Hetti Poukka

Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

ABSTRACT 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.

Ubc9 Interacts with the Androgen Receptor and Activates Receptor-dependent Transcription

Ubc9, a homologue of the class E2 ubiquitin-conjugating enzymes, has recently been shown to catalyze conjugation of a small ubiquitin-like molecule-1 (SUMO-1) to a variety of target proteins. SUMO-1 modifications have been implicated in the targeting of proteins to the nuclear envelope and certain intranuclear structures and in converting proteins resistant to ubiquitin-mediated degradation. In the present work, we find that Ubc9 interacts with the androgen receptor (AR), a member of the steroid receptor family of ligand-activated transcription factors. In transiently transfected COS-1 cells, AR-dependent but not basal transcription is enhanced by the coexpression of Ubc9. The N-terminal half of the AR hinge region containing the C-terminal part of the bipartite nuclear localization signal is essential for the interaction with Ubc9. Deletion of this part of the nuclear localization signal, which does not completely prevent the transfer of AR to the nucleus, abolishes the AR-Ubc9 interaction and attenuates the transcriptional response to cotransfected Ubc9. The C93S substitution of Ubc9, which prevents SUMO-1 conjugation by abrogating the formation of a thiolester bond between SUMO-1 and Ubc9, does not influence the capability of Ubc9 to stimulate AR-dependent transactivation, implying that Ubc9 is able to act as an AR coregulator in a fashion independent of its ability to catalyze SUMO-1 conjugation.

A TESTIS-SPECIFIC ANDROGEN RECEPTOR COREGULATOR THAT BELONGS TO A NOVEL FAMILY OF NUCLEAR PROTEINS

We have characterized a novel partner for androgen receptor (AR), termed ARIP3, that interacts with the DNA-binding domain/zinc finger region of AR and is predominantly expressed in the testis. Rat ARIP3 is a nuclear protein comprising 572 amino acids. It modulates AR-dependent but not basal transcription, suggesting that ARIP3 acts as an AR transcriptional coregulator. Except for the C-terminal AR-interacting domain, ARIP3 contains distinct regions that are also present in two recently described proteins, a protein inhibitor of activated Stat3 and an RNA helicase II-interacting protein (Gu/RH-II binding protein). Conserved structural features of these proteins indicate the existence of a gene family involved in the regulation of various transcription factors. Collectively, ARIP3 belongs to a novel nuclear protein family and is perhaps the first tissue-specific coregulator of androgen receptor.

Small ubiquitin-related modifier-1 (SUMO-1) modification of the glucocorticoid receptor.

Small ubiquitin-related modifier-1 (SUMO-1) is covalently attached to many cellular targets to regulate protein-protein and protein-DNA interactions, as well as localization and stability of the target protein. The SUMO-1-conjugating E2 enzyme Ubc9 is known to interact with the glucocorticoid receptor (GR), a ligand-dependent transcription factor. In the present study, we show that GR is post-translationally modified by SUMO-1 (sumoylated) in a ligand-enhanced fashion. We identify experimentally three consensus SUMO attachment sites, two in the N-terminal transactivation region and one in the ligand-binding domain of GR. The two N-terminal sites are the major acceptor sites for SUMO-1 attachment. Mutation of these sites enhances transcriptional activity of GR on minimal promoters, but has no clear effect on the more complex mouse mammary tumour virus promoter. Thus SUMO-1 modification of GR influences receptor function in a promoter context-dependent fashion.

Coregulator Small Nuclear RING Finger Protein (SNURF) Enhances Sp1- and Steroid Receptor-mediated Transcription by Different Mechanisms

The small nuclear RING finger protein SNURF is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroid-independent promoters. In this work, we show that SNURF, via the RING finger domain, enhances protein binding to Sp1 elements/GC boxes and interacts and cooperates with Sp1 in transcriptional activation. The activation of androgen receptor (AR) function requires regions other than the RING finger of SNURF, and SNURF does not influence binding of AR to cognate DNA elements. The zinc finger region (ZFR) together with the hinge region of AR are sufficient for contacting SNURF. The nuclear localization signal in the boundary between ZFR and the hinge region participates in the association of AR with SNURF, and a receptor mutant lacking the C-terminal part of the bipartite nuclear localization signal shows attenuated response to coexpressed SNURF. Some AR ZFR point mutations observed in patients with partial androgen insensitivity syndrome or male breast cancer impair the interaction of AR with SNURF and also render AR refractory to the transcription-activating effect of SNURF. Collectively, SNURF modulates the transcriptional activities of androgen receptor and Sp1 via different domains, and it may act as a functional link between steroid- and Sp1-regulated transcription.

Transcription Activating and Repressing Functions of the Androgen Receptor Are Differentially Influenced by Mutations in the Deoxyribonucleic Acid-Binding Domain1

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.

Androgen Receptor in Transcriptional Regulation

Male sex steroids (androgens) sculpture a male body in a number of ways, such as development of male sex organs; growth of facial, body, and pubic hair; enlargement of vocal cords (deepening of the voice); loss of hair at temples; production of sperm; development of muscle strength; growth of prostate, and development of masculine behavior. Each of these events is regulated by tissue-specific mechanisms through the androgen receptor that activates and/or represses gene networks to elicit its distinct physiological actions. The principal events leading to hyperplastic and/or hypertrophic responses to androgens are, however, believed to be similar, if not identical, in reproductive and nonreproductive tissues (Berger and Watson 1989). Recent studies in a number of laboratories have delineated the ways by which androgen receptor interacts with specific DNA elements — located within and/or around responsive genes — to increase the rate of gene transcription. Much less information is available, however, about mechanisms by which androgens bring about gene repression. In order to elicit defined biological (androgenic) responses, the receptor protein ought to communicate not only with DNA motifs, but also with other transcription factors or coactivators. Finally, practically nothing is known about the role of androgen receptor protein in the stabilization of specific mRNA species.