Identification of a novel RING finger protein as a coregulator in steroid receptor-mediated gene transcription.

Using the DNA-binding domain of androgen receptor (AR) as a bait in a yeast two-hybrid screening, we have identified a small nuclear RING finger protein, termed SNURF, that interacts with AR in a hormone-dependent fashion in both yeast and mammalian cells. Physical interaction between AR and SNURF was demonstrated by coimmunoprecipitation from cell extracts and by protein-protein affinity chromatography. Rat SNURF is a highly hydrophilic protein consisting of 194 amino acid residues and comprising a consensus C3HC4 zinc finger (RING) structure in the C-terminal region and a bipartite nuclear localization signal near the N terminus. Immunohistochemical experiments indicated that SNURF is a nuclear protein. SNURF mRNA is expressed in a variety of human and rat tissues. Overexpression of SNURF in cultured mammalian cells enhanced not only androgen, glucocorticoid, and progesterone receptor-dependent transactivation but also basal transcription from steroid-regulated promoters. Mutation of two of the potential Zn2+ coordinating cysteines to serines in the RING finger completely abolished the ability of SNURF to enhance basal transcription, whereas its ability to activate steroid receptor-dependent transcription was maintained, suggesting that there are separate domains in SNURF that mediate interactions with different regulatory factors. SNURF is capable of interacting in vitro with the TATA-binding protein, and the RING finger domain is needed for this interaction. Collectively, we have identified and characterized a ubiquitously expressed RING finger protein, SNURF, that may function as a bridging factor and regulate steroid receptor-dependent transcription by a mechanism different from those of previously identified coactivator or integrator proteins.

Activation of androgen receptor function by a novel nuclear protein kinase.

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

Disruption of the murine PIASx gene results in reduced testis weight.

PIASx belongs to the PIAS protein family, the members of which modulate activities of several transcription factors and act as E3 ligases in the sumoylation pathway. The PIASx gene is highly expressed in testis, suggesting a role in spermatogenesis. To investigate the function of PIASx in vivo, we have disrupted the PIASx gene in mice. Interestingly, the knockout mice were viable and fertile. Despite the normal fertility, the testis weight of the mutant animals was reduced and their number of apoptotic testicular cells was increased. Also, the sperm count of mutant mice tended to be reduced, but the quality of their sperm cells was normal. No significant changes were observed in the serum levels of LH and FSH or in the intratesticular testosterone concentration between the knockout animals and their wild-type littermates. Compensatory increases in other PIAS protein mRNAs were not observed in the knockout mice. These results imply that PIASx is required quantitatively rather than qualitatively for normal spermatogenesis.

Disrupted amino- and carboxyl-terminal interactions of the androgen receptor are linked to androgen insensitivity.

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.

Androgen receptor-mediated transcriptional regulation in the absence of direct interaction with a specific DNA element.

Androgen receptor (AR) brings about a ligand-dependent inhibition of low-affinity neurotrophin receptor (p75) promoter constructs in cultured cells, with the greatest inhibition being achieved with a reporter gene containing 1050 nucleotides (nt) of the promoter. The receptor domain critical for trans-repression localizes to the same region (amino acids 147-296) as that mandatory for transactivation. In contrast to trans-activation, AR does not interact directly with specific DNA elements to elicit trans-repression of p75 promoter constructs, although an intact DNA-binding domain of the receptor is required for both actions. In a search for interacting partners, both extensively purified full-length AR and AR-DNA binding domain were found to inhibit c-Jun/AP-1 site interaction without themselves binding to the AP-1 element. Prior binding of c-Jun to the AP-1 element protected the complex from the receptor's interference. Repression was not mutual, as c-Jun did not inhibit AR-androgen response element interaction or trans-activation through an androgen response element-containing promoter. The 1050-nt-long p75 promoter sequence does not contain an AP-1 element; an AP-1-like site in the vector backbone mediates the trans-repression by the AR in recipient cells. Intriguingly, an AR form with a large N-terminal deletion (the delta 46-408 mutant) behaved as a transcriptional activator of the p75 promoter through a mechanism that was also independent of specific DNA binding. Collectively, these data indicate that, in a proper context, AR is able to elicit both transrepression and trans-activation without interacting directly with specific DNA elements. Sequences responsible for the down-regulation of p75 mRNA by androgens in vivo are, however, not located in the proximal 1050 nt of the p75 promoter.

ARIP3 (androgen receptor-interacting protein 3) and other PIAS (protein inhibitor of activated STAT) proteins differ in their ability to modulate steroid receptor-dependent transcriptional activation.

Steroid receptors mediate their actions by using various coregulatory proteins. We have recently characterized ARIP3/PIASx alpha as an androgen receptor (AR)-interacting protein (ARIP) that belongs to the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] protein family implicated in the inhibition of cytokine signaling. We have analyzed herein the roles that four different PIAS proteins (ARIP3/PIASx alpha, Miz1/PIASx beta, GBP/PIAS1, and PIAS3) play in the regulation of steroid receptor- or STAT-mediated transcriptional activation. All PIAS proteins are able to coactivate steroid receptor-dependent transcription but to a differential degree, depending on the receptor, the promoter, and the cell type. Miz1 and PIAS1 are more potent than ARIP3 in activating AR function on minimal promoters. With the natural probasin promoter, PIAS proteins influence AR function more divergently, in that ARIP3 represses, but Miz1 and PIAS1 activate it. Miz1 and PIAS1 possess inherent transcription activating function, whereas ARIP3 and PIAS3 are devoid of this feature. ARIP3 enhances glucocorticoid receptor-dependent transcription more efficiently than Miz1 or PIAS1, and all PIAS proteins also activate estrogen receptor- and progesterone receptor-dependent transcription but to a dissimilar degree. The same amounts of PIAS proteins that modulate steroid receptor-dependent transcription influence only marginally transactivation mediated by various STAT proteins. It remains to be established whether the PIAS proteins play a more significant physiological role in steroid receptor than in cytokine signaling.

Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.

A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.

Mutant and wild-type androgen receptors exhibit cross-talk on androgen-, glucocorticoid-, and progesterone-mediated transcription.

Androgen, glucocorticoid, and progesterone receptors (ARs, GRs, and PRs) often can regulate transcription via composite hormone response elements in target genes. We have used artificial and natural mutant ARs from patients with androgen resistance to study their effects on dominant negative activity on wild type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and tyrosine aminotransferase (TAT) promoters. Artificial ARs that contained internal deletions within the amino-terminal region had minimal transcriptional activity but blocked ligand-mediated transcription by wild type AR. Mutants containing deletions of the DNA-binding and ligand-binding domains had minimal or weak dominant negative activity. We then tested the ability of wild type and mutant ARs to modulate GR- and PR-mediated transcriptional activity. The amino-terminal deletion mutants exerted dominant negative effects on GR- and PR-mediated activity, both in the absence and presence of testosterone. Surprisingly, wild type AR, which had approximately 20% of the maximal transcriptional activity of GR on the MMTV promoter, also had dominant negative activity on dexamethasone-regulated transcription mediated by GR. This dominant negative activity likely involves DNA binding because a point mutation in the DNA-binding domain abrogated such activity of an amino-terminal deletion mutant. Additionally, natural human AR mutants from patients with androgen resistance, which do not bind either DNA or ligand, did not block dexamethasone-mediated transcription. In summary, these studies suggest that mutant and wild type ARs can display dominant negative activity on other steroid hormone receptors that bind to a composite hormone response element This cross-regulation may be important in regulating maximal transcriptional activity in tissues where these receptors are coexpressed and may contribute to the phenotype of patients with steroid hormone resistance.

Cyclin D1 binds the androgen receptor and regulates hormone-dependent signaling in a p300/CBP-associated factor (P/CAF)-dependent manner.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

Effects of mitogens on androgen receptor-mediated transactivation.

The effects of mitogens and agents affecting tyrosine phosphorylation signaling on androgen-regulated transcription were investigated. CV-1 and HeLa cells were cotransfected with an androgen receptor (AR) expression vector and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse mammary tumor virus promoter. Growth factors [epidermal growth factor (EGF) and insulin-like growth factor I] that activate receptor tyrosine kinases, an inhibitor of phosphotyrosine phosphatases (vanadate), or an inhibitor of tyrosine kinases (genistein) did not influence basal promoter activity or that of unliganded AR. However, EGF, insulin-like growth factor I, and vanadate enhanced AR-dependent transactivation by 1.5- to 2.5-fold, and genistein diminished it by two thirds in the presence of androgen. None of the treatments affected pRSV-CAT or pSV-beta-galactosidase expression, suggesting that gross activation of the transcription machinery was not involved. A reporter with two androgen response elements (AREs) in front of the thymidine kinase promoter (p delta ARE2tk-CAT) was used to examine promoter specificity. EGF activated this reporter even in the absence of androgen. However, when EGF was used concomitantly with testosterone, it augmented the action of androgen. Vanadate enhanced androgen-induced transactivation 2-fold without altering basal promoter activity. Neither EGF nor vanadate altered immunoreactive AR content or elicited changes in the receptor's DNA-binding properties. The intracellular content of hormone-binding AR was not influenced by EGF, but was decreased by vanadate and increased by genistein, as judged by [3H]mibolerone binding assays. An AR form lacking the hormone-binding domain (delta 641-902 mutant) transactivated p delta ARE2tk-CAT reporter similar to or better than the wild-type receptor in the presence of androgen. The transactivation by the delta 641-902 mutant was augmented by EGF and vanadate, but was attenuated by genistein, implying that the steroid-binding region is not critical for regulatory events initiated by tyrosine phosphorylation. Collectively, these data indicate that there is cross-talk between androgen-mediated signaling systems and growth factor/receptor tyrosine kinase pathways.

Comparison of androgen regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase gene expression in rodent kidney and accessory sex organs.

Androgen regulation of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities and accumulation of the mRNAs encoding these two enzymes in rodent kidney and accessory sex organs were studied. The ODC mRNA concentration and enzyme activity were increased by androgens in kidney, prostate, and seminal vesicle of 3-day castrated mice and rats, whereas AdoMetDC mRNA and protein levels were androgen inducible only in rodent accessory sex organs. ODC and AdoMetDC mRNAs were regulated in prostate and seminal vesicles in a coordinate fashion, with the maximal levels reached within 24-48 h of steroid exposure. The extent of induction was similar for the two gene products, and ODC and AdoMetDC mRNA accumulation occurred primarily in epithelial cells of accessory sex organs, as shown by in situ hybridization studies. The murine ODC promoter contains an androgen-responsive element (ARE)-like sequence at about -910 nucleotides from the cap site; this element binds to androgen receptor in vitro, albeit with much lower affinity than some other AREs. In transient expression studies with CV-1 cells, an ODC promoter construct (pODCCAT) conferred androgen responsiveness upon the reporter gene. ODC mRNA accumulation is androgen regulated in epithelial cells of proximal tubules in both murine and rat kidney; however, different subtypes of the proximal tubular cells respond in the two species, as revealed by in situ hybridization studies. Expression of the ODC gene was induced more markedly and for a longer duration in the murine than in the rat kidney, but the initial response occurred faster in the rat kidney. The relatively slow kinetics of ODC mRNA accumulation in mouse kidney were not due to recruitment of new cells to respond; rather, in situ hybridization studies indicated that there was progressive accumulation of the mRNA in the responding cells. Collectively, these data indicate that the genes for two key enzymes in polyamine biosynthesis are not regulated in an identical fashion in different androgen target tissues, such as rodent kidney and accessory sex organs.

Agonists, but not antagonists, alter the conformation of the hormone-binding domain of androgen receptor.

Androgen receptors synthesized by translation in vitro form dimeric aporeceptor complexes with an androgen response element (ARE). Physiological and synthetic androgens elicit a conformational change in the receptor, which increases the mobility of receptor-ARE complexes in gel retardation assays. Neither a steroidal (cyproterone acetate) nor a non-steroidal (casodex) antiandrogen brings about the same effect and, at high concentrations, reverse the action of androgen agonists. When receptor-agonist complexes are subjected to a limited trypsin or chymotrypsin digestion, a protease-resistant 30-kDa fragment corresponding to the entire ligand-binding domain is formed. A similar fragment is not protected in the presence of antiandrogens. The C-terminal origin of the protected region was verified using mutated receptor forms: a mutant with a large N-terminal deletion behaves like the wild-type protein, but the properties of a hormone binding-negative receptor, due to a single-base substitution at codon 807, are not influenced by androgen agonists or antagonists.

Transcription activating and repressing functions of the androgen receptor are differentially influenced by mutations in the deoxyribonucleic acid-binding domain.

Despite the wide spectrum of androgen receptor (AR) mutants described in androgen insensitivity syndromes (AIS), their influence on transactivating and, in particular, transrepressing functions of AR are poorly defined. Rat AR mutants with substitutions in the DNA-binding domain, corresponding to several mutations in AIS patients, were examined for these activities. AR variants (G551V and C562G) with mutations in the first zinc finger (ZF) exhibited reduced DNA binding activity and attenuated transactivation. An R590Q substitution in the second ZF diminished transcriptional activity only from a promoter with a single androgen response element, whereas activation at multiple androgen response element sites was unaffected, despite the poor DNA-binding affinity of R590Q. Another substitution in the second ZF, A579T, yielded similar findings. In comparison to wild-type AR, G551V, and C562G variants had markedly reduced ability to repress an NF-kappaB/RelA-activated promoter but R590Q behaved like the native receptor. AP1 function was repressed not only by wild-type AR but also by the transactivating mutants A579T and R590Q as well as by the transcriptionally inactive mutants G551V and C562G. Furthermore, a Lys-to-Ala substitution in codon 563 of the first ZF switched AR into a ligand-dependent activator at AP1 sites but maintained the ability to repress NF-kappaB/RelA function. Taken together, DNA-binding domain mutations in AIS patients influence transcriptional activating and repressing functions of AR in a selective fashion, which probably contributes to the complexity in the presentation of the AIS phenotype.

Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation.

The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.

Novel assay for determination of androgen bioactivity in human serum.

We have developed a mammalian cell (COS-1) bioassay, which can measure androgen bioactivity directly from a small amount (10 microL) of human serum. The recombinant assay is based on androgen-dependent interaction between the ligand-binding domain and the N-terminal region of the androgen receptor (AR), which were fused to Gal4 DNA-binding domain of Saccharomyces cerevisiae and transcriptional activation domain of herpes simplex VP16 protein, respectively. The interaction is amplified by coexpression of AR-interacting protein 3 in the cells. The reporter plasmid contains 5 Gal4-binding sites upstream of the luciferase gene; luciferase activity in cell lysates is derived from androgen bioactivity in human serum. Saturating concentration of testosterone in FCS induced more than 700-fold induction in relative luciferase activity. The sensitivity was less than 1.0 nmol/L testosterone in FCS. The intra- and interassay coefficients of variation were 8.3% and 21%, respectively. Interaction between the AR termini was blocked by nonsteroidal antiandrogens, and the assay exhibited minimal cross-reactivity with 17 beta-estradiol. Serum androgen bioactivity was studied in 23 boys (13.9--16.8 yr old) with constitutional delay of puberty and in 9 prepubertal boys with cryptorchidism (1.0--6.4 yr old). Androgen bioactivity was detectable in 15 boys with constitutional delay of puberty and in all boys with cryptorchidism during treatment with human CG (range, 1.0-14.5 nmol/L testosterone equivalents). Serum androgen bioactivity measured by the bioassay correlated strongly with serum testosterone concentration (r = 0.93, P < 0.0001, n = 22) but not to 5 alpha-dihydrotestosterone, dehydroepiandrosterone, or androstenedione levels. We conclude that our novel bioassay enables quantitation of mammalian cell response to bioactive androgens in human serum, even in pediatric patients with relatively low androgen levels.

Characterization of the cystatin B gene promoter harboring the dodecamer repeat expanded in progressive myoclonus epilepsy, EPM1.

Mutations in the gene encoding cystatin B (CSTB) are responsible for the primary defect in progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1). A novel and unique type of disease-causing mutation, an unstable dodecamer repeat expansion, accounts for the majority of EPM1 patients world-wide. This minisatellite repeat expansion, located in the putative promoter of CSTB 175 bp upstream from the translation initiation codon, appears to downregulate CSTB gene expression in vivo. We report here the characterization of the CSTB promoter using different promoter-luciferase gene constructs. Transient transfections of cultured mammalian cells suggest that the region from -670 to -1 bp from the translation initiation codon functions as the CSTB promoter. Active binding to five Sp1 and four AP1 sites as well as weak binding to an androgen response element (ARE) half site was demonstrated by electrophoretic mobility shift assays. The effect of the minisatellite expansion on the promoter activity was evaluated by comparing the activity of constructs containing wild-type and expanded alleles. An increase in the number of dodecamer units from three to 19 repeats lowered transcription in vitro by 10-fold. Northern analysis of lymphoblastoid RNA from individuals with 'premutation' length dodecamer repeat (12-17 copies) expansions showed decreased levels of CSTB mRNA expression. These data indicate that expansion of the dodecamer repeat located in the proximal promoter of CSTB severely disrupts the function of the promoter and thereby reduces transcription of CSTB.

Heterodimerization is mainly responsible for the dominant negative activity of amino-terminally truncated rat androgen receptor forms.

Rat androgen receptor (rAR) mutants devoid of the amino-terminal transactivation domain are able to behave as dominant negative regulators of wild-type rAR. To address the underlying mechanisms of the trans-dominant negative action, we have examined the roles of the DNA-binding domain (DBD) and the ligand-binding domain (LBD) in this process. Transactivation experiments in CV-1 cells complemented by electrophoretic mobility shift assays revealed that the dominant negative receptor forms repress the function of wild-type rAR mainly through heterodimer formation, rather than through competition for binding to cognate DNA elements. Heterodimerization of receptor forms containing LBDs may take place even in the absence of specific DNA binding.

Pure antiandrogens disrupt the recruitment of coactivator GRIP1 to colocalize with androgen receptor in nuclei.

We have used confocal microscopy to elucidate the effects of antiandrogens on nuclear localization of the androgen receptor (AR) with its transcriptional coactivator GRIP1. We show that the agonist-activated AR recruits GRIP1 to colocalize with the receptor in the nucleoplasm. By contrast, AR complexed to the antiandrogens hydroxyflutamide and bicalutamide fails to influence nuclear distribution of GRIP1. Likewise, the non-steroidal antiandrogens prevent the agonist-induced AR-GRIP1 colocalization from occurring. Androgen antagonists affect nuclear redistribution of AR-GRIP1 in a fashion that parallels their effects on the transcriptional activity of AR, in that the pure antagonists block GRIP1-dependent activation of AR function, whereas the mixed antagonist/agonist cyproterone acetate promotes both AR-driven redistribution of GRIP1 and activation of AR by GRIP1.

Nuclear factor Y activates the human xanthine oxidoreductase gene promoter.

To study the regulation of the human xanthine oxidoreductase (XOR) gene, we cloned 1.9 kb of the promoter region. In reporter gene assays, a construct encompassing nucleotides between 142 to +42 conferred maximal basal activity of the XOR promoter in 293T cells, in comparison with shorter (-92 to +42) or longer (up to -1937 to +42) constructs. The promoter activity was low in NIH-3T3 cells. The most active construct contained a putative CCAAT motif at -119 to -123. Electrophoretic mobility shift assays showed that this sequence binds the ubiquitous nuclear factor Y (NF-Y). Mutation of the CCAAT motif (CTGAT) abolished the NF-Y binding and considerably reduced the promoter activity. Our data suggest an important functional role for NF-Y in the transcriptional activation of the human XOR gene.

Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity.

SNURF/RNF4 has been implicated in transcriptional regulation and growth inhibition in a RING finger-dependent fashion. In this work, we show that SNURF mediates its own ubiquitination in vitro in a ubiquitin-conjugating enzyme (E2)-selective manner: SNURF acts as an E3 ligase with UbcH5A and B, HHR6B (RAD6B), E2-25K, MmUbc7 and UbcH13, but not with UbcH3, UbcM4, MmUbc6 or E2-20K. In contrast, the well-characterized RING E3, AO7, functions only with members of the UbcH5 family. Furthermore, depending on the E2 used, the ubiquitin modification manifests as mono- or multi-ubiquitination. Mutation of conserved cysteine residues within the RING finger motif of SNURF abolishes the ubiquitination in vitro and in intact cells. Size fractionation of murine embryonal carcinoma F9 cell proteins shows that the majority of endogenous SNURF resides in salt-resistant > or =500-kDa complexes, suggesting that SNURF functions as a RING component in a multiprotein complex. Taken together, SNURF/RNF4 functions as an E3 ligase and this activity is closely linked to its transcription regulatory functions.