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The protein inhibitor of activated STAT (PIAS) family proteins act as the important negative regulators in janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which can be also involved in regulating the expression of interferon-stimulated genes (ISGs). In the present study, a PIAS homologue (designated as CgPIAS) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgPIAS cDNA was of 1887 bp encoding a peptide of 628 amino acid residues. The CgPIAS protein contains a conserved scaffold attachment factor A/B/acinus/PIAS (SAP) domain, a Pro-Ile-Asn-Ile-Thr (PINIT) motif, a RING-finger-like zinc-binding domain (RLD) and two SUMO-interaction Motifs (SIMs). The deduced amino acid sequence of CgPIAS shared 74.58-81.36% similarity with other PIAS family members in the RLD domain. The mRNA transcripts of CgPIAS were detected in all the tested tissues with highest level in haemocytes (32.98-fold of mantles, p 0.001). After poly (I:C) and recombinant Interferon-like protein (rCgIFNLP) stimulation, the mRNA expression of CgPIAS in haemocytes significantly up-regulated to the highest level at 48 h (7.38-fold, p 0.001) and at 24 h (13.08-fold, p 0.01), respectively. Moreover, the nuclear translocation of CgPIAS was observed in haemocytes after poly (I:C) stimulation. Biolayer Interferometry (BLI) assay revealed that the recombinant protein CgPIAS-RLD could interact with the recombinant protein CgSTAT in vitro with the K

Background Protein inhibitors of activated STAT (PIAS) proteins are regarded as negative regulators of cytokine-signaling and potent immunosuppressive proteins. However, their role in the process of organ transplant rejection has not been elucidated. Methods In the current study, we compared transcript levels of PIAS1 to 4 in the peripheral blood of renal transplant recipients who experienced transplant rejection with those having normal transplant functions. Expression of PIAS1 was significantly higher in nonrejected group compared with the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other PIAS genes were not different between study groups. Significant pairwise correlations were found between expression levels of PIAS genes in all study subgroups. The current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.

Aim: Protein inhibitor of activated STAT ( PIAS) family includes transcriptional regulator proteins with SUMO E3 ligase activity. They regulate expression of several genes involved in cell proliferation, differentiation and survival. Method: We evaluated expression of PIAS1–4 genes in 54 breast cancer tissues and their paired adjacent noncancerous tissues. Results: PIAS2 and PIAS3 genes were significantly downregulated in tumoral tissues compared with adjacent noncancerous tissues. PIAS1–3 expressions were significantly lower in estrogen receptor (ER+) samples compared with ER- samples while PIAS4 had the opposite trend. PIAS3 expression was significantly higher in grade 1 samples compared with grade 2 samples. Conclusion: These findings highlight the role of PIAS genes in the pathogenesis of breast cancer and their association with determinants of response to antihormone therapies.

Protein inhibitors of activated STAT (PIAS) are involved in the regulation of the JAK/STAT signaling pathway and have interactions with NF-κB, p73 and p53. These proteins regulate immune responses; therefore dysregulation in their expression leads to several immune-mediated disorders. In the present study, we examined expression of PIAS1-4 in peripheral blood of patients with acute/chronic inflammatory demyelinating polyradiculoneuropathy (AIDP/CIDP) compared with healthy subjects. We demonstrated down-regulation of all PIAS genes in both AIDP and CIDP cases compared with controls. Similarly, comparisons in gender-based groups revealed down-regulation of these gene0s in patients of each gender compared with gender-matched controls. There was no significant difference in expression of PIAS genes between AIDP and CIDP cases. Based on the area under the receiver operating characteristic curves, PIAS1-4 genes could distinguish between inflammatory demyelinating polyradiculoneuropathy and healthy status with accuracy values of 0.87, 0.87, 0.79 and 0.80, respectively. In differentiation between AIDP cases and healthy controls, these values were 0.92, 0.92, 0.83 and 0.86, respectively. Finally, PIAS1-4 genes could discriminate CIDP from healthy status with accuracy values of 0.82, 0.83, 0.75 and 0.75, respectively. The current study underscores the role of PIAS genes in the pathogenesis of inflammatory demyelinating polyradiculoneuropathy and their potential usage as biomarkers.

Objectives Protein inhibitors of activated STAT (PIAS) are transcription co-regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway as well as nuclear factor-κB family of transcription factors. Both of them are involved in cytokine release during inflammatory response. Patients and methods Considering the role of cytokine imbalance in the pathogenesis of multiple sclerosis (MS), we compared blood expressions of PIAS1-4 genes in 48 interferon β (IFNβ)-treated MS patients with those of healthy subjects by means of real time PCR. Results Although the expression levels of these genes were not significantly different between MS patients and healthy subjects, significant inverse correlations have been found between PIAS1 expression and age at disease onset. PIAS2 and PIAS3 expressions were inversely correlated with Expanded Disability Status Scale (EDSS) in patients. Moreover, PIAS3 expression was correlated with disease duration in patients and with age in controls. In addition, PIAS4 expression was inversely correlated with EDSS and age at disease onset while it was positively correlated with disease duration. Conclusion The present study provides evidences for altered expression of PIAS genes in IFNβ-treated MS patients compared with healthy subjects. However, future studies are needed for elaboration of their exact function in this disorder.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.

Background Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the pestivius virus group. BVDV is responsible for significant economic loss in cattle industry worldwide because of reducing reproductive performance, increasing incidence of other diseases and mortality among young stock. The core (C) protein of the Flaviviridae family member is involved in host antiviral immune response through activation of related signaling pathways that affect the viral replication. However, the influence of C protein-interaction partners in BVDV infections is poorly defined. Methods To explore C-protein-interacting partners, yeast two-hybrid was used to screen the interaction protein of C protein using bovine peripheral blood mononuclear cell (PBMC) cDNA library. The co-immunoprecipitation and confocal assays were manipulated to determine the interaction between potential partners and C protein. Knockdown and overexpression of the partner were used to examine whether the C-protein-interacting partner plays a role in BVDV proliferation and virulence. Meanwhile, qRT-PCR and western blot assays were used to investigate the effect of C protein and C-protein-interacting partner on the immune response of host cells. Results We identified protein inhibitor of activated STAT 4 (PIAS4) as a novel interacting partner of the BVDV C protein. Co-immunoprecipitation and confocal assays demonstrated a strong interaction between C protein and PIAS4. Silencing of PIAS4 with small interfering RNA suppressed C protein expression and BVDV growth, while overexpression of PISA4 increased C protein expression and BVDV growth. The overexpression of PIAS4 increased the cell apoptosis. Meanwhile, the expressions of STAT4, SOCS3, IFITM, IFN-α were negatively regulated by the expression of PIAS4. The expression of C protein suppressed the antiviral proteins expression, and the inhibition effect was enhanced by interaction of PIAS4 and C protein. These results highlighted the beneficial properties of cellular PIAS4 for BVDV protein expression and growth. Conclusions This study provides reliable clues for understanding the roles of PIAS4 in the regulation of BVDV growth.

Oncolytic viruses exploit tumor-specific cellular changes for selective replication, inducing cancer cell death. Our previous research showed that Ras/mitogen-activated protein kinase kinase (MEK) downregulation of interferon regulatory factor 1 (IRF1) is a major mechanism underlying viral oncolysis. Protein inhibitor of activated STAT 3 (PIAS3) is known as the E3 ligase of IRF1 sumoylation. The objective of this study was to identify if, and how, PIAS3 modulates cellular sensitivity to oncolytic viruses via regulation of IRF1. By conducting co-immunoprecipitation, I found that IRF1 has no direct interaction with PIAS3 found within HT1080 cells. However, CRISPR knockdown of PIAS3 increases IRF1 expression as well as transcription of IRF1-responsive anti-viral genes. Furthermore, PIAS3 knockdown HT1080 cells were equally sensitive to viral infection as their parent HT1080 cells. Together, these results demonstrate that PIAS3 does not directly interact with IRF1 but regulates expression and transcriptional activity of IRF1. Moreover, as CRISPR knockdown of PIAS3 did not change cellular sensitivity to viral infection, IRF1 modulation via PIAS3 does not play very critical roles in host innate antiviral responses.

Excessive nutrition promotes the pathogenesis of non-alcoholic steatohepatitis (NASH), characterized by the accumulation of pro-inflammation mediators in the liver. In the present study we investigated the regulation of pro-inflammatory transcription in hepatocytes by protein inhibitor of activated STAT 4 (PIAS4) in this process and the underlying mechanisms. We report that expression of the class III deacetylase SIRT1 was down-regulated in the livers of NASH mice accompanied by a simultaneous increase in the expression and binding activity of PIAS4. Exposure to high glucose stimulated the expression PIAS4 in cultured hepatocytes paralleling SIRT1 repression. Estrogen, a known NASH-protective hormone, ameliorated SIRT1 trans-repression by targeting PIAS4. Over-expression of PIAS4 enhanced, while PIAS4 knockdown alleviated, repression of SIRT1 transcription by high glucose. Lentiviral delivery of short hairpin RNA (shRNA) targeting PIAS4 attenuated hepatic inflammation in NASH mice by restoring SIRT1 expression. Mechanistically, PIAS4 promoted NF-κB-mediated pro-inflammatory transcription in a SIRT1 dependent manner. In conclusion, our study indicates that PIAS4 mediated SIRT1 repression in response to nutrient surplus contributes to the pathogenesis of NASH. Therefore, targeting PIAS4 might provide novel therapeutic strategies in the intervention of NASH.

S mall u biquitin-like mo difier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. IMPORTANCE Posttranslational modifications with s mall u biquitin-like mo difier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The p rotein i nhibitor of a ctivated S TAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection.

Previous studies have shown that protein inhibitor of activated STAT (PIAs)xα is crucial in protein sumoylation and is associated with cancer cell progression. However, the mechanism underlying the inhibitory effect on cancer cells, which may assist in developing novel treatment strategies in cancer remains to be elucidated. In present study, the expression levels of PIAsxα from tissue samples of osteosarcoma and adjacent tissues from 25 patients were analyzed using reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemical analyses. In addition, techniques using an overexpression vector and small interfering (si)RNAs were used to examine the effect of PIAsxα on osteosarcoma cells. Finally, using xenograft U2-OS osteosarcoma cells overexpressing PIAsxα, the effect of PIAsxα on osteosarcoma formation was determined. The results revealed low expression of PIAsxα in osteosarcoma tissues. In addition, following overexpression of PIAsxα, the apoptotic rates were significantly increased. The rate of G2/M arrest was at the highest level in the overexpression group, compared with other groups assessed. Furthermore, the expression levels of cyclin D1 and cyclin D3 were inhibited following PIAsxα increase, indicating the repressive effects of PIAsxα on cell cycle. Accordingly, cyclin D kinase (CDK) genes, including CDK4, CDK6 and CDK8, increased markedly following treatment with PIAsxα siRNAs. The expression levels of CDK4, CDK6 and CDK8 decreased significantly in the overexpression group, compared to the other groups. Furthermore, high expression levels of PIAsxα inhibited tumor formation in a nude mouse model. Taken together, these findings provide evidence for the effects of PIAsxα and its mechanism on osteosarcoma progression, which offers novel insight into sumoylation and the cell cycle in osteosarcoma.

Obesity is associated with chronic low-level inflammation, especially in fat tissues, which contributes to insulin resistance and type 2 diabetes mellitus (T2DM). Protein inhibitor of activated STAT 1 (PIAS1) modulates a variety of cellular processes such as cell proliferation and DNA damage responses. Particularly, PIAS1 functions in the innate immune system and is a key regulator of the inflammation cascade. However, whether PIAS1 is involved in the regulation of insulin sensitivity remains unknown. Here, we demonstrated that PIAS1 expression in white adipose tissue (WAT) was downregulated by c-Jun N-terminal kinase in prediabetic mice models. Overexpression of PIAS1 in inguinal WAT of prediabetic mice significantly improved systemic insulin sensitivity, whereas knockdown of PIAS1 in wild-type mice led to insulin resistance. Mechanistically, PIAS1 inhibited the activation of stress-induced kinases and the expression of nuclear factor-κB target genes in adipocytes, mainly including proinflammatory and chemotactic factors. In doing so, PIAS1 inhibited macrophage infiltration in adipose tissue, thus suppressing amplification of the inflammation cascade, which in turn improved insulin sensitivity. These results were further verified in a fat transplantation model. Our findings shed light on the critical role of PIAS1 in controlling insulin sensitivity and suggest a therapeutic potential of PIAS1 in T2DM.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of various signaling pathways as a negative regulator. To further understand the role of PIAS in immune and stress response in mud crab ( Scylla paramamosain ), in the present study, a full-length cDNA of PIAS from S. paramamosain (SpPIAS) was cloned for the first time, which contained an open reading frame of 2364 bp encoding a polypeptide of 788 amino acids. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the tissue distribution of PIAS in mud crab. The results revealed that SpPIAS was expressed in hemocytes, heart, hepatopancreas, gills, stomach, intestines, muscle, connective tissue and gonad of S. paramamosain , and the higher expression levels were found in connective tissue and gonad. The expression levels of SpPIAS in hemocytes were up-regulated after challenge with Staphylococcus aureus , Vibrio harveyi , and white spot syndrome virus (WSSV) for 2–24 h, which suggested that SpPIAS was involved in the pathogen-resistant activities of mud crab. Furthermore, the RING-like domain (RLD) and Serine/Threonine rich repeat C-terminal domain (S/T) of SpPIAS were expressed by prokaryotic expression system and purified by glutathione Sepharose 4B chromatography. This study contributed to define the biological characteristics of SpPAIS and further demonstrate the critical role of SpPAIS in vivo during the pathogen infection.

It is known that the LIM homeodomain transcription factor Isl1 is highly expressed in all pancreatic endocrine cells and functions in regulating pancreatic development and insulin secretion. The Isl1 mutation has been found to be associated with type 2 diabetes, but the mechanism responsible for Isl1 regulation of insulin synthesis and secretion still needs to be elucidated. In the present study, the protein inhibitor of activated STAT Y (PIASy) was identified as a novel Isl1-interacting protein with a yeast two-hybrid system, and its interaction with Isl1 was further confirmed by a co-immunoprecipitation experiment. PIASy and Isl1 colocalize in human and mouse pancreas and NIT beta cells. Furthermore, PIASy and Isl1 upregulate insulin gene expression and insulin secretion in a dose-dependent manner by activating the insulin promoter. PIASy and Isl1 mRNA expression levels were also increased in type 2 diabetic db/db mice. In addition, our results demonstrate that PIASy and Isl1 cooperate to activate the insulin promoter through the Isl1 homeodomain and PIASy ring domain. These data suggest that that PIASy regulates insulin synthesis and secretion by interacting with Isl1 and provide new insight into insulin regulation, although the detailed molecular mechanism needs to be clarified in future studies.

It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein β (C/EBPβ), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPβ has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPβ to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPβ began to decline. PIAS1 was found to interact with C/EBPβ through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPβ. C/EBPβ became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPβ. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPβ.

The PIAS proteins (protein inhibitor of activated STAT) were originally identified as inhibitors of the JAK-STAT pathway. Subsequently, their roles on transcriptional regulation have been identified in modulation of the androgen receptor (AR) and other nuclear hormone receptor-mediated actions. Zimp7, also named Zmiz2, is a novel PIAS-like protein and functions as a transcriptional co-activator. In this study, we demonstrate an interaction between Zimp7 and PIAS proteins with higher preference for PIAS3. A modified mammalian one-hybrid assay showed that the NH(2)-terminal proline-rich domain of Zimp7 and the region spanning amino acids 321-486 of PIAS3 were the primary interaction segments. The interaction between Zimp7 and PIAS3 proteins was further confirmed by in vitro protein pull-down and co-immunoprecipitation assays with both exogenous and endogenous proteins. Expression of exogenous PIAS3 further enhances Zimp7-mediated augmentation of AR transcription. Knockdown of the endogenous PIAS3 protein using a specific PIAS3 small hairpin RNA reduced the augmentation of Zimp7 on AR-mediated transcription. Co-localization of Zimp7 and PIAS3 proteins was observed in the nuclei of cells by immunostaining. Exogenous PIAS3 expression enhances the stability of the Zimp7 protein. Using chromatin immunoprecipitation assays, we showed that PIAS3 is involved in the AR- and Zimp7-formed protein complex(es) in the AR downstream target promoter to facilitate androgen-induced transcription. Finally, we further demonstrated that loss of Zimp7 significantly impaired PIAS3-mediated enhancement on AR activity in mouse Zimp7 null (zimp7(-/-)) embryonic fibroblasts. Taken together, these results demonstrate a novel interaction between PIAS and PIAS-like proteins and elucidate a novel regulatory mechanism for PIAS proteins in AR-mediated transcription.

Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.

Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP co-immunoprecipitated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBP(CORE) and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors.

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

Review on PIAS1, with data on DNA/RNA, on the protein encoded and where the gene is implicated.