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Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood-testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3'-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.

RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2O2/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2O2/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.

RING finger protein 4 (RNF4) is a multifunctional small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase (STUbL) ubiquitously expressed in all tissues, and which mainly participates in DNA repair and in chromatin and transcriptional regulation. Although RNF4 has been implicated in hematopoietic disorders, its ontogenic role during hematopoietic development remains undiscovered. We generated a zebrafish rnf4 knockout line by using transcription activator-like effector nucleases technology to address the impact of rnf4 during hematopoiesis. Rnf4-deficient zebrafish embryos exhibited sharply decreased neutrophils numbers during both primitive and definitive hematopoiesis. Mechanistic studies revealed that repression of the key granulocytic activator, CCAAT/enhancer-binding protein α ( c/ebpα), via promoter hypermethylation by SUMOylated DNA methyltransferase 1 (DNMT1) was the main cause of impaired granulopoiesis in rnf4-deficient zebrafish. In addition, for the first time, we identified DNMT1 as a potential new STUbL substrate of RNF4, with knockdown of dnmt1 largely restoring primitive and definitive granulopoiesis in rnf4-deficient zebrafish. Collectively, RNF4 is indispensable for zebrafish granulopoiesis through regulation of the DNMT1-C/EBPα functional axis.-Wang, L., Liu, X., Wang, H., Yuan, H., Chen, S., Chen, Z., de The, H., Zhou, J., Zhu, J. RNF4 regulates zebrafish granulopoiesis through the DNMT1-C/EBPα axis.

Single zinc finger domain containing proteins are very few in number. Of numerous zinc finger proteins in eukaryotes, only three of them like GAGA, Superman and DNA binding by one finger (Dof) have single zinc finger domain. Although few zinc finger proteins have been described in eubacteria, no protein with single C4 zinc finger has been described in details in anyone of them. In this article, we are describing two novel C-terminal C4 zinc finger proteins-Msmeg_0118 and Msmeg_3613 from Mycobacterium smegmatis. We have named these proteins as Mszfp1 (Mycobacterial Single Zinc Finger Protein 1) and Mszfp2 (Mycobacterial Single Zinc Finger Protein 2). Both the proteins are expressed constitutively, can bind to DNA and regulate transcription. It appears that Mszfp1 and Mszfp2 may activate transcription by interacting with RNA polymerase.

The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres.

The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y)) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells.

Androgen signaling plays a crucial role in spermatogenesis, yet few downstream targets for this signaling pathway have been identified. In the current study, we found that the expression of heat-shock transcription factor 1 (Hsf1) was increased in the testes of Sertoli cell-selective androgen receptor knockout (S-AR(-/y) ) mice compared with wild-type mice by quantitative real-time PCR, and the expression of HSF1 in the S-AR(-/y) Sertoli cells was significantly increased, based on immunofluorescence analysis. In vitro cell-culture studies showed that testosterone repressed the expression of Hsf1 in TM4 cells, a mouse Sertoli cell line. Moreover, a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to the Hsf1 promoter. Our experiments also demonstrated that testosterone-mediated inhibition of Hsf1 transcription down-regulated the expression of heat-shock proteins HSP105 and HSP60. Taken together, these results reveal that Hsf1 is a novel target of androgen receptor in mouse Sertoli cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting Hsf1 expression.

Androgens and oestrogens have been implicated in prostatic carcinogenesis and tumour progression. Although the actions of androgens have been studied extensively, the mechanisms underlying oestrogen signalling in prostate cancer are not fully understood. In the present study, we analyzed the effect of androgens and oestrogens on the expression of anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), comprising two highly-related genes encoding secretory proteins that are expressed in prostate cancer and one of which (AGR2) has been associated with tumour metastasis. Quantitative reverse-transcriptase PCR and western blot analysis showed androgen induction of AGR2 and AGR3 in three androgen receptor positive cell lines, starting at concentrations of 0.1 nm. Both AGR genes were also transcriptionally activated by ≥ 5 nM oestradiol but not by isotype selective or nonselective oestrogen receptor agonists in DUCaP cells that harbour a high-level of wild-type androgen receptor. A functional androgen receptor but not oestrogen receptor turned out to be required for both androgen and oestrogen regulation. This pattern of androgen and oestrogen regulation was confirmed in VCaP cells and was also observed for FKBP5, a well-characterized androgen-regulated gene. Genome-wide chromatin-immunoprecipitation studies coupled with deep sequencing identified androgen receptor binding sites localized in the distal promoter and intron regions of the AGR2 and AGR3 genes, respectively. The androgen responsiveness of these enhancers was verified by luciferase reporter gene assays and site-directed mutagenesis analysis. Androgen treatment also induced p300 and RNA Pol II recruitment to androgen receptor enhancers of AGR2 and initiated local chromatin remodelling and the formation of RNA Pol II-containing androgen receptor transcription complexes.

Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO-targeted ubiquitin ligases (STUbLs). STUbLs bind to SUMOylated proteins via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. In this study, we show that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho (Gro/transducin-like enhancer (TLE)) during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO-independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. Our findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes.

PML nuclear bodies (NBs) were first described by electron microscopy and rediscovered through their treatment-reversible disruption in a rare leukaemia. They recruit multiple partner proteins and now emerge as interferon- and oxidative stress-responsive sumoylation factories. NBs mediate interferon-induced viral restriction, enhance proteolysis, finely tune metabolism and enforce stress-induced senescence. Apart from being markers of cellular stress, PML NBs could be harnessed pharmacologically in a number of conditions, including cancer, viral infection or neurodegenerative diseases. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Post-translational modification by small ubiquitin-like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin-proteasome degradation system by the action of SUMO-targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly-SUMO chain through their SUMO-interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO-like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a beta-grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO-binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly-SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9-mediated poly-SUMO formation.

PA-TM-RING proteins have an N-terminal protease-associated domain, a structure found in numerous proteases and implicated in protein binding, and C-terminal RING finger and PEST domains. Homologous proteins include GRAIL (gene related to anergy in leukocytes), which controls T-cell anergy, and AtRMR1 (receptor homology region-transmembrane domain-RING-H2 motif protein), a plant protein storage vacuole sorting receptor. Another family member, chicken RING zinc finger (C-RZF), was identified as being upregulated in embryonic chicken brain cells grown in the presence of tenascin-C. Despite algorithm predictions that the cDNA encodes a signal peptide and transmembrane domain, the protein was found in the nucleus. We showed that RING finger protein 13 (RNF13), the murine homolog of C-RZF, is a type I integral membrane protein localized in the endosomal/lysosomal system. By quantitative real-time RT-PCR analysis, we demonstrated that expression of RNF13 is increased in adult relative to embryonic mouse tissues and is upregulated in B35 neuroblastoma cells stimulated to undergo neurite outgrowth. We found that RNF13 is very labile, being subject to extensive proteolysis that releases both the protein-associated domain and the RING domain from the membrane. By analyzing microsomes, we showed that the ectodomain is shed into the lumen of vesicles, whereas the C-terminal half, which possesses the RING finger, is released to the cytoplasm. This C-terminal fragment of RNF13 has the ability to mediate ubiquitination. Proteolytic release of RNF13 from a membrane anchor thus provides unique spatial and temporal regulation that has not been previously described for an endosomal E3 ubiquitin ligase.

Muscle atrophy results from a range of conditions, including immobilization, spinal cord damage, inflammation and aging. An investigation designed to identify changes in gene expression in skeletal...

Androgen action in prostate and prostate cancer cells is dependent upon the androgen receptor (AR) protein that transcriptionally regulates the expression of androgen-dependent genes in the presence of a steroid ligand. Whereas the overall schema of androgen action mediated by this receptor protein appears to be relatively simple, androgen signaling is now known to be influenced by several other cell signal transduction pathways and here we review the evidence that the canonical Wnt signaling pathway also modulates androgen signaling at multiple levels. Wnt is a complex signaling pathway whose endpoint involves activation of transcription from LEF-1/TCF transcription factors and it is known to be involved in the development and progression of numerous human epithelial tumors including prostate cancer. beta-catenin protein, a particularly critical molecular component of canonical Wnt signaling is now known to promote androgen signaling through its ability to bind to the AR protein in a ligand-dependent fashion and to enhance the ability of liganded AR to activate transcription of androgen-regulated genes. Under certain conditions, glycogen synthase kinase-3beta (GSK-3beta), a protein serine/threonine kinase that regulates beta-catenin degradation within the Wnt signaling pathway, can also phosphorylate AR and suppress its ability to activate transcription. Finally, it was recently found that the human AR gene itself is a target of LEF-1/TCF-mediated transcription and that AR mRNA is highly upregulated by activation of Wnt signaling in prostate cancer cells. Paradoxically, Wnt activation also appears to stimulate Akt activity promoting an MDM-2-mediated degradation process that reduces AR protein levels in Wnt-stimulated prostate cancer cells. Collectively, this information indicates that the multifaceted nature of the interaction between the Wnt and the androgen signaling pathways likely has numerous consequences for the development, growth, and progression of prostate cancer.

The RING finger motif exists in E3 ligases of the ubiquitination pathway. These ubiquitin ligases bind to target proteins, leading to their modification by covalent addition of ubiquitin peptides. Current databases contain hundreds of proteins with RING finger motifs. This study investigates the role of RING finger genes in embryogenesis of the nematode, Caenorhabditis elegans. We expand the previous list of RING finger-containing genes and show that there are 103 RING finger-containing genes in the C. elegans genome. DNA microarrays of these 103 genes were probed with various RNA samples to identify 16 RING finger genes whose expression is enriched in the germline. RNA interference (RNAi) analysis was then used to determine the developmental role of these genes. One RING finger gene, C32D5.10, showed a dramatic larval arrest upon RNAi. Three RING finger genes exhibited embryonic lethality after RNAi. These three genes include par-2, and two small RING finger proteins: F35G12.9 (an ortholog of APC11) and ZK287.5 (an ortholog of rbx1). Embryos from RNAi of the APC11 and rbx1 orthologs were arrested in the cell cycle, confirming the role of these particular RING finger proteins in regulation of the cell cycle. genesis 38:1–12, 2004. © 2003 Wiley-Liss, Inc.

The MDM2 oncoprotein is overexpressed in many human tumors and cancers. MDM2 functions as an E3 ligase for p53 and for itself. MDM2 also interacts with the retinoblastoma protein (RB) and the transcription factor E2F1 to promote cell cycle S-phase entry. Here, we report that MDM2 protein expression is cell cycle-regulated, which is dependent on its RING finger domain and requires Lys446. We show that MDM2 protein is stabilized at S phase. In addition, overexpression of MDM2 results in stimulation of E2F activity and accumulation of cells in S phase. These data suggest that ubiquitination of MDM2 is cell cycle-regulated and that MDM2 may play a role in cell cycle progression.

Background: The myeloid zinc finger protein 2A (MZF-2A) is a Kruppel-type C2H2 zinc finger transcription factor expressed in myeloid cells and involved in the growth, differentiation and tumorigenesis of myeloid progenitors. Previously we identified a 180 amino acid domain in MZF-2A which is responsible for the transcriptional activation of MZF-2A. To understand the mechanism of the MZF-2A-dependent transcriptional activation, we screened for molecules that interact with the transactivation domain (TAD) of MZF-2A. Results: By using the yeast Ras recruitment two-hybrid screening, we identified a novel SWI2/SNF2-related protein, termed mammalian Domino (mDomino), as an MZF-2A-binding partner. The mDomino protein, which shows a marked similarity to the Drosophila Domino protein, contains a SWI2/SNF2-type ATPase/helicase domain, a SANT domain, and a glutamine-rich (Q-rich) domain. The C-terminal Q-rich domain of mDomino physically associates with the TAD of MZF-2A in mammalian cells as well as in yeast. Expression of the mDomino Q-rich domain, together with MZF-2A in myeloid LGM-1 cells, enhanced the MZF-2A-mediated activation of a reporter gene. Conclusions: These results strongly suggest that an ATP-dependent chromatin-remodelling complex containing mDomino interacts with MZF-2A to regulate gene expression in myeloid cells.

Background: The RING finger proteins function in a variety of fundamental cellular processes. The products of some members of the Polycomb group (PcG) bear ring finger domains and are defined as a subclass of RING finger proteins. Among them are Drosophila posterior sex combs and suppressor 2 of zeste, whose RING fingers are conserved in vertebrate PcG proteins Mel18 and Bmi1. Results: We have identified a new mammalian RING finger protein, termed MBLR due to its structural similarity to Mel18 and Bmi1 (Mel18 and Bmi1-like RING finger protein). MBLR interacts with some PcG proteins: in vitro biochemical data support the idea of a direct interaction of MBLR’s RING finger domain with Ring1B, which is highly homologous to one of the mammalian PcG genes, Ring1A. We also show that MBLR acts as a transcriptional repressor in transiently transfected cells, as is the case for other PcG proteins. Immunocytochemical analysis reveals that MBLR protein is localized in a fine-grained distribution throughout the nucleoplasm in interphase cultured cells and in a fainter diffuse cytoplasmic distribution in mitotic cells. In addition, we find that serine 32 of MBLR is specifically phosphorylated during mitosis, most likely by CDK7, a component of the basal transcriptional machinery. Conclusion: Similarities to previously defined PcG proteins suggest that MBLR should be included in the same subclass of RING finger proteins as Mel18 and Bmi1. Although the biological relevance of the cell cycle-related phosphorylation remains to be demonstrated, serine 32 phosphorylation could nevertheless be functionally important.

Accumulating evidence indicates that androgens and the androgen receptor modulate the development and progression of breast adenocarcinoma; however, the precise role and actions remain poorly defined. We examined previously the steroid hormone regulation of 2 known androgen-regulated kallikreins, KLK3 (encoding PSA) and KLK2 (encoding human kallikrein 2 or hK2) in BT-474, T-47D, ZR75-1, MCF-7, MFM-223 and BT-20 human breast cancer cells and found that they were differentially regulated, with the cells showing variable responses to androgen. To determine if this variable response was reflected by differences in androgen receptor, we characterized the expression of androgen receptor in these cells by Western blot analysis and saturation binding analysis. In addition, we sequenced androgen receptor cDNA from each of these cell lines to check whether any androgen receptor mutations were present. The expression of 11 nuclear receptor co-regulatory factors (SRC-1, AIB1, ARA24, ARA54, ARA55, ARA70, ARA160, FHL2, PDEF, NCoR1, SMRT) was compared in these cell lines by semi-quantitative RT-PCR to determine if the pattern of receptor co-activators or -repressors expressed in these cells might explain the differential regulation of KLK2 and KLK3. The levels of androgen receptor varied among the cell lines, but did not correlate with hK2 and PSA secretion determined previously. No mutations within the coding regions of the receptor were detected. With the exception of receptor expressed by MCF-7 cells, the polymorphic CAG repeat length was in the normal range. Every breast cancer cell line exhibited a distinct expression pattern of the nuclear receptor co-regulators examined raising the possibility that the relative levels of these co-activators/-repressors might differentially modulate androgen receptor transcriptional activity within the promoter/enhancer region of KLK2 and KLK3 of these cells. © 2002 Wiley-Liss, Inc.

The cellular abundance of the transcription factor Nrf2 is largely controlled by Keap1, an inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf2 by the Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex, thereby targeting Nrf2 for proteasome-dependent degradation in the cytoplasm. It is becoming increasingly apparent that degradation of Nrf2 can also occur by Keap1-independent mechanisms. Recently, we showed that Nrf2 is a SUMO-modifiable protein and that polysumoylated Nrf2 can be polyubiquitylated by the poly-SUMO-specific E3 ubiquitin ligase known as RING finger protein 4 (RNF4) and subsequently degraded in PML-NBs. The role of sumoylation in the turnover of Nrf2, independent of Keap1, is a novel aspect of the biology of Nrf2. Here, we explored whether sumoylated Nrf2 is a substrate for Arkadia, another polySUMO-specific E3 ubiquitin ligase (also known as RING finger protein 111 [RNF111]). Our results indicate that Arkadia can ubiquitylate polysumoylated Nrf2 in PML...

Recent results from several laboratories suggest that the interaction of E2 ubiquitin-conjugating enzymes with the RING finger domain has a central role in mediating the transfer of ubiquitin to proteins. Here we present a mutational analysis of the interaction between the E2 enzyme UbcM4/UbcH7 and three different RING finger proteins, termed UIPs, which, like Parkin, contain a RING1-IBR-RING2 motif. The results show that the E2 enzyme binds to the RING1 domain but not to the other cysteine/histidine-rich domains of the RING1-IBR-RING2 motif. Three regions within the UbcM4 molecule are involved in this interaction: the H1 alpha helix, loop L1, connecting the third and fourth strand of the beta sheet, and loop L2, located between the fourth beta strand and the second alpha helix. Loop L2 plays an important role in determining the specificity of interaction. The effects of L2 mutations on UbcM4/UIP interaction are different for each UIP, indicating that RING finger domains can vary considerably in their structural requirements for binding to E2 enzymes. The result that single amino-acid changes can regulate binding of E2 enzymes to different RING finger proteins suggests a novel approach to experimentally manipulate proteolytic pathways mediated by RING finger proteins.

To identify proteins that interact with Huntingtin-interacting protein-2 (Hip-2), a ubiquitin-conjugating enzyme, a yeast two-hybrid screen system was used to isolate five positive clones. Sequence analyses showed that, with one exception, all Hip-2-interacting proteins contained the RING finger motifs. The interaction of Hip-2 with RNF2, one of the clones, was further confirmed through in vitro and in vivo experiments. Mutations in the RING domain of RNF2 prevented the clone from binding to Hip-2, an indication that the RING domain is the binding determinant. RNF2 showed a ubiquitin ligase (E3) activity in the presence of Hip-2, suggesting that a subset of RING finger proteins may have roles as E3s.

Gonadal steroids are potent modulators of gonadotropin releasing hormone (GnRH) secretion, and androgen binding sites and 5alpha-reductase activity have been found in the immortalized GnRH secreting cell line GT1-1, suggesting the existence of a direct androgenic control of GnRH dynamics. Two isoforms of the 5alpha-reductase have been cloned with very different biochemical/functional properties: 5alpha-reductase type 1 (widely distributed in the body) and 5alpha-reductase type 2 (confined in androgen target structures). We have analysed whether, in GT1-1, androgen binding sites are linked to "classical" androgen receptor, and which 5alpha-reductase isoform is active. Reverse transcriptase-polymerase chain reaction analysis showed that the mRNAs coding for androgen receptor and for the two 5alpha-reductase isoforms are all expressed in GT1-1 cells. However, the 5alpha-reductase enzymatic reaction showed a peak of activity at a narrow pH around 5.5, the optimum for the 5alpha-reductase type 2. The affinity for testosterone, of the enzyme present in GT1-1 cells, was very similar to that observed for the recombinant type 2 isozyme expressed in yeasts. The data indicate that GT1-1 cells (i) express a "classical" androgen receptor and (ii) contain the 5alpha-reductase type 2 isoform, a specific marker of androgen-responsiveness.

A yeast 2-hybrid screen was performed to identify possible transcriptional modulators interactive with goosecoid-like (gscl), a transcription factor with suppressive activity, expressed during early brain and gonad development. The screen resulted in the identification of a RING protein known as rnf4 or snrf. Gscl/rnf4 interactions were confirmed by affinity chromatography and by immunoprecipitation. Northern analysis confirmed earlier reports of ubiquitous rnf4 expression in adult tissues. Immunohistochemical analysis of mouse embryos revealed expression primarily in the developing nervous system, with strong expression in the dorsal root ganglia and developing gonads. In contrast to previous reports, both cytoplasmic and nuclear expression of rnf4 was documented. The results reported here confirm and extend earlier reports of rnf4 expression. They suggest for the first time, that in addition to acting as a modulator of transcriptional activity, rnf4 may function, as do other RING proteins, to promote the formation of intracytoplasmic complexes involved in shuttling information between the cytoplasm and the nucleus. Dev Dyn;218:102–111. © 2000 Wiley-Liss, Inc.

The RET finger protein (RFP), which belongs to the B box zinc finger protein family, has a tripartite motif consisting of a Ring finger, a B box finger and a coiled-coil domain. The RET finger protein becomes oncogenic when its tripartite motif is fused with the tyrosine kinase domain of the RET protein. This study examined the RFP expression in normal and tumor tissues by immunohistochemistry. RFP was detected in the nuclei of various cells, including peripheral and central neurones, hepatocytes, adrenal chromaffin cells and male germ cells. Among them, RFP was expressed at high levels in male germ cells such as primary spermatocytes and round spermatids, and formed a perinuclear cap structure in primary spermatocytes. On the other hand, high levels of cytoplasmic expression of RFP were observed in some plasma cells as well as solitary plasmacytoma and multiple myeloma. These results suggested that different nuclear/cytoplasmic distributions of RFP might play a role in the regulation of growth or differentiation of different cell types.

The zinc finger protein A20 is encoded by an immediate early response gene whose expression is induced by different inflammatory stimuli, including interleukin-1 (IL-1). Gene induction by IL-1 is mediated by activation of the transcription factor NF-kappaB, and requires the signal adapter protein TRAF6. The latter interacts with the NF-kappaB-inducing kinase NIK, which is believed to be part of the IkappaB kinase complex. Expression of A20 potently inhibits IL-1-induced NF-kappaB activation by an unknown mechanism. Inhibition of IL-1-induced NF-kappaB activation was found to be mediated by the C-terminal zinc finger-containing domain of A20. More importantly, we present evidence that A20 interferes with IL-1-induced NF-kappaB activation at the level of TRAF6, upstream of NIK. Moreover, A20 was shown to directly interact with TRAF6.

To shed light on the nature and evolution of structure-function relations in the androgen receptor (AR), we have undertaken a comparative analysis of all available AR and other steroid receptor sequences. We have identified a group of amino acids that "diagnose" the clade of androgen receptors--residues that are strictly conserved among the ARs but not shared with other receptors. We hypothesize that these amino acids, clustered in a few regions of the protein, confer upon the androgen receptor its unique functions, including recognition of specific DNA response elements and affinity for androgens, rather than other steroid hormones. The four domains of the AR display markedly different rates of evolutionary divergence; conserved portions of the sequence, including small stable stretches within otherwise divergent regions, may be essential to receptor function. Current data from experimental, crystallographic, and clinical studies support these hypotheses, which can now be further tested in the laboratory.

We have cloned two isoforms of cDNAs encoding novel zinc finger proteins. One form encodes a 274-amino acid protein containing an acidic amino acid and serine-rich domain and a zinc finger domain which shows high sequence homology to that of Drosophila Ovo protein. The other form encodes a 179-amino acid protein containing only the zinc finger domain. Expression of both proteins possessing an antigenic epitope in COS cells revealed that they are localized in the nucleus. The 1.3-kbp mRNAs are predominantly expressed in testis, and the expression increases from 3 weeks postnatal, implying that these proteins may play important roles in the development of the testes.

The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin-dependent and SUMO-dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

The RING finger is a form of zinc finger motif found in proteins of widely varying biological function. The Dictyostelium RNGB protein contains a RING finger and also a K-box, a sequence motif found in several plant homeotic proteins. The rngB mRNA is present at low concentration in growing cells and gradually increases in abundance throughout development. However, the RNGB protein is not detected until culmination and we present evidence that suggests it is translationally regulated. The protein is specifically localised in maturing spore cells and is cytoplasmic, suggesting that the RING finger does not function as a DNA binding domain.

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression.

The androgen receptor (AR) belongs to the superfamily of nuclear receptors that employ complex genetic mechanisms to guide the development and physiological functions of different target tissues. Upon interaction with its cognate hormone, AR activates or represses gene transcription through association with specific DNA elements and/or proteins. This review summarize briefly our current view of androgen action, with a special emphasis on genetic factors that may modulate the response.

Zfy1 is a mouse Y chromosomal gene encoding a zincfinger protein which is thought to have some function during spermatogenesis. Here we show that, when introduced into tissue culture cells, Zfy1 is targeted to the nucleus. Two independent signals are present within the protein for nuclear localization. This nuclear Zfy1 protein is able to bind strongly to DNA-cellulose and, using site-selection assays, we have identified specific Zfy1 DNA binding sites. Taken together these results suggest that Zfy1 is a nuclear-located sequence-specific DNA binding protein which functions during spermatogenesis.

In the course of a comprehensive genomic screen for cell cycle regulated genes in the yeast Saccharomyces cerevisiae (Price et al., 1991) we identified and characterized a transcriptional unit encoding a putative zinc finger protein named FZF1 (EMBL accession number: X67787). This gene encodes a protein containing five zinc fingers of the Cys2His2 class, three of which are positioned in tandem at the N-terminus. The fourth and fifth finger follow after an interruption of 61 and 66 amino acids, respectively. While FZF1 is constitutively expressed at a very low level, its deletion is not essential for growth. Its similarity with known transcription factors, however, suggests that the FZF1 gene product may serve as a transcription factor in yeast.

OBJECTIVE Determine the sequence of the androgen receptor gene in men with impaired responsiveness to androgens in order to identify the molecular basis of their under-virilization. DESIGN Blood samples were used as the source of genomic DNA. Portions of the androgen receptor gene were amplified by polymerase chain reaction and sequenced. PATIENTS Samples were obtained from three patients and five normal fertile controls. Patients were all 46 XY and were undervirilized with ambiguous external genitalia, gynaecomastla and infertility. MEASUREMENTS Total cellular DNA was purified from peripheral blood leucocytes. Pairs of ollgonucleotide primers designed to flank the individual exons of the androgen receptor gene were synthesized. The specific regions of the androgen receptor were amplified from the samples of cellular DNA by polymerase chain reaction. Amplified DNA was purified, sequenced and compared to the published sequence. RESULTS In all three patients point mutations in the androgen receptor gene were detected but no defects were detected in samples from normal controls. In two of the patients, an identical single nucleotide change from G to T was detected. This nucleotide was within the codon for amino acid 866 and would change it from valine to leucine. Amino acid 866 is found within an area of the steroid binding domain thought to be involved in receptor dimerization. Within the repetitive sequence of exon I patient 1 had 21 glutamine residues and patient 2 had 25. In the third patient a single change of G to A would result in incorporation of lysine in place of a conserved arginine residue at position 607 within the second zinc finger of the DNA binding domain. The sequence of the androgen receptor gene of the mother of the third patient revealed her to be heterozygous for the same defect. CONCLUSION Patients 1 and 2 are unrelated although they have an identical point mutation in their androgen receptor gene. A patient with complete androgen insensitivity syndrome has been reported to have a defect at the same position causing the amino acid substitution of methlonine for valine. Therefore we confirm that the nature of the amino acid change in the peptide sequence of the androgen receptor as well as its location within the protein, can have a profound effect on the phenotypic severity of androgen resistance. Studies on mutated receptors from individuals with a wide range of degrees of androgen resistance may enable us to construct a map of the key amino acids in the different domains of the protein.

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.