SUMOylation mediates CtIP's functions in DNA end resection and replication fork protection.

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.

SUMO E3 ligase PIAS1 is a potential biomarker indicating stress susceptibility.

Prior studies suggest that individual differences in stress responses contribute to the pathogenesis of neuropsychiatric disorders. In the present study, we investigated the role of small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) in mediating stress responses to chronic social defeat stress (CSDS). We found that mRNA and protein levels of PIAS 1 were decreased in the hippocampus of high-susceptibility (HS) mice but not in low-susceptibility (LS) mice after CSDS. Local overexpression of PIAS1 in the hippocampus followed by CSDS exposure promoted stress resilience by attenuating social avoidance and improving anxiety-like behaviors. Viral-mediated gene transfer to generate a conditional knockdown of PIAS1 in the hippocampus promoted social avoidance and stress vulnerability after subthreshold microdefeat. HS mice displayed decreased levels of glucocorticoid receptor (GR) expression, and GR SUMOylation in the hippocampus was associated with stress vulnerability. Furthermore, cytokine/chemokine levels were changed predominantly in the hippocampus of HS mice. These results suggest that hippocampal PIAS1 plays a role in the regulation of stress susceptibility by post-translational modification of GRs.

DPPA2/4 and SUMO E3 ligase PIAS4 opposingly regulate zygotic transcriptional program.

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.

The Role of PIAS SUMO E3-Ligases in Cancer.

SUMOylation modifies the interactome, localization, activity, and lifespan of its target proteins. This process regulates several cellular machineries, including transcription, DNA damage repair, cell-cycle progression, and apoptosis. Accordingly, SUMOylation is critical in maintaining cellular homeostasis, and its deregulation leads to the corruption of a plethora of cellular processes that contribute to disease states. Among the proteins involved in SUMOylation, the protein inhibitor of activated STAT (PIAS) E3-ligases were initially described as transcriptional coregulators. Recent findings also indicate that they have a role in regulating protein stability and signaling transduction pathways. PIAS proteins interact with up to 60 cellular partners affecting several cellular processes, most notably immune regulation and DNA repair, but also cellular proliferation and survival. Here, we summarize the current knowledge about their role in tumorigenesis and cancer-related processes. Cancer Res; 77(7); 1542-7. ©2017 AACR.

Pias1 is essential for erythroid and vascular development in the mouse embryo.

The protein inhibitor of activated STAT-1 (PIAS1) is one of the few known SUMO E3 ligases. PIAS1 has been implicated in several biological processes including repression of innate immunity and DNA repair. However, PIAS1 function during development and tissue differentiation has not been studied. Here, we report that Pias1 is required for proper embryonic development. Approximately 90% of Pias1 null embryos die in utero between E10.5 and E12.5. We found significant apoptosis within the yolk sac (YS) blood vessels and concomitant loss of red blood cells (RBCs) resulting in profound anemia. In addition, Pias1 loss impairs YS angiogenesis and results in defective capillary plexus formation and blood vessel occlusions. Moreover, heart development is impaired as a result of loss of myocardium muscle mass. Accordingly, we found that Pias1 expression in primary myoblasts enhances the induction of cardiac muscle genes MyoD, Myogenin and Myomaker. PIAS1 protein regulation of cardiac gene transcription is dependent on transcription factors Myocardin and Gata-4. Finally, endothelial cell specific inactivation of Pias1 in vivo impairs YS erythrogenesis, angiogenesis and recapitulates loss of myocardium muscle mass. However, these defects are not sufficient to recapitulate the lethal phenotype of Pias1 null embryos. These findings highlight Pias1 as an essential gene for YS erythropoiesis and vasculogenesis in vivo.

ZBTB17 (MIZ1) Is Important for the Cardiac Stress Response and a Novel Candidate Gene for Cardiomyopathy and Heart Failure.

BACKGROUND: Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. METHODS AND RESULTS: We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. CONCLUSIONS: We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene.

Starvation promotes REV1 SUMOylation and p53-dependent sensitization of melanoma and breast cancer cells.

Short-term starvation or fasting can augment cancer treatment efficacy and can be effective in delaying cancer progression in the absence of chemotherapy, but the underlying molecular mechanisms of action remain elusive. Here, we describe the role of REV1, a specialized DNA polymerase involved in DNA repair, as an important signaling node linking nutrient sensing and metabolic control to cell fate. We show that REV1 is a novel binding partner of the tumor suppressor p53 and regulates its activity. Under starvation, REV1 is modified by SUMO2/3, resulting in the relief of REV1's inhibition of p53 and enhancing p53's effects on proapoptotic gene expression and apoptosis in breast cancer and melanoma cells. Thus, fasting in part through its effect on REV1 is a promising nontoxic strategy to increase p53-dependent cell death and to enhance the efficacy of cancer therapies.

Smad3 signaling in the regenerating liver: implications for the regulation of IL-6 expression.

Liver regeneration is vital for graft survival and adequate organ function. Smad activation regulates hepatocyte proliferation and macrophage function. The aim of the current study was to evaluate the impact of Smad3 signaling during liver regeneration in the mouse. Male C57Bl/6 wild-type (wt) mice or mice deficient in Smad3 (Smad3(-/-) ) were subjected to a 70% partial hepatectomy (pHx) or sham surgery and sacrificed 24, 42, or 48 h later. Tissue was analyzed for TGF-ß signaling, the mitogenic cytokine response [i.e., tumor necrosis factor alpha, TNF-α; interleukin (IL)-6], and liver regeneration. Partial hepatectomy stimulated a strong regenerative response measured by proliferating cell nuclear antigen-positive hepatocytes 42 and 48 h post-pHx in conjunction with an increased expression of IL-6, TNF-α, and Smad2/3 phosphorylation 24 h post-pHx in both hepatocytes and nonparenchymal cells. Surprisingly, Smad3 deficiency led to reduced hepatocyte proliferation 42 h post-pHx which recovered by 48 h, a process that correlated with and was preceded by significant reductions in IL-6 expression and signal transducer and activator of transcription 3 phosphorylation, and cyclin D1 induction 24 h post-pHx. Loss of Smad3 signaling suppresses the expression of key mitogenic cytokines and delays hepatocellular regeneration. Therapies directed at finely regulating Smad3 activation early within the regenerating liver may prove useful in promoting liver cell proliferation and restoration of liver mass.

PIAS adds methyl-bias to HSC-differentiation.

Epigenetic modifications of stem cell genome including DNA methylation and histone modifications are critical for the regulation of stem cell gene expression and maintenance of stem cell pool and their differentiation. Although the importance of epigenetic modifications specifically DNA methylation to adult hematopoietic stem cells (HSC) has been established, the identity of specific modulators and precise mechanism of integration of methylation events remain to be uncovered. In this issue, Shuai and colleagues identify the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT1) as a key regulator of DNA methylation of HSC required for their maintenance and lineage commitment (Liu et al, 2014).

PIAS3 activates the intrinsic apoptotic pathway in non-small cell lung cancer cells independent of p53 status.

Protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In this study, we sought to determine whether PIAS3 inhibits cell growth in non-small cell lung cancer cell lines by inducing apoptosis. Our results demonstrate that overexpression of PIAS3 promotes mitochondrial depolarization, leading to cytochrome c release, caspase 9 and 3 activation and poly (ADP-ribose) polymerase cleavage. This intrinsic pathway activation was associated with decreased Bcl-xL expression and increased Noxa expression and was independent of p53 status. Furthermore, PIAS3 inhibition of STAT3 activity was also p53 independent. Microarray experiments were performed to discover STAT3-independent mediators of PIAS3-induced apoptosis by comparing the apoptotic gene expression signature induced by PIAS3 overexpression with that induced by STAT3 siRNA. The results showed that a subset of apoptotic genes was uniquely expressed only after PIAS3 expression. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy and its potential to synergize with Bcl-2 targeted inhibitors.

PIAS1 SUMO ligase regulates the self-renewal and differentiation of hematopoietic stem cells.

The selective and temporal DNA methylation plays an important role in the self-renewal and differentiation of hematopoietic stem cells (HSCs), but the molecular mechanism that controls the dynamics of DNA methylation is not understood. Here, we report that the PIAS1 epigenetic pathway plays an important role in regulating HSC self-renewal and differentiation. PIAS1 is required for maintaining the quiescence of dormant HSCs and the long-term repopulating capacity of HSC. Pias1 disruption caused the abnormal expression of lineage-associated genes. Bisulfite sequencing analysis revealed the premature promoter demethylation of Gata1, a key myeloerythroid transcription factor and a PIAS1-target gene, in Pias1(-/-) HSCs. As a result, Pias1 disruption caused the inappropriate induction of Gata1 in HSCs and common lymphoid progenitors (CLPs). The expression of other myeloerythroid genes was also enhanced in CLPs and lineage-negative progenitors, with a concurrent repression of B cell-specific genes. Consistently, Pias1 disruption caused enhanced myeloerythroid, but reduced B lymphoid lineage differentiation. These results identify a novel role of PIAS1 in maintaining the quiescence of dormant HSCs and in the epigenetic repression of the myeloerythroid program.

Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1.

Smoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

PIASγ enhanced SUMO-2 modification of Nurr1 activation-function-1 domain limits Nurr1 transcriptional synergy.

Nurr1 (NR4A2) is a transcription factor that belongs to the orphan NR4A group of the nuclear receptor superfamily. Nurr1 plays key roles in the origin and maintenance of midbrain dopamine neurons, and peripheral inflammatory processes. PIASγ, a SUMO-E3 ligase, represses Nurr1 transcriptional activity. We report that Nurr1 is SUMOylated by SUMO-2 in the lysine 91 located in the transcriptional activation function 1 domain of Nurr1. Nurr1 SUMOylation by SUMO-2 is markedly facilitated by overexpressing wild type PIASγ, but not by a mutant form of PIASγ lacking its first LXXLL motif (PIASγmut1). This PIASγmut1 is also unable to interact with Nurr1 and to repress Nurr1 transcriptional activity. Interestingly, the mutant PIASγC342A that lacks SUMO ligase activity is still able to significantly repress Nurr1-dependent transcriptional activity, but not to enhance Nurr1 SUMOylation. A SUMOylation-deficient Nurr1 mutant displays higher transcriptional activity than the wild type Nurr1 only in promoters harboring more than one Nurr1 response element. Furthermore, lysine 91, the major target of Nurr1 SUMOylation is contained in a canonical synergy control motif, indicating that SUMO-2 posttranslational modification of Nurr1 regulates its transcriptional synergy in complex promoters. In conclusion, PIASγ can exert two types of negative regulations over Nurr1. On one hand, PIASγ limits Nurr1 transactivation in complex promoters by SUMOylating its lysine 91. On the other hand, PIASγ fully represses Nurr1 transactivation through a direct interaction, independently of its E3-ligase activity.

NMDA receptor signaling mediates the expression of protein inhibitor of activated STAT1 (PIAS1) in rat hippocampus.

Protein inhibitor of activated STAT1 (PIAS1) was shown to play an important role in inflammation and innate immune response, but how PIAS1 is regulated is not known. We have recently demonstrated that PIAS1 enhances spatial learning and memory performance in rats. In this study, we examined the signaling pathway and neural mechanism that regulate PIAS1 expression in the brain by using pharmacological and molecular approaches. Our results revealed that pias1 gene expression is rapidly induced upon NMDA receptor activation in rat hippocampus, but this effect is blocked by transfection of sub-threshold concentrations of ERK1 siRNA/ERK2 siRNA or CREB siRNA. Pias1 gene expression is similarly induced by overexpression of the ERK1/ERK2 plasmids in rat hippocampus, and this effect is also blocked by sub-threshold concentration of CREB siRNA transfection. On the other hand, transfection of ERK1 siRNA/ERK2 siRNA or CREB siRNA at a higher concentration is sufficient to down-regulate PIAS1 expression. Inhibition of PI-3 kinase signaling and CaMKII signaling, which both result in CREB inactivation, similarly decreases PIAS1 expression. But NMDA and MK-801 do not affect the expression of IL-6 and TNFα. NMDA also did not affect the expression of PIAS2, PIAS3 and PIAS4. Further, pias1 mRNA has a similar degradation rate to that of the zif268 gene. These results together suggest that pias1 may function as an immediate early gene in an activity-dependent manner and PIAS1 expression is regulated by the NMDA-MAPK/ERK-CREB signaling pathway implicated in neuronal plasticity.

Forkhead box protein A2 (FOXA2) protein stability and activity are regulated by sumoylation.

The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

SUMOylation of GTF2IRD1 regulates protein partner interactions and ubiquitin-mediated degradation.

GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxß, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxß. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxß itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell.

PIAS1 is increased in human prostate cancer and enhances proliferation through inhibition of p21.

Prostate cancer development and progression are associated with alterations in expression and function of elements of cytokine networks, some of which can activate multiple signaling pathways. Protein inhibitor of activated signal transducers and activators of transcription (PIAS)1, a regulator of cytokine signaling, may be implicated in the modulation of cellular events during carcinogenesis. This study was designed to investigate the functional significance of PIAS1 in models of human prostate cancer. We demonstrate for the first time that PIAS1 protein expression is significantly higher in malignant areas of clinical prostate cancer specimens than in normal tissues, thus suggesting a growth-promoting role for PIAS1. Expression of PIAS1 was observed in the majority of tested prostate cancer cell lines. In addition, we investigated the mechanism by which PIAS1 might promote prostate cancer and found that down-regulation of PIAS1 leads to decreased proliferation and colony formation ability of prostate cancer cell lines. This decrease correlates with cell cycle arrest in the G0/G1 phase, which is mediated by increased expression of p21(CIP1/WAF1). Furthermore, PIAS1 overexpression positively influences cell cycle progression and thereby stimulates proliferation, which can be mechanistically explained by a decrease in the levels of cellular p21. Taken together, our data reveal an important new role for PIAS1 in the regulation of cell proliferation in prostate cancer.

SUMO E3 ligases are expressed in the retina and regulate SUMOylation of the metabotropic glutamate receptor 8b.

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 ligase PIAS1 restricts natural regulatory T cell differentiation by epigenetic repression.

CD4(+)Foxp3(+) regulatory T (T(reg)) cells are important for maintaining immune tolerance. Understanding the molecular mechanism that regulates T(reg) differentiation will facilitate the development of effective therapeutic strategies against autoimmune diseases. We report here that the SUMO E3 ligase PIAS1 restricts the differentiation of natural T(reg) cells by maintaining a repressive chromatin state of the Foxp3 promoter. PIAS1 acts by binding to the Foxp3 promoter to recruit DNA methyltransferases and heterochromatin protein 1 for epigenetic modifications. Pias1 deletion caused promoter demethylation, reduced histone H3 methylation at Lys(9), and enhanced promoter accessibility. Consistently, Pias1(-/-) mice displayed an increased natural T(reg) cell population and were resistant to the development of experimental autoimmune encephalomyelitis. Our studies have identified an epigenetic mechanism that negatively regulates the differentiation of natural T(reg) cells.