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The expressions and roles of protein inhibitor of activated STAT (PIAS) proteins, a group of proteins with STAT inhibition and SUMOylation E3 ligase activity, are rarely revealed in endometrial cancer (EC). In this study, we analyzed the expressions of PIASs and their relationships with clinical features by mining online data through web servers, including UALCAN and Gene Expression Profiling Interactive Analysis (GEPIA) in EC. The expressions of PIASs in EC tissues were further validated by immunohistochemistry (IHC). The online analyses revealed only PIAS1 was consistently downregulated both at mRNA and protein level in EC, which was validated by the IHC. Subsequently, the mechanism of PIAS1 downregulation was explored with online tools like UALCAN, cBioPortal, LinkedOmics, and the Encyclopedia of RNA Interactomes (ENCORI). The results indicated that the mutation rate of PIAS1 was extremely low and not associated with PIAS1 expression. The promoter methylation level of PIAS1 was comparable between normal and EC tissues. miR-182-5p and miR-96-5p with negative association with PIAS1 in EC were predicted to target PIAS1. Dual luciferase reporter assay confirmed miR-182-5p and miR-96-5p could target PIAS1 in EC. MiR-182-5p and miR-96-5p inhibitors could upregulate PIAS1 in EC cells. Moreover, ectopic PIAS1 expression and STAT3 inhibitor treatment significantly inhibited STAT3's activity and the levels of miR-182-5p and miR-96-5p in EC cells. Collectively, our findings revealed PIAS1 was downregulated in EC, which was caused by upregulation of miR-182-5p and miR-96-5p, and PIAS1 downregulation further activated STAT3 and increased the expression of miR-182-5p and miR-96-5p, confirming miR-182-5p and miR-96-5p mediated the negative feedback regulatory loop between PIAS1 and STAT3 in EC.

Oxidative stress serves important roles in cardiac injury during the process of ischemia/reperfusion (I/R). Y‑box protein 1 (YB1), a member of the highly conserved Y‑box protein family, is closely associated with inflammation and stress responses by regulating gene transcription, RNA splicing and mRNA translation. However, the roles of YB1 in oxidative stress‑induced myocardial‑I/R (M‑I/R) injury are unknown. The aim of the present study was to examine the effects of YB1 on H2O2‑induced cardiomyocyte injury and its underlying mechanism. The results demonstrated that YB1 expression was upregulated during H2O2‑induced myocardial injury. YB1 knockdown through transfection of small interfering RNA significantly aggravated cardiac cell apoptosis. Furthermore, YB1 knockdown significantly reversed the H2O2‑mediated increase in phosphorylated signal transducer and activator of transcription (STAT)3, but did not affect the phosphorylation of P38, extracellular signal‑regulated kinases 1/2, c‑Jun N‑terminal kinases, P65, Janus kinase 1 and 2 or STAT1. Moreover, protein co‑immunoprecipitation and RNA‑binding protein immunoprecipitation assays revealed that YB1 interacted with protein inhibitor of activated STAT 3 (PIAS3) mRNA but not its translated protein. YB1 overexpression may have promoted PIAS3 mRNA decay, decreasing PIAS3 protein levels, and therefore increased the levels of phosphorylated STAT3. Finally, YB1 knockdown, mediated by a lentivirus carrying YB1 targeted short hairpin RNA, significantly decreased left ventricle percentage fractional shortening and ejection fraction values, while increasing the infarct sizes in a rat model of M‑I/R injury. These results demonstrated for the first time (to the best of our knowledge) that YB1 may protect cardiac myocytes against H2O2 or M‑I/R‑induced injury by binding to PIAS3 mRNA and resulting in the phosphorylation of STAT3.

Ribosome biogenesis is critical for proliferating cells and requires the coordinated activities of three eukaryotic RNA polymerases. We recently showed that the small ubiquitin-like modifier (SUMO) system controls the global level of RNA polymerase II (Pol II)–controlled transcription in mammalian cells by regulating cyclin-dependent kinase 9 activity. Here, we present evidence that the SUMO system also plays a critical role in the control of Pol I transcription. Using an siRNA-based knockdown approach, we found that multiple SUMO E3 ligases of the PIAS (protein inhibitor of activated STAT) family are involved in SUMO-mediated repression of ribosomal DNA (rDNA) gene transcription. We demonstrate that endogenous SUMO represses rDNA transcription primarily by repressing upstream-binding factor and proto-oncogene c-Myc expression and that ectopic overexpression of SUMO-associated enzymes additionally represses rDNA transcription via c-Myc SUMOylation and its subsequent degradation. The results of our study reveal a critical role of SUMOylation in the control of rDNA transcription, uncover the underlying mechanisms involved, and indicate that the SUMO system coordinates Pol I– and Pol II–mediated transcription in mammalian cells.

Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) β. Protein S-glutathionylation decreased the interaction of C/EBPβ and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier (SUMO) E3 ligase that facilitates C/EBPβ degradation. Experiments with truncated mutant C/EBPβ demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPβ. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPβ. The C201S, C296S double mutant C/EBPβ prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPβ, stabilizing and increasing C/EBPβ protein levels.

CoREST family of transcriptional co-repressors regulates gene expression and cell fate determination during development. CoREST co-repressors recruit with different affinity the histone demethylase LSD1 (KDM1A) and the deacetylases HDAC1/2 to repress with variable strength the expression of target genes. CoREST protein levels are differentially regulated during cell fate determination and in mature tissues. However, regulatory mechanisms of CoREST co-repressors at the protein level have not been studied. Here, we report that CoREST (CoREST1, RCOR1) and its homologs CoREST2 (RCOR2) and CoREST3 (RCOR3) interact with PIASγ (protein inhibitor of activated STAT), a SUMO (small ubiquitin-like modifier)-E3-ligase. PIASγ increases the stability of CoREST proteins and facilitates their SUMOylation by SUMO-2. Interestingly, the SUMO-conjugating enzyme, Ubc9 also facilitates the SUMOylation of CoREST proteins. However, it does not change their protein levels. Specificity was shown using the null enzymatic form of PIASγ (PIASγ-C342A) and the SUMO protease SENP-1, which reversed SUMOylation and the increment of CoREST protein levels induced by PIASγ. The major SUMO acceptor lysines are different and are localized in nonconserved sequences among CoREST proteins. SUMOylation-deficient CoREST1 and CoREST3 mutants maintain a similar interaction profile with LSD1 and HDAC1/2, and consequently maintain similar repressor capacity compared with wild-type counterparts. In conclusion, CoREST co-repressors form protein complexes with PIASγ, which acts both as SUMO E3-ligase and as a protein stabilizer for CoREST proteins. This novel regulation of CoREST by PIASγ interaction and SUMOylation may serve to control cell fate determination during development.

As a conserved posttranslational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Of importance, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1 and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.

(AP) is a kind of inflammatory misorder existing in pancreas. Non-coding RNAs (ncRNAs) have been reported to play important roles in development of AP. The current study was designed to explore the role of circular RNA zinc finger protein 644 (circRNA circ_ZFP644) in caerulein-induced AR42J cells. AP model in vitro was established by exposure of rat pancreatic acinar AR42J cells to caerulein. Amylase activity was measured using a kit. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the levels of several inflammatory factors. The expression of circ_ZFP644, microRNA (miR)-106b and protein inhibitor of activated STAT 3 (Pias3) was detected by quantitative real-time PCR (qRT-PCR) or western blot assay. And flow cytometry was employed to monitor cell apoptosis. Western blot assay was also conducted to analyze the expression of apoptosis-related proteins. The association among circ_ZFP644, miR-106b and Pias3 was validated by dual-luciferase reporter assay. Caerulein treatment activated amylase activity and promoted the secretion of inflammatory cytokines in AR42J cells. Circ_ZFP644 and Pias3 were downregulated, but miR-106b was upregulated in caerulein-induced AR42J cells. Enforced expression of circ_ZFP644 or miR-106b inhibition could reduce amylase activity and inflammatory cytokine secretion, while promote apoptosis in caerulein-induced AR42J cells, which was almost reversed by Pias3 knockdown. Circ_ZFP644 targeted miR-106b to upregulate Pias3 expression. Circ_ZFP644 might exert its anti-inflammation and pro-apoptosis roles in caerulein-induced AR42J cells by regulating miR-106b/Pias3 axis.

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms.

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.

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.

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.

Growth hormone (GH) plays a key role regulating somatic growth and in controlling metabolism and other physiological processes in humans and other animal species. GH acts by binding to the extracellular part of its transmembrane receptor, leading to induction of multiple intracellular signal transduction pathways that culminate in changes in gene and protein expression. A key agent in GH-stimulated growth is the latent transcription factor signal transducer and activator of transcription (STAT) 5B, one of four STAT proteins induced by the GH receptor in cultured cells and in vivo. As shown by genetic and biochemical studies, GH-activated STAT5B promotes transcription of the gene encoding the critical growth peptide, insulin-like growth factor-I (IGF-I), and natural null mutations of STAT5B in humans lead to growth failure accompanied by diminished IGF-I expression. Here we have examined the possibility that other GH-activated STATs can enhance IGF-I gene transcription, and thus potentially contribute to GH-regulated somatic growth. We find that human STAT5A is nearly identical to STAT5B in its biochemical and functional responses to GH but that STAT1 and STAT3 show a weaker profile of in vitro binding to STAT DNA elements from the IGF-I gene than STAT5B, and are less potent inducers of gene transcription through these elements. Taken together, our results offer a molecular explanation for why STAT5B is a key in vivo mediator of GH-activated IGF-I gene transcription and thus of GH-regulated somatic growth.

The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.

The long-term objective of this study was to produce STAT synthetic protein in chicken during growth period resulting from the increase of growth hormone (GH) as growth promoter. This study used ten male chicken Lohman from PT. Multibreeder Indonesia. The chicken were kept within batteried cage, with a capacity of one chicken in each cage. The chickens were fed twice a day, at 6 a.m. and 6 p.m. with the amount of feed 10% less than standard. On day 21 the chicken were slaughtered to obtain the samples, i.e., adipose, liver and muscles for the following examinations (1) isolation of STAT-3 signaling protein from adipose, liver and muscles of the chicken, (2) analysis of STAT-3 signaling protein using SDS-PAGE method, and (3) identification of STAT-3 signaling protein using Western blot method by means of protein detection using electrophoresis with polyacrylamide gels. Results of examination on protein in hepatic, muscle and adipose of chickens in growth period revealed that STAT protein was positively pre...

The Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway is one of a limited number of signaling cascades that is extensively utilized for many developmental and homeostatic functions. The JAK/STAT pathway is evolutionarily conserved from insects to mammals, with homologous transduction machinery in each. Yet the mammalian pathway is composed of multiple members for each family of proteins, while flies have only a single homologue of most pathway components. This simplicity and the abundance of genetic, biochemical, and developmental tools make Drosophila an attractive model to investigate this important signaling pathway. This review provides a basic description of the Drosophila JAK/STAT cascade and summarizes currently available reagents and tools to study and manipulate the pathway.

// Samadhi Aparicio-Siegmund 1 , Jan Sommer 1 , Niloufar Monhasery 1 , Ralf Schwanbeck 2 , Eric Keil 3 , David Finkenstadt 3 , Klaus Pfeffer 3 , Stefan Rose-John 2 , Jurgen Scheller 1 and Christoph Garbers 1,4 1 Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Dusseldorf, Germany; 2 Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany; 3 Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine University, Dusseldorf, Germany 4 Present address: Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany Correspondence: Jurgen Scheller, email: // Christoph Garbers, email: // Keywords : STAT3, cytokines, tumor, oncogene, signal transduction Received : February 11, 2014 Accepted : March 22, 2014 Published : March 23, 2014 Abstract The Janus kinase / signal transducer and activator of transcription (Jak/STAT) pathway can be activated by many different cytokines, among them all members of the Interleukin (IL-)6 family. Dysregulation of this pathway, resulting in its constitutive activation, is associated with chronic inflammation and cancer development. In the present study, we show that activity of protein kinase II (CK2), a ubiquitously expressed serine/threonine kinase, is needed for induced activation of STAT1 and STAT3 by IL-6 classic and trans-signaling, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). Inhibition of CK2 efficiently prevented STAT phosphorylation and inhibited cytokine-dependent cell proliferation in a Jak1-dependent manner. Conversely, forced activation of CK2 alone was not sufficient to induce activation of the Jak/STAT signaling pathway. Inhibition of CK2 in turn inhibited Jak1-dependent STAT activation by oncogenic gp130 mutations. Furthermore, CK2 inhibition diminished the Jak1- and Src kinase-dependent phosphorylation of a constitutively active STAT3 mutant recently described in human large granular lymphocytic leukemia. In conclusion, we characterize CK2 as an essential component of the Jak/STAT pathway. Pharmacologic inhibition of this kinase is therefore a promising strategy to treat human inflammatory diseases and malignancies associated with constitutive activation of the Jak/STAT pathway.

Impaired phosphatase activity leads to the persistent activation of signal transducers and activators of transcription (Stat). In mammals, Stat family members are often phosphorylated or dephosphorylated by the same enzymes. To date, only one Stat similar to mammalian Stat5a/b has been found in crustaceans and there have been few studies in Stat signal regulation in crustaceans. Here, we report that β-arrestin1 interacts with TC45 (45-kDa form of T cell protein tyrosine phosphatase) in the nucleus to attenuate Stat signaling by promoting dephosphorylation of Stat. Initially, we showed that Stat translocates into the nucleus to induce antimicrobial peptide (AMP) expression after bacterial infection. βArr1 enters the nucleus of hemocytes and recruits TC45 to form the βarr1-TC45-Stat complex, which dephosphorylates Stat efficiently. The interaction of TC45 with Stat decreased and Stat phosphorylation increased in βarr1-silenced shrimp (Marsupenaeus japonicus) after challenge with Vibrio anguillarum. βArr1 directly interacts with Stat in nucleus and accelerates Stat dephosphorylation by recruiting TC45 after V. anguillarum challenge. Further study showed that βarr1 and TC45 also affect AMP expression, which is regulated by Stat. Therefore, βarr1 and TC45 are involved in the anti-V. anguillarum immune response by regulating Stat activity negatively to decrease AMP expression in shrimp.

The molecular mechanism controlling the zygotic genome activation (ZGA) in mammals remains poorly understood. The 2-cell (2C)-like cells spontaneously emerging from cultures of mouse embryonic stem cells (ESCs) share some key transcriptional and epigenetic programs with 2C-stage embryos. By studying the transition of ESCs into 2C-like cells, we identified developmental pluripotency associated 2 and 4 (Dppa2/4) as important regulators controlling zygotic transcriptional program through directly up-regulating the expression of double homeobox (Dux). In addition, we found that DPPA2 protein is sumoylated and its activity is negatively regulated by small ubiquitin-like modifier (Sumo) E3 ligase protein inhibitor of activated STAT 4 (PIAS4). PIAS4 is down-regulated during ZGA process and during transitioning of ESCs into 2C-like cells. Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program, whereas depleting Dppa2/4 or forced expression of Pias4 or Sumo2–Dppa2 inhibits 2C-like transcriptional program. Furthermore, ectopic expression of Pias4 or Sumo2–Dppa2 impairs early mouse embryo development. In summary, our study identifies key molecular rivals consisting of transcription factors and a Sumo2 E3 ligase that regulate zygotic transcriptional program upstream of Dux., Analysis of cultured cells resembling those from 2-cell-stage embryos identifies transcription factors Dppa2/4 and the Sumo2 E3 ligase PIAS4 as key antagonistic regulators of the zygotic transcriptional program upstream of Dux.

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 JAK/STAT signaling pathway plays a critical role in different processes of ontogenesis in higher eukaryotes. The fruit fly drosophila is a convenient model used to study this pathway, since, in this system, its major components are represented by unique factors. This work describes the use of Drosophila melanogaster S2 cells to study the activation of JAK/STAT target genes. It was shown that S2 cells contain large quantities of STAT protein, which migrates into the nucleus in response to the treatment of the cell with pervanadate. In response to pervanadate, STAT, along with other transcription factors, is recruited onto the regulatory sequences of target genes and activates their transcription.

Protein expression abnormalities have been implicated in the pathophysiology of schizophrenia, but the underlying cause of these changes is not known. We sought to investigate ubiquitin and ubiquitin-like (UBL) systems (SUMOylation, NEDD8ylation, and Ufmylation) as putative mechanisms underlying protein expression abnormalities seen in schizophrenia. For this, we performed western blot analysis of total ubiquitination, free ubiquitin, K48- and K63-linked ubiquitination, and E1 activases, E2 conjugases, and E3 ligases involved in ubiquitination and UBL post-translational modifications in postmortem brain tissue samples from persons with schizophrenia (n=13) and comparison subjects (n=13). We studied the superior temporal gyrus (STG) of subjects from the Mount Sinai Medical Center brain collection that were matched for age, tissue pH, and sex. We found an overall reduction of protein ubiquitination, free ubiquitin, K48-linked ubiquitination, and increased K63 polyubiquitination in schizophrenia. Ubiquitin E1 activase UBA (ubiquitin activating enzyme)-6 and E3 ligase Nedd (neural precursor cell-expressed developmentally downregulated)-4 were decreased in this illness, as were E3 ligases involved in Ufmylation (UFL1) and SUMOylation (protein inhibitor of activated STAT 3, PIAS3). NEDD8ylation was also dysregulated in schizophrenia, with decreased levels of the E1 activase UBA3 and the E3 ligase Rnf7. This study of ubiquitin and UBL systems in schizophrenia found abnormalities of ubiquitination, Ufmylation, SUMOylation, and NEDD8ylation in the STG in this disorder. These results suggest a novel approach to the understanding of schizophrenia pathophysiology, where a disruption in homeostatic adaptation of the cell underlies discreet changes seen at the protein level in this illness.

Liver X receptors (LXRs) are nuclear receptors that function to modulate lipid metabolism as well as immune and inflammatory responses. Upon activation by their ligands, LXRs up-regulate a spectrum of gene transcription programs involved in cholesterol and fatty acid homeostasis. However, the mechanisms by which LXR-mediated transcriptional activation is regulated remain incompletely understood. Here, we show that PIAS1, a member of the protein inhibitor of the activated STAT family of proteins with small ubiquitin-like modifier (SUMO) E3 ligase activity, acts to suppress LXR ligand-dependent transcriptional activation of the lipogenic program in hepatocytes. We found that liver mRNA expression levels of Pias1 and Pias3 were inversely associated with those of genes involved in lipogenesis in mouse models with diet-induced or genetic obesity. Overexpression of PIAS1 in primary hepatocytes resulted in a reduction of LXR ligand-induced fatty acid synthesis and suppression of the expression of lipogenic genes, including Srebp1c and Fas. Moreover, PIAS1 was able to interact with LXRβ and repress its transcriptional activity upon ligand stimulation, which did not require PIAS1-promoted SUMO modification of LXRβ. In addition, PIAS1 could also interact with PGC-1β and attenuate its association with LXRβ, blunting the ability of PGC-1β to co-activate LXRβ. Importantly, PIAS1 impaired LXRβ binding to its target DNA sequence. Taken together, our results suggest that PIAS1 may serve as a lipogenic regulator by negatively modulating LXRs in a SUMOylation-independent manner.

Signal transducer and activator of transcription (STAT) proteins are key signaling molecules in response to cytokines and in regulating T cell biology. However, there are contradicting reports on whether STAT is involved in T-cell antigen receptor (TCR) signaling. To better define the role of STAT in TCR signaling, we activated the CD4/CD8-associated Lck kinase by co-crosslinking TCR and CD4/CD8 co-receptors in human peripheral blood T cells. Sequential STAT1, STAT3 and STAT5 activation was observed 1 hour after TCR stimulation suggesting that STAT proteins are not the immediate targets in the TCR complex. We further identified interferon-gamma as the key cytokine in STAT1 activation upon TCR engagement. In contrast to transient STAT activation in cytokine response, this autocrine/paracrine-induced STAT activation was sustained. It correlated with the absence of two suppressor of cytokine signaling (SOCS) proteins, SOCS3 and cytokine-inducible SH2 containing protein, that are negative feedback regulators of STAT signaling. Moreover, enforced expression of SOCS3 inhibited tyrosine phosphorylation of zeta-associated protein kinase of 70 kD in TCR-stimulated human Jurkat T cells. This is the first report demonstrating delayed and prolonged STAT activation coordinated with the loss of SOCS expression in human primary T cells after co-crosslinking of TCR and CD4/CD8 co-receptors.

ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function.

Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway.

In their journey to acquire the ability to fertilize the egg, numerous intracellular signaling systems are activated in spermatozoa, leading to an increase in protein tyrosine phosphorylation. Although the JAK/STAT signaling pathway is usually associated with the activation of transcription of specific genes, our laboratory previously demonstrated the presence of the IL6 receptor (IL6R) and the Janus kinase 1 (JAK1) in human spermatozoa, a cell that is mostly transcriptionally inactive. In order to determine the importance of the JAK/STAT signaling pathway, our objectives were to identify and characterize the mediators of this system in human sperm. Cell fractionation and surface biotinylation assays clearly demonstrated that IL6R is expressed at the sperm membrane surface. The kinase JAK1 is enriched in membrane fractions and is activated during human sperm capacitation as suggested by its increase in phosphotyrosine content. Many signal transducer and activator of transcription (STAT) proteins are expressed in human sperm, including STAT1, STAT3, STAT4, STAT5, and STAT6. Among them, only STAT1 and STAT5 were detected in the cytosolic fraction. All the detected STAT proteins were enriched in the cytoskeletal structures. STAT4 was present in the perinuclear theca, whereas JAK1, STAT1, and STAT5 were detected in the fibrous sheath. Indirect immunofluorescence studies showed that JAK1 and STAT1 colocalized in the neck region and that STAT4 is present at the equatorial segment and flagella. The presence of STAT proteins in sperm structural components suggests that their role is different from their well-known transcription factor activity in somatic cells, but further investigations are required to determine their role in sperm function.

Metastasis tumor antigen 1 (MTA1), a component of the Mi-2·nucleosome remodeling and deacetylase complex, plays a crucial role in gene transcription, but the mechanism involved remains largely unknown. Here, we report that MTA1 is a substrate for small ubiquitin-related modifier 2/3 (SUMO2/3) in vivo. Protein inhibitor of activated STAT (PIAS) proteins enhance SUMOylation of MTA1 and may participate in paralog-selective SUMOylation, whereas sentrin/SUMO-specific protease 1 (SENP1) and 2 may act as deSUMOylation enzymes for MTA1. Moreover, MTA1 contains a functional SUMO-interacting motif (SIM) at its C terminus, and SIM is required for the efficient SUMOylation of MTA1. SUMO conjugation on Lys-509, which is located within the SUMO consensus site, together with SIM synergistically regulates the co-repressor activity of MTA1 on PS2 transcription, probably by recruiting HDAC2 onto the PS2 promoter. Interestingly, MTA1 may up-regulate the expression of SUMO2 via interaction with RNA polymerase II and SP1 at the SUMO2 promoter. These findings not only provide novel mechanistic insights into the regulation of the transcriptional repressor function of MTA1 by SUMOylation and SIM but also uncover a potential function of MTA1 in modulating the SUMOylation pathway.

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain.

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development.

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.

The central nervous system regulates neuronal excitability by macromolecular signalling complexes that consist of functionally related proteins, including neurotransmitter receptors, enzymes and scaffolds. The composition of these signal complexes is regulated by post-translational modifications, such as phosphorylation and SUMOylation (SUMO is small ubiquitin-related modifier). In the present study, we searched for proteins interacting with the intracellular C-termini of the metabotropic glutamate receptors mGluR8a and mGluR8b and identified proteins of the SUMOylation and NEDDylation machinery. The SUMO E3 ligases Pias1 [Pias is protein inhibitor of activated STAT (signal transducer and activator of transcription)] and Pias3L interacted strongly with mGluR8b, and were co-localized with the E2-conjugating Ubc9, SUMO1 and mGluR8b in cell bodies present in the ganglion cell layer of the mammalian retina. SUMO1 conjugation of Lys882, present in a bona fide consensus sequence for SUMOylation (VKSE) in the mGluR8b C-terminus, was enhanced by addition of Pias1, consistent with an interaction between both proteins. Mutation of Lys882 to arginine reduced, but did not abolish, mGluR8b SUMOylation. Co-mutating a second lysine residue (Lys903) located in the mGluR8b isoform-specific C-terminus largely prevented SUMO1 conjugation by Ubc9. Modelling studies suggested that Lys903 contacts Ubc9 and thus is part of the non-canonical SUMOylation site VKSG. In summary, the results of the present study show in vivo SUMOylation of the complete mGluR8b and co-localize proteins of the SUMOylation machinery in the retina.

The protein inhibitor of activated STAT (PIAS) family proteins regulates innate immune responses by controlling transcription induced by Toll-like receptor, RIG-I-like receptor signaling, and JAK/STAT pathways. Here, we show that PIASy negatively regulates type I interferon (IFN) transcription. Virus infection led to enhanced type I IFN induction in PIASy null cells, and conversely PIASy overexpression reduced IFN transcription. A mutation in the LXXLL motif of the SAP domain abolished inhibition of IFN-stimulated gene expression but did not affect virus or Toll-like receptor/RIG-I-like receptor-stimulated IFN transcription, indicating that PIASy employs distinct mechanisms to inhibit virus-induced and IFN-stimulated transcription. SUMO E3 activity was not required for PIASy inhibition of IFN transcription; however, PIASy relied on the SUMO modification mechanism to inhibit IFN transcription, because the activity of the SUMO-interacting motif was required for inhibition, and knockdown of SUMO E2 enzyme UBC9 decreased inhibitory activity of PIASy. Our results demonstrate that PIASy negatively regulates both IFN transcription and IFN-stimulated gene expression through multiple mechanisms utilizing the function of different domains.

The signal transducers and activators of transcription (STAT) proteins are a small family of signaling proteins that are crucial for cytokine and growth factor receptor-mediated signaling in various blood cell types. Despite their central role in immune and hematopoietic cellular regulation, there are relatively few options for monitoring receptor-mediated JAK/STAT signaling events in a cell-based format, without the need for cellular transfections or labor intensive methodology. Indeed, traditional methods such as the Western blot or ELISA remain a standard method for determining the phosphorylation status of endogenous STAT proteins. Here we present data for the rapid detection of endogenous receptor-mediated phosphorylation of multiple STAT proteins using the bead-based AlphaScreen SureFire technology. With three different cell lines (human acute monocytic leukemia THP-1 cells, human erythroleukemic TF-1 cells, and human T lymphocytic Jurkat cells), we have optimized a rapid and homogeneous methodology for monitoring endogenous, receptor-mediated signaling via STAT 1, STAT 3, or STAT 5 phosphorylation, in response to several agonists. These assays, which can be tailored for both standard research applications or high-throughput drug screening applications, afford quantitative data for receptor-mediated signaling mechanisms in an endogenous, cellular environment.

Special AT-rich sequence-binding protein 1 (SATB1) is a tissue-restricted genome organizer that provides a key link between DNA loop organization, chromatin modification/remodeling, and transcription factor association at matrix attachment regions (MARs). The SUMO E3 ligase PIAS1 enhances SUMO conjugation to SATB1 lysine-744, and this modification regulates caspase-6 mediated cleavage of SATB1 at promyelocytic leukemia nuclear bodies (PML NBs). Since this regulated caspase cleavage occurs on only a subset of SATB1, and the products are relatively stable, proteolysis likely mediates cellular processes other than programmed cell death. However, the mechanism for the spatial and temporal regulation of SATB1 sumoylation and caspase cleavage is not known. Here we report that these processes are controlled by SATB1 phosphorylation; specifically, PIAS1 interaction with SATB1 is inhibited by phosphorylation. Mutagenesis studies identified interaction of the PIAS SAP (scaffold attachment factor-A/B/acinus/PIAS) motif with SATB1 N-terminal sequences. Notably, phosphorylation of SATB1 at threonine-188 regulates its interaction with PIAS1. Sequences near this phosphorylation site, LXXLL (residues 193 to 197), appear to be conserved among a subset of SUMO substrate proteins. Thus, this motif may be commonly involved in interaction with the PIAS SAP, and phosphorylation may similarly inhibit some of these substrates by preventing their interaction with the ligase.

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.

The family of STAT proteins consists of seven members that mediate highly specific functions in cytokine signaling. STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. In this report, we show that the coacitivator p100 protein can interact with STAT6 through its SN domain both in vivo and in vitro, resulting in enhancement of STAT6-mediated gene transcriptional acitivation. Consistent with our previous reports, we identified intracellular localization of p100 and STAT-6 by confocal microscopy examined in response to IL-4. Moreover, in consideration of STAT molecules sharing significant homology in structure and function, we detected whether p100 can associate with STAT-1. In conclusion, this study found no evidence that p100 functions as a transcriptional coactivator for STAT1-dependent gene regulation. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.

Members of the PIAS (protein inhibitor of activated STAT) family perform essential functions in modulating the activity of transcriptional regulators. Zimp7 and Zimp10 are two proteins that together form a subfamily of the PIAS. Like the other members of this family, they contain the zinc-binding SP-RING/Miz domain, which confers SUMO-conjugating activity. Both proteins have been shown to stimulate androgen receptor-mediated transcription. Previously, we reported that both Zimp7 and Zimp10 genes are extensively expressed and dynamically regulated in the developing mouse embryo. In this work, we investigated the expression of these genes during gonadal development. We found that their expression is sex-specific. Both genes initiate their transcription at early stages in the embryonic male gonad, reaching their peak at 13.5 days post coitum, which coincides with the process of sex-specific germ cell mitotic arrest. Zimp7 is expressed in germ cells of the embryonic gonad and the adult testis. Immunofluorescence of spermatogenic cells revealed that Zimp7 protein localizes to nuclear territories in meiotic spermatocytes, including the XY bodies. On the other hand, Zimp10 is found in somatic cells, outside the testis cords and ceases to be expressed in the adult testis.

The orphan nuclear receptor LRH-1 (liver receptor homologue-1; NR5A2) plays a critical role in development, bile acid synthesis and cholesterol metabolism. LRH-1 is also expressed in the ovary where it is implicated in the regulation of steroidogenic genes for steroid hormone synthesis. In the present study, we investigated the molecular mechanisms of the transcriptional regulation of CYP11A1 by LRH-1 and found that LRH-1-mediated transactivation was markedly repressed by PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) y], the shortest member of the PIAS family. The suppression of LRH-1 activity requires the N-terminal repression domain. Although PIAS proteins also function as E3 SUMO (small ubiquitin-related modifier) ligases and enhance SUMO conjugation, PIASy-mediated repression was independent of LRH-1 SUMOylation status. In addition, histone deacetylase activity was not involved in the inhibition of LRH-1 by PIASy. Immunoprecipitation and mammalian two-hybrid analyses indicated that PIASy interacted with LRH-1 through the C-terminal region, including the AF-2 (activation function-2) motif, which was also involved in the interaction between LRH-1 and the co-activator SRC-1 (steroid receptor co-activator-1). PIASy inhibited the binding of SRC-1 to LRH-1, although overexpression of SRC-1 partially overcame the PIASy inhibition of LRH-1 induction of the CYP11A1 promoter. The results of the present study suggest that competition with co-activators may be an important mechanism underlying the PIASy repression of LRH-1-mediated transactivation.

Objective— TGFβ and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) α-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFβ activates SM α-actin through PIAS1. Methods and Results— An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFβ-induced expression of SM α-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM α-actin promoter activity in a TGFβ control element (TCE)-dependent manner. Because the TCE within the SM α-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM α-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. Conclusions— These results provide evidence that PIAS1 promotes TGFβ-induced activation of SM α-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1’s corepressor activity.Structured summaryMINT-6787569:PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with HDAC3 (uniprotkb:O15379) by anti tag coimmunoprecipitation (MI:0007)MINT-6787767:PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with SUMO-1 (uniprotkb:P63165) by anti tag coimmunoprecipitation (MI:0007)

Covalent modification by small ubiquitin-related modifiers (SUMOs) is an important means to regulate dynamic residency of transcription factors within nuclear compartments. Here, we identify a multi-C(2)H(2)-type zinc finger protein (ZNF), ZNF451, as a novel nuclear protein that can be associated with promyelocytic leukemia bodies. In keeping with its interaction with SUMO E2 conjugase Ubc9 and SUMOs, ZNF451 is covalently modified by SUMOs (sumoylated) at several, albeit nonconsensus, sites. Interestingly, noncovalent SUMO-binding activity of ZNF451 (SUMO-interacting motif) is also important for its sumoylation. SUMO modifications regulate the nuclear compartmentalization of ZNF451, since coexpression of ZNF451 with SUMO-specific proteases SENP1 or SENP2, both capable of desumoylating the protein, redistributes ZNF451 from nuclear domains to speckles and nucleoplasm. Interaction of ZNF451 with PIAS1 (protein inhibitor of activated STAT 1) is not manifested as PIAS1's E3 SUMO ligase activity towards ZNF451 but results in disintegration of ZNF451 nuclear domains and recruitment of ZNF451 to androgen receptor (AR) speckles. ZNF451 interacts weakly, but in a SUMO-1-enhanced fashion, with AR. ZNF451 does not harbor an intrinsic transcription activation function, but interestingly, ablation of endogenous ZNF451 in prostate cancer cells significantly decreases expression of several AR target genes. Thus, we suggest that ZNF451 exerts its effects via SUMO modification machinery and trafficking of transcription regulators between promyelocytic leukemia bodies and nucleoplasm.

The JAK/STAT signal transduction pathway has traditionally been viewed as a cytokine-stimulated activator of gene expression consisting of a straightforward receptor/JAK kinase/STAT transcription factor cascade. Recent studies in Drosophila, have, however consistently identified a range of chromatin-remodelling factors as regulators of in vivo JAK/STAT signalling. Now, the detailed analysis of one of these, heterochromatin protein 1 (HP1), has provided an insight into an unexpected non-canonical in vivo role for STAT. In this model, unphosphorylated STATs associate with and maintain the stability of transcriptionally repressed heterochromatin--an effect countered by the recruitment of STAT to the canonical pathway. We examine the background of this new model and its implications for JAK/STAT pathway requirements in stem cell maintenance and cancer.

7 páginas, 5 figuras., In vertebrates, seven signal transducer and activator of transcription (STAT) proteins bind to palindromic sites separated by spacers of two or three nucleotides (STAT1), four nucleotides (STAT6) or three nucleotides (STAT2 to STAT5a/b). This diversity of binding sites provides specificity to counter semiredundancy and was thought to be a recent evolutionary acquisition. Here, we examine the natural DNA-binding sites of the single Drosophila Stat and show that this is not the case. Rather, Drosophila Stat92E is able to bind to and activate target gene expression through both 3n and 4n spaced sites. Our experiments indicate that Stat92E has a higher binding affinity for 3n sites than for 4n sites and suggest that the levels of target gene expression can be modulated by insertion and/or deletion of single bases. Our results indicate that the ancestral STAT protein had the capacity to bind to 3n and 4n sites and that specific STAT binding preferences evolved with the radiation of the vertebrate STAT family., J.C.-G.H. was supported by Junta de Andalucía, Ministerio de Educación y Ciencia, Consolider and the European Regional Development Fund (ERDF); M.P.Z. by the Deutsche Forschungsgemeinschaft (DFG), the Max Planck Society and The Royal Society. M.P.Z. is a Cancer Research-UK Senior Cancer Research Fellow.

Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins regulates multiple processes in the eukaryotic cell. In numerous cases sumoylation is facilitated by protein inhibitor of activated STAT (PIAS) proteins, characterized by the presence of a SP-RING domain related to the RING finger of many ubiquitin E3 ligases. The importance of SP-RING relies on its capacity to bind the E2 enzyme of the pathway. Additional domains may participate in SUMO ligase function and target selection. We have studied the Arabidopsis SUMO ligase AtSIZ1, belonging to the PIAS family, and describe self-sumoylation and AtSIZ1-mediated sumoylation of the E2 enzyme AtSCE1 and GTE3, a bromodomain protein interacting with AtSIZ1. Modification of GTE3 modulates its capacity to bind acetyl-histone H3 in vitro. Interestingly, AtSIZ1, as other plant PIAS proteins, also includes a PHD domain. We found that the PHD domain binds AtSCE1 and contributes to the SUMO ligase function, being partially and absolutely required for AtSCE1 and GTE3 sumoylation, respectively. Based on the capacity of AtSCE1 and GTE3 to associate with both the PHD and SP-RING domains, we propose a model of interactions to explain AtSIZ1-mediated sumoylation of GTE3 and ligase function of the PHD domain.

Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.

Although the JAK/STAT signaling pathway is usually involved in antiviral defense, a recent study suggested that STAT might be annexed by WSSV (white spot syndrome virus) to enhance the expression of a viral immediate early gene in infected shrimps. In the present study, we clone and report the first full-length cDNA sequence for a crustacean STAT from Penaeus monodon. Alignment and comparison with the deduced amino acid sequences of other STATs identified several important conserved residues and functional domains, including the DNA binding domain, SH2 domain and C-terminal transactivation domain. Based on these conserved sequences, a phylogenetic analysis suggested that shrimp STAT belongs to the ancient STAT family, while the presence of the functional domains suggested that shrimp STAT might share similar functions and regulating mechanisms with the well-known STATs isolated from model organisms. Real-time PCR showed a decreased transcription level of shrimp STAT after WSSV infection, but a Western blot analysis using anti-phosphorylated STAT antibody showed an increased level of phosphorylated (activated) STAT in the lymphoid organ of shrimp after WSSV infection. We further show that a primary culture of lymphoid organ cells from WSSV-infected shrimp resulted in activated STAT being translocated from the cytoplasm to the nucleus. This report provides experimental evidence that shrimp STAT is activated in response to WSSV infection. Our results support an earlier finding that WSSV does not disrupt JAK/STAT pathway, but on the contrary benefits from STAT activation in the shrimp host.

Transcriptional activity of signal-dependent transcription factors, including nuclear receptors, relies on interacting co-regulator proteins, many of which possess protein-modifying activity. SUMOs (small ubiquitin-related modifiers) and their conjugation pathway components act as co-regulator proteins for numerous transcription factors that also are often targets for SUMO modification. PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] proteins promote SUMOylation in a manner that resembles the action of RING-type ubiquitin E3 ligases. PIAS proteins were initially named for their ability to interact with STAT proteins and inhibit their activity, but their interactions and functions are not restricted to the STATs. Moreover, PIAS proteins do not operate merely as SUMO E3s, since their co-regulator effects are often independent of their RING finger but dependent on their SIM (SUMO-interacting motif) or SAP (scaffold attachment factor-A/B/acinus/PIAS) domain capable of interacting with DNA. The modulator activity imparted by the PIAS/SUMO system involves altered subnuclear targeting and/or assembly of transcription complexes. PIAS proteins may act as platforms that facilitate both removal and recruitment of other regulatory proteins in the transcription complexes.