3 or transcortin (1). Plasma CBG also binds several synthetic glucocorticoids, such as prednisolone (2), and influences the half-life, distribution, and efficacy of these drugs in the same way as for natural steroids (3). The steroid binding characteristics of CBG from many species have been documented (1), and a benchmark set of CBG steroid binding characteristics has be used to develop three-dimensional quantitative structure activity relationship (3D-QSAR) computational methods (4, 5) aimed at predicting the binding affinities of ligands to target proteins. Although residues within CBG sequences critical for steroid binding have been identified through studies of naturally occurring variants (6 -10), photo-affinity labeling (11), and mutagenesis (12), a precise picture of its structure and steroidbinding site has been lacking.The primary structure of CBG defines it as a clade A member of the serine proteinase inhibitor (serpin) superfamily, together with the related transport protein for thyroxin, thyroxin-binding globulin (TBG), and the prototypical serpin members ␣1-antitrypsin (AAT) and ␣1-antichymotrypsin (ACT), among others (13,14). A hallmark of serpin structures is that they undergo conformational rearrangements as part of their biological function. The conformations they adopt are highly dependent on whether a surface-exposed loop, known as the reactive center loop (RCL) or "proteinase bait" domain, is intact. Cleavage of the RCL segment by proteinases usually causes a typical stressed to relaxed (S 3 R) transition in structure (15). This involves insertion of the entire N-terminal segment of t...

The transcriptional coactivator p/CIP(SRC-3/AIB1/ACTR/RAC3) binds liganded nuclear hormone receptors and facilitates transcription by directly recruiting accessory factors such as acetyltransferase CBP/p300 and the coactivator arginine methyltransferase CARM1. In the present study, we have established that recombinant p/CIP (p300/CBP interacting protein) is robustly methylated by CARM1 in vitro but not by other protein arginine methyltransferase family members. Metabolic labeling of MCF-7 breast cancer cells with S-adenosyl-L-[methyl-3 H]methionine and immunoblotting using dimethyl arginine-specific antibodies demonstrated that p/CIP is specifically methylated in intact cells. In addition, methylation of full-length p/CIP is not supported by extracts derived from CARM1 ؊/؊ mouse embryo fibroblasts, indicating that CARM1 is required for p/CIP methylation. Using mass spectrometry, we have identified three CARM1-dependent methylation sites located in a glutamine-rich region within the carboxy terminus of p/CIP which are conserved among all steroid receptor coactivator proteins. These results were confirmed by in vitro methylation of p/CIP using carboxyterminal truncation mutants and synthetic peptides as substrates for CARM1. Analysis of methylation site mutants revealed that arginine methylation causes an increase in full-length p/CIP turnover as a result of enhanced degradation. Additionally, methylation negatively impacts transcription via a second mechanism by impairing the ability of p/CIP to associate with CBP. Collectively, our data highlight coactivator methylation as an important regulatory mechanism in hormonal signaling.

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