PIAS Proteins Research Articles

A UL26-PIAS1 complex antagonizes anti-viral gene expression during Human Cytomegalovirus infection.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

PIAS Factors from Rainbow Trout Control NF-κB- and STAT-Dependent Gene Expression.

Four ‘protein inhibitors of activated STAT’ (PIAS) control STAT-dependent and NF-κB-dependent immune signalling in humans. The genome of rainbow trout (Oncorhynchus mykiss) contains eight pias genes, which encode at least 14 different pias transcripts that are differentially expressed in a tissue- and cell-specific manner. Pias1a2 was the most strongly expressed variant among the analysed pias genes in most tissues, while pias4a2 was commonly low or absent. Since the knock-out of Pias factors in salmonid CHSE cells using CRISPR/Cas9 technology failed, three structurally different Pias protein variants were selected for overexpression studies in CHSE-214 cells. All three factors quenched the basal activity of an NF-κB promoter in a dose-dependent fashion, while the activity of an Mx promoter remained unaffected. Nevertheless, all three overexpressed Pias variants from trout strongly reduced the transcript level of the antiviral Stat-dependent mx gene in ifnγ-expressing CHSE-214 cells. Unlike mx, the overexpressed Pias factors modulated the transcript levels of NF-κB-dependent immune genes (mainly il6, il10, ifna3, and stat4) in ifnγ-expressing CHSE-214 cells in different ways. This dissimilar modulation of expression may result from the physical cooperation of the Pias proteins from trout with differential sets of interacting factors bound to distinct nuclear structures, as reflected by the differential nuclear localisation of trout Pias factors. In conclusion, this study provides evidence for the multiplication of pias genes and their sub-functionalisation during salmonid evolution.

Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins

The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.

Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins

The human PIAS proteins are SUMO E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C-terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how posttranslational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation ismediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that inDrosophila the small ubiquitin-like protein SUMOand the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

Chordate PIAS proteins act as conserved repressors of the TRAF6 self-polyubiquitination

In mammals, PIAS proteins are important SUMO E3 ligases and act as versatile regulators of over sixty different proteins, including components from the NF-κB pathways. But the PIAS functions are not well-understood due to complicated molecular mechanisms and multiple gene paralogs with overlapping roles, which is especially true in lower vertebrates where dedicated studies are scarce. As a basal chordate with a single PIAS gene, amphioxus is a convenient model to study PIAS from the evolutionary perspective. TRAF6 is a critical adaptor of the NF-κB pathways but it is not known whether TRAF6 is regulated by PIAS. Here we discover that in mammalian cells, amphioxus PIAS inhibited NF-κB activation by co-localizing and binding with TRAF6. The interaction relied on the N-terminal SAP and PINIT domains of PIAS. TRAF6 is an E3 ubiquitin ligase, which initiates downstream NF-κB signaling by promoting its self-ubiquitination. Both amphioxus SUMO1 and Ubc9 (SUMO E2 ligase) could suppress TRAF6 self-ubiquitination and NF-κB activation, suggesting that the SUMOylation activity competed away the ubiquitination activity of TRAF6. However, we show that the wild-type PIAS and the mutant PIAS without SUMO E3 ligase activity both could inhibit TRAF6-mediated NF-κB activation by reducing TRAF6 self-ubiquitination. This implies that SUMO ligase activity is not the only mechanism for PIAS to negatively regulate TRAF6. Finally, we tested the interactions between human PIAS1-4 and TRAF6. It reveals that human PIAS1, 3 and 4, but not 2, were able to repress NF-κB activation by reducing TRAF6 self-ubiquitination. Taken together, our study discovers a conserved regulatory interaction between chordate PIAS and TRAF6. It therefore sheds light on the complicated role of PIAS in immune regulation, and may help to understand the PIAS functions in other lower chordate taxa, such as jawless and jawed fishes.

Sumoylation of a small isoform of NFATc1 is promoted by PIAS proteins and inhibits transactivation activity

The NFAT family of transcription factors plays an important role in immune system development and function. NFATc1 and NFATc2 are highly expressed in peripheral T cells, and several isoforms are produced via the use of different promoters and polyadenylation sites. The specific isoforms with relatively long C-termini, NFATc1/C and NFATc2/A, have been shown to be modified by SUMO within their specific C-terminal regions, which regulates NFAT protein localization and transactivation activity. Here, we demonstrate that an isoform NFATc1/A, which has a short C-terminus and does not contain the sumoylation sites found in the long isoforms, is also modified by SUMO. NFATc1/A sumoylation increased with low level expression of SUMO E3 ligases, specifically PIAS1, PIAS3, and PIASy, in co-transfected cells. PIAS3 interacted with NFATc1/A and an active site mutant failed to promote NFATc1/A sumoylation, indicating a role for PIAS3 as a SUMO E3 ligase. A lysine residue at 351 within the central regulatory domain was identified as the major SUMO attachment site in both co-transfection and in vitro assays. Sumoylation of NFATc1/A did not affect nuclear translocation upon ionomycin and phorbol 12-myristate 13-acetate treatment. However, although sumoylation of NFATc1/A slightly increased protein stability, it inhibited transactivation activity for reporter genes driven by promoters containing NFAT sites. Our results indicate that the transactivation activity of NFATc1/A is negatively regulated by PIAS protein-mediated sumoylation, and that SUMO is a general regulator of NFAT family members with either long or short C-termini.

Chromatin remodeling: demethylating H3K4me3 of type I IFNs gene by Rbp2 through interacting with Piasy for transcriptional attenuation.

Type I IFNs (IFNIs) are involved in the course of antiviral and antimicrobial activities; however, robust inductions of these can lead to host immunopathology. We have reported that the Pias (protein inhibitor of activated signal transducer and activator of transcription) family member, Piasy, possesses the ability to suppress IFNI transcriptions in mouse embryonic fibroblasts (MEFs), yet the specific molecular mechanism by which it acts remains elusive. Here, we identify that the H3K4me3 levels, one activation mark of genes, in MEFs that were stimulated by poly(I:C) were impaired by Piasy in the IFN-β gene. Piasy bound to the promoter region of the IFN-β gene in MEFs. Meanwhile, retinoblastoma binding protein 2 (Rbp2) was proven to be the only known and novel H3K4me3 demethylase that interacted with Piasy. Overexpression of Rbp2, but not its enzymatically inactive mutant Rbp2H483G/E485Q, retarded the transcription activities of IFNI, whereas small interfering RNA-mediated or short hairpin RNA-mediated knockdown of Rbp2 enhanced IFNI promoter responses. Above all, coexpression of Piasy and Rbp2 led to statistically less IFNI induction than overexpression of either Piasy or Rbp2 alone. Mechanistically, Piasy bound to the Jmjc domain (451-503 aa) of Rbp2 via its PINIT domain (101-218 aa), which is consistent with the domain required for their attenuation of transcription and H3K4me3 levels of IFNI genes. Our study demonstrates that Piasy may prevent exaggerated transcription of IFNI by Rbp2-mediated demethylation of H3K4me3 of IFNI, avoiding excessive immune responses.-Yu, X., Chen, H., Zuo, C., Jin, X., Yin, Y., Wang, H., Jin, M., Ozato, K., Xu, S. Chromatin remodeling: demethylating H3K4me3 of type I IFNs gene by Rbp2 through interacting with Piasy for transcriptional attenuation.

SUMO Modification Reverses Inhibitory Effects of Smad Nuclear Interacting Protein-1 in TGF-β Responses

SNIP1 (Smad nuclear interacting protein 1) is a transcription repressor for the TGF-β and NF-κB signaling pathways through disrupting the recruitment of co-activator p300. However, it is unclear how the functions of SNIP1 in the TGF-β signaling pathway are controlled. Our present studies show that SNIP1 is covalently modified by small ubiquitin-like modifier (SUMO) in vitro and in vivo at three lysine sites: Lys5, Lys30, and Lys108, with Lys30 being the major SUMO modification site. SUMOylation of SNIP1 is enhanced by SUMO E3 ligase PIAS proteins and inhibited by SUMO proteases SENP1/2. Furthermore, we find that SUMOylation of SNIP1 attenuates its inhibitory effect in TGF-β signaling because the SUMO-conjugated form of SNIP1 exhibits impaired ability to disrupt the formation of Smad complex and the interaction between p300 and Smads. Subsequently, SUMOylation of SNIP1 leads to the loss of SNIP1-mediated inhibition on expression of the TGF-β target genes PAI-1 and MMP2 and eventually enhances TGF-β-regulated cell migration and invasion.

SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1.

ABSTRACTAspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1) as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signaling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signaling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.

Treating a Dysregulated JAK/STAT Pathway in Cancer Cells

The JAK/STAT pathway is induced by the binding of a cytokine to its cognate receptor. The receptor’s engagement with the cytokine recruits a JAK protein, which activates itself via auto/trans-phosphorylation. In turn, the activated JAKs recruit and phosphorylate STAT proteins. The phosphorylated STAT proteins form a dimer, translocate to the cell nucleus and acts as a transcription factor to induce gene expression. In this way, the JAK/STAT pathway can mediate a cell’s response to extracellular signals. The proteins ultimately induced by the JAK/STAT pathway contribute to processes such as inflammatory response, differentiation, proliferation, and apoptosis. When the JAK/STAT pathway becomes dysregulated, proto-oncogenes and/or tumor-suppressor genes are often inappropriately expressed, commonly resulting in oncogenesis. This review discusses how SOCS, PIAS, and PTPS proteins modulate the JAK/STAT pathway ensuring that it remains cyclic and transient. The use of jakibins, STAT inhibitors, decoy oligonucleotides, RNA interference and genome editing to synthetically regulate a dysregulated JAK/STAT pathway in cancer cells are also considered.

PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status

The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a “protein inhibitor of activated c-Myb” in the absence of SUMOylation while, in its presence, PIAS behaves as a “protein activator of repressed c-Myb”.

The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-like modifiers.

Mutations affecting the transcription factor FOXP2 cause a rare form of severe speech and language disorder. Although it is clear that sufficient FOXP2 expression is crucial for normal brain development, little is known about how this transcription factor is regulated. To investigate post-translational mechanisms for FOXP2 regulation, we searched for protein interaction partners of FOXP2, and identified members of the PIAS family as novel FOXP2 interactors. PIAS proteins mediate post-translational modification of a range of target proteins with small ubiquitin-like modifiers (SUMOs). We found that FOXP2 can be modified with all three human SUMO proteins and that PIAS1 promotes this process. An aetiological FOXP2 mutation found in a family with speech and language disorder markedly reduced FOXP2 SUMOylation. We demonstrate that FOXP2 is SUMOylated at a single major site, which is conserved in all FOXP2 vertebrate orthologues and in the paralogues FOXP1 and FOXP4. Abolishing this site did not lead to detectable changes in FOXP2 subcellular localization, stability, dimerization or transcriptional repression in cellular assays, but the conservation of this site suggests a potential role for SUMOylation in regulating FOXP2 activity in vivo.

Ubiquitously expressed transcript is a novel interacting protein of protein inhibitor of activated signal transducer and activator of transcription 2.

Protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is a member of the PIAS protein family. This protein family modulates the activity of several transcription factors and acts as an E3 ubiquitin ligase in the sumoylation pathway. To improve understanding of the physiological roles of PIAS2, the current study used a yeast two-hybrid system to screen mouse stem cell cDNA libraries for proteins that interact with PIAS2. The screening identified an interaction between PIAS2 and ubiquitously expressed transcript (UXT). UXT, also termed androgen receptor trapped clone-27, is an α-class prefoldin-type chaperone that acts as a coregulator for various transcription factors, including nuclear factor-κB and androgen receptor (AR). A direct interaction between PIAS2 and UXT was confirmed by direct yeast two-hybrid analysis. In vitro evidence of the association of UXT with PIAS2 was obtained by co-immunoprecipitation. Colocalization between PIAS2 and UXT was identified in the nucleus and cytoplasm of HEK 293T and human cervical carcinoma HeLa cells. The results of the current study suggested that UXT is a binding protein of PIAS2, and interaction between PIAS2 and UXT may be important for the transcriptional activation of AR.

A26 Basic Understanding: The Role Of Abnormal Htt Accumulation

<h3></h3> Diverse cellular processes are impacted by expression and accumulation of mutant HTT (mHTT) and post-translational modifications of HTT contribute to normal and aberrant protein levels and function. A number of cellular processes are involved in accumulation of mutant HTT. One of these, SUMO modification, involves the covalent attachment of SUMO (Small Ubiquitin-like Modifier) to specific lysine residues and modulates protein activity and clearance. The SUMO pathway is highly conserved and is implicated in HD and other neurodegenerative diseases including spinocerebellar ataxias, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS). We have shown that a single E3 SUMO ligase, PIAS1, modulates both SUMO-1 and 2/3 modification of mHTT in cells, raising the possibility that modulation of this enzyme could provide a selective therapeutic target. In recent studies, we show that instrastriatal viral delivery of PIAS1 miRNA in R6/2 mice significantly reduces HD-like behavioural phenotypes. Biochemical analysis reveals a reduction of accumulated SUMO-1/2, Ubiquitin modified proteins, and insoluble HMW HTT species. PIAS proteins are relevant in transcriptional regulation (mainly negative) of pathways including proinflammatory cytokine signalling and innate immune response. PIAS1 interacts with a wide range of proteins and structurally diverse molecules and we show that it may act as an important negative regulator of signalling cascades that regulate inflammation and protein clearance networks in HD. The pathways identified here in the context of HD and targeting gene-specific regulators such as PIAS1 may represent novel therapeutic strategies that impact mutant HTT levels and provide insight into mechanisms underlying HD.

Protein Inhibitors of Activated STAT (Pias1 and Piasy) Differentially Regulate Pituitary Homeobox 2 (PITX2) Transcriptional Activity

Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and β-catenin. Lef-1, β-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.

Abstract 3115: Sumoylation enhances the stability of the proto-oncogene EVI1 however negatively regulates its DNA binding and transcriptional activity.

Abstract Aberrant expression of the proto-oncogene EVI1 (Ecotropic virus integration site1) has been implicated not only in lymphoid or myeloid malignancies but also in several other solid tumors like colon, ovarian and breast cancer. Despite its crucial role in oncogenesis, the regulatory factors and mechanisms that control the function of EVI1 are partially known. Post translational modifications play a crucial role in regulating various cellular processes by controlling the function of several proteins. Here we show that EVI1, a transcription factor is post-translationally modified by SUMO1. Indirect immunofluorescence studies revealed that EVI1 and SUMO1 colocalized in the nuclear speckles in the cells transiently transfected with EVI1 and SUMO1 as well as cells that constitutively expresses EVI1. Sumoylation is known to stabilize their target protein which was also observed in case of EVI1 through pulse chase assay with cycloheximide. Reporter gene assay revealed that sumoylation impaired EVI1 mediated transactivation of Bcl-xL and SIRT1 promoters and also reduced the DNA binding efficiency of EVI1 which was confirmed through ChIP assay. PIAS proteins are known as SUMO E3 ligase which enhances the sumoylation of the proteins. PIAS proteins are also known to regulate the function of several proteins independent of its ligase activity. Co-immunoprecipiation assay revealed that EVI1 directly interacts with SUMO E3 ligase PIASy, which was also found to enhance EVI1 sumoylation. Our study also demonstrates that PIASy negatively regulated EVI1 transcription activity which is independent of its ligase activity. Thus PIASy was found to be an interacting partner that regulates the function of EVI1 in sumoylation dependent as well as independent manner. In summary, we present a new modifier of EVI1 which controls its stability and deregulates its transcriptional activity. Further study in context with EVI1-SUMO1-PIASy may give a better idea in understanding the functions and the regulation of EVI1 in normal and diseased cells. Citation Format: Sneha Singh, Soumen Chakraborty. Sumoylation enhances the stability of the proto-oncogene EVI1 however negatively regulates its DNA binding and transcriptional activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2013-3115

The transcriptional activity of co‐activator AIB1 is regulated by the SUMO E3 Ligase PIAS1

Amplified in breast cancer 1 (AIB1) is a transcriptional coactivator of nuclear receptors and other transcription factors. It is required for animal growth and reproductive development, and has also been implicated in breast carcinogenesis. Although AIB1 is known to be covalently modified by SUMO-1, which serves to regulate its stability and transcriptional activity, the exact SUMO E3 ligase involved in its sumoylation has not been determined. In order to resolve this question, we investigated the interaction between AIB1 and different members of PIAS proteins (all are SUMO E3 ligases) through immunoprecipiation. Among the five different PIAS proteins, only PIAS1 co-immunoprecipitated with AIB1 in extract prepared from breast cancer cells (MCF-7). Over-expression of PIAS1 together with AIB1 in MCF-7 cells led to increased sumoylation of AIB1, resulting in repression of its transcriptional activity. In contrast, the PIAS1 mutant (C350S) lacking E3 ligase activity appeared to have no effect on the sumoylation of AIB1. Through sumoylation of AIB1, PIAS1 also promoted the stability of AIB1 and attenuated its interaction with estrogen receptor α (ERα), resulting in repression of the transactivation activity of ERα. In addition, MCF-7 cells co-transfected with wild-type PIAS1 and AIB1 showed about 40% reduction in cell growth, while cells co-transfected with wild-type PIAS1 and mutant AIB1 resistant to sumoylation showed about 34% increase in cell growth compared to cells transformed with wild-type AIB1 only. Taken together, these results suggested that PIAS1 may play a crucial role in the regulation of AIB1 transcriptional activity through sumoylation.

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.

PIASy inhibits LRH-1-dependent CYP11A1 expression by competing for SRC-1 binding

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.