Search

G-Protein Pathway Suppressor 2 (GPS2) is an inhibitor of non-proteolytic K63 ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. Previous studies have associated GPS2-mediated restriction of ubiquitination with the regulation of insulin signaling, inflammatory responses and mitochondria-nuclear communication across different tissues and cell types. However, a detailed understanding of the targets of GPS2/Ubc13 activity is lacking. Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation on the outer mitochondrial membrane. Characterization of the RNA-binding protein PABPC1 as a target of GPS2-regulated ubiquitination within the mitochondrial compartment revealed a strategy for regulating the mitochondrial proteome via MUL1-mediated ubiquitination. Removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the mitochondria-associated translation of selected mRNAs leading to increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.

Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies. We captured hundreds of differential features, which map to distinct molecular mechanisms modulating mitochondrial homeostasis at the earliest stages of pathobiology, as well as stage-specific metabolic and excitation-coupling maladaptation. Collectively, this study fills in gaps from previous studies by expanding knowledge of the initial responses to mutations that protect cells against the early stress prior to contractile dysfunction and overt disease.

The PI3K/AKT signaling pathway is a known oncogenic pathway in breast cancer, involved in essential cellular processes necessary for cancer development and progression, including altered proliferation, metabolism, and cell survival. Mutations in this pathway occur in 20% of all TNBC cases, making it the most deregulated pathway in the only subclass of breast cancer with no targeted therapy. Few clinical trials testing PI3K/AKT pathway inhibition in these breast cancers have shown limited long term efficacy; the heterogeneity of tumors and variable response rates in clinical studies underscore the necessity to better understand the mechanisms governing TNBC and the PI3K/AKT pathway in order to predict patient response to targeted therapies. In particular, appropriate biomarkers are required to identify the tumor subtypes that may be more responsive to PI3K pathway inhibition. While regulation of AKT activation through a classic phosphorylation signaling cascade is well understood, recent studies have revealed a non-canonical pathway contributing to regulate AKT activation and possibly providing an unexpected mechanism for resistance to PI3K inhibition. Non-canonical regulation of AKT is provided by non-proteolytic ubiquitination of AKT favoring its recruitment to the plasma membrane and leading to hyperactivation, but a full understanding of the mechanism and players involved in this process is missing. Our lab has recently identified G-Protein Pathway Suppressor 2 (GPS2) as a novel regulator of the insulin signaling pathway through the inhibition of AKT ubiquitination in the adipose tissue. Here, we explore the role of GPS2 as a tumor suppressor in the context of breast cancer. Deletion of GPS2 in aggressive MDA-MB231 TNBC cells results in increased AKT phosphorylation associated with greater proliferation, migration and invasion in vitro, with all phenotypes mitigated in presence of the allosteric AKT inhibitor MK2206.Transcriptomic profiling of wild-type MB231, MB231-GPS2-KO, and myristoylated AKT (MB231-myrAKT) revealed a significant overlap between the MB231-GPS2-KO and myrAKT signatures. Conversely, deletion of GPS2 in MDA-MB468, a TNBC cell line with an activating mutation in the PI3K/AKT pathway, do not show any increase in proliferation, migration or invasion, but remain sensitive to AKT inhibition. Together these results indicate that loss of GPS2-mediated inhibition of AKT ubiquitination is sufficient to drive activation of the PI3K/AKT pathway in absence of other activating mutations. Furthermore, preliminary analysis of an orthotopic model of MB231-GPS2-KO cells in NOD scid gamma (NSG) mice resulted in greater breast tumor sizes when compared to the parental cell line, confirming GPS2 putative tumor suppressor role in vivo. Further studies are required to investigate GPS2 expression levels and mutational status in tumor samples, and to confirm GPS2 relevance as a predictive biomarker for treatment outcome, thus exposing an additional category of tumors that could be responsive to PI3K/AKT class inhibition and/or would benefit from alternative strategies to prevent resistance. Citation Format: Stefanie Chan, Emma Smith, Stefano Monti, M Dafne Cardamone, Valentina Perissi. Novel tumor suppressor regulating the PI3K/AKT pathway in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-05.

ADP-ribosylation is a reversible post-translational modification where an ADP-ribose moiety is covalently attached to target proteins by ADP-ribosyltransferases (ARTs). Although best known for its nuclear roles, ADP-ribosylation is increasingly recognized as a key regulatory strategy across cellular compartments. ADP-ribosylation of mitochondrial proteins has been widely reported, but the exact nature of mitochondrial ART enzymes is debated. We have identified neuralized-like protein 4 (NEURL4) as a mitochondrial ART enzyme and show that most ART activity associated with mitochondria is lost in the absence of NEURL4. The NEURL4-dependent ADP-ribosylome in mitochondrial extracts from HeLa cells includes numerous mitochondrial proteins previously shown to be ADP-ribosylated. In particular, we show that NEURL4 is required for the regulation of mtDNA integrity via poly-ADP-ribosylation of mtLIG3, the rate-limiting enzyme for base excision repair (BER). Collectively, our studies reveal that NEURL4 acts as the main mitochondrial ART enzyme under physiological conditions and provide novel insights in the regulation of mitochondria homeostasis through ADP-ribosylation.

Oxidative stress and increased ROS production, as observed in aging and obesity, commonly lead to the accumulation of mitochondrial dysfunctions. This is met by the activation of a robust mitochondria-to-nucleus stress response promoting the rewiring of nuclear gene expression to limit cellular and tissue damage and promote organelle adaptation. Here we will review previous work uncovering the transcriptional cofactor G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondria retrograde signaling and a key nuclear regulator of nuclear-encoded mitochondrial genes, including mitochondrial chaperones/proteases, mitokines, and other protective enzymes such as the ADP-ribosyltransferase NEURL4. We will also discuss unpublished work showing that shuttling of GPS2 between organelles plays a role in coordinating the transcriptional and translational regulation of antioxidant factors and pro-apoptotic genes by promoting the ubiquitination of mitochondria-associated translation factors.

G-Protein Pathway Suppressor 2 (GPS2) was recently identified as an endogenous inhibitor of non-proteolytic ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. GPS2-mediated restriction of K63 ubiquitination is associated with the regulation of insulin signaling, inflammation and mitochondria-nuclear communication, however a detailed understanding of the targets of GPS2/Ubc13 activity is currently lacking, Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation. Characterization of putative targets, including the RNA-binding protein PABPC1 and translation factor eiF3m, revealed a strategy for regulating the mitochondria-associated translation of selected mRNAs via Mul1-mediated ubiquitination. Our data indicate that removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the ubiquitination of mitochondria-associated translation factors leading to increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.

ADP-ribosylation is a reversible post-translational modification where an ADP-ribose moiety is covalently attached to amino acid side-chains of target proteins either as mono-ADP-ribose (MARylation or MAR) or poly-ADP-ribose chains (PARylation or PAR) by a class of enzymes called ADP-ribosyltransferases (ARTs). Although ADP-ribosylation is best known for its nuclear roles, ADP-ribosylation of extra nuclear proteins is increasingly recognized as a key regulatory strategy across cellular compartments. ADP-ribosylation of mitochondrial proteins, in particular, has been widely reported, even though the extent to which ADP-ribosylation of specific proteins regulates mitochondrial functions is unclear and the exact nature of mitochondrial ART enzymes is debated.Here, we have identified Neuralized-like protein 4 (NEURL4) as a mitochondrial ART enzyme and profiled the NEURL4-dependent ADP-ribosylome in mitochondrial extracts from Hela cells by LC-MS/MS, using isobaric tandem mass tag (TMT) labeling for relative quantification. Comparison of WT and NEURL4-KO cells generated by CRISPR/Cas9 genome editing revealed that most ART activity associated with mitochondria is lost in absence of NEURL4. Putative NEURL4 targets include numerous mitochondrial proteins previously shown to be ADP-ribosylated. In particular, we show that NEURL4 enzymatic activity is required for the regulation of mtDNA integrity via poly-ADP-ribosylation of mitochondrial specific Ligase III (mtLIG3), the rate-limiting enzyme for mitochondrial DNA (mtDNA) Base Excision Repair (BER).Collectively, our studies reveal that NEURL4 acts as the main mitochondrial ART enzyme under physiological conditions and provide novel insights in the regulation of mitochondria homeostasis through ADP-ribosylation.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system., Graphical Abstract, Highlights • SARS-CoV-2 infection in induced lung cells is characterized by phosphoproteomics • Analysis of response reveals host cell signaling and protein expression profile • Comparison to studies in undifferentiated cell lines shows unique pathology in iAT2s • Systems-level predictions find druggable pathways that can impede viral life cycle, Hekman et al. describe how a layer of primary stem cells (iAT2s) recapitulating lung biology responds to infection with SARS-CoV-2. They compare their work to previous studies with immortalized cell lines. Their data predict what effect the virus has on a lung cell and which drugs may slow infection.

Cellular homeostasis in eukaryotic cells requires synchronized coordination of multiple organelles. A key role in this stage is played by mitochondria, which have recently emerged as highly interconnected and multifunctional hubs that process and coordinate diverse cellular functions. Beyond producing ATP, mitochondria generate key metabolites and are central to apoptotic and metabolic signaling pathways. Because most mitochondrial proteins are encoded in the nuclear genome, the biogenesis of new mitochondria and the maintenance of mitochondrial functions and flexibility critically depend upon effective mitonuclear communication. This review addresses the complex network of signaling molecules and pathways allowing mitochondria-nuclear communication and coordinated regulation of their independent but interconnected genomes, and discusses the extent to which dynamic communication between the two organelles has evolved for mutual benefit and for the overall maintenance of cellular and organismal fitness.

Visceral white adipose tissue (vWAT) expands and undergoes extensive remodeling during diet-induced obesity. Much is known about the contribution of various stromal vascular cells to the remodeling process, but less is known of the changes that occur within the adipocyte as it becomes progressively dysfunctional. Here, we performed a transcriptome analysis of isolated vWAT adipocytes to assess global pathway changes occurring in response to a chronic high fat diet (HFD). The data demonstrate that the adipocyte responds to the HFD by adopting a fibroblast-like phenotype, characterized by enhanced expression of ECM, focal adhesion and cytoskeletal genes and suppression of many adipocyte programs most notably those associated with mitochondria. This study reveals that during obesity the adipocyte progressively becomes metabolically dysfunctional due to its acquisition of fibrogenic functions. We propose that mechano-responsive transcription factors such as MRTFA and SRF contribute to both upregulation of morphological genes as well as suppression of mitochondrial programs.

Breast cancer affects approximately 1 in 8 women over the course of their lifetime. Of the many genes and pathways deregulated by this heterogeneous disease, alterations of members in the PI3K/AKT pathway are present in approximately 20% of all cases, making it one of the more frequently mutated pathways in breast cancer. The PI3K/AKT pathway is well known to be involved in essential cellular processes necessary for cancer development and progression, including altered proliferation, metabolism, and cell survival. Previous studies have made it well known that the activation of AKT is regulated through its phosphorylation, but recent studies have also shown that AKT can also be regulated via ubiquitination, leading to AKT hyperactivation. However, the mechanism and players involved in this process are not well understood. Better understanding of these pathways could inform on better potential therapeutics to target this disease. Recent work in our lab has identified GPS2, a member of the NCoR/SMRT complex, as a regulator of the insulin-signaling pathway through the inhibition of AKT ubiquitination by Ubc13. As many human cancers frequently have an increase in activated AKT, here we explore the potential of GPS2 as a tumor suppressor in the context of breast cancer and hope to elucidate the mechanism by which AKT ubiquitination regulates its activation. Citation Format: Chan S, Cardamone MD, Perissi V. Novel tumor suppressor regulating the PI3K/AKT pathway in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-02.

BAT-controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet-induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold-stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1-cre (BAT-Mfn2-KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold-stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender-specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP-synthesizing fat oxidation, whereas in BAT from males, complex I-driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT-Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole-body cold-stimulated thermogenesis.

The ability to respond to fluctuations of reactive oxygen species (ROS) within the cell is a central aspect of mammalian physiology. This dynamic process depends on the coordinated action of transcriptional factors to promote the expression of genes encoding for antioxidant enzymes. Here, we demonstrate that the transcriptional coregulators, PGC-1α and NCoR1, are essential mediators of mitochondrial redox homeostasis in skeletal muscle cells. Our findings reveal an antagonistic role of these coregulators in modulating mitochondrial antioxidant induction through Sod2 transcriptional control. Importantly, the activation of this mechanism by either PGC-1α overexpression or NCoR1 knockdown attenuates mitochondrial ROS levels and prevents cell death caused by lipid overload in skeletal muscle cells. The opposing actions of coactivators and corepressors, therefore, exert a commanding role over cellular antioxidant capacity.

Summary As most of the mitochondrial proteome is encoded in the nucleus, mitochondrial functions critically depend on nuclear gene expression and bidirectional mito-nuclear communication. However, mitochondria-to-nucleus communication pathways in mammals are incompletely understood. Here, we identify G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondrial retrograde signaling and a transcriptional activator of nuclear-encoded mitochondrial genes. GPS2-regulated translocation from mitochondria to nucleus is essential for the transcriptional activation of a nuclear stress response to mitochondrial depolarization and for supporting basal mitochondrial biogenesis in differentiating adipocytes and brown adipose tissue (BAT) from mice. In the nucleus, GPS2 recruitment to target gene promoters regulates histone H3K9 demethylation and RNA POL2 activation through inhibition of Ubc13-mediated ubiquitination. These findings, together, reveal an additional layer of regulation of mitochondrial gene transcription, uncover a direct mitochondria-nuclear communication pathway, and indicate that GPS2 retrograde signaling is a key component of the mitochondrial stress response in mammals.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes. To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models. Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNFα pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment.

Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.

Objective Insulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT. Methods The molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice). Results Our results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo. As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved. Conclusions Our findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance., Graphical abstract, Highlights • Ubc13-mediated ubiquitination of AKT is required for activation of the insulin signaling pathway. • GPS2 regulates insulin signaling by inhibiting AKT ubiquitination and activation. • Adipo-specific deletion of GPS2 results in increased adiposity and altered lipid flux in the adipocytes. • GPS2-AKO mice have higher levels of circulating adiponectin and are insulin sensitive despite being obese.

The association between hyper-inflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, non-transcriptional role of GPS2 as a guardian against hyperstimulation of the TNFα-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNFα target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the non-transcriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis.

A crucial aspect of development, homeostasis and prevention of disease is the strict maintenance of patterns of gene repression. Gene repression is largely achieved by the combinatorial action of various enzymatic complexes - known as co-repressor complexes - that are recruited to DNA by transcription factors and often act through enzymatic modification of histone protein tails. Our understanding of how co-repressors act has begun to change over recent years owing to the increased availability of genome-scale data. Here, we consider specific strategies that underlie repression events - for example, those mediated by the nuclear receptor co-repressor (NCoR, also known as NCOR1) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT, also known as NCOR2) co-repressor complexes - and discuss emerging themes in gene repression.

G-protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of different metabolic organs. First identified as part of the N-CoR corepressor complex, GPS2 has been...

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has essential roles in adipogenesis and glucose homeostasis, and is a molecular target of insulin-sensitizing drugs. Although the ability of PPAR-gamma agonists to antagonize inflammatory responses by transrepression of nuclear factor kappa B (NF-kappaB) target genes is linked to antidiabetic and antiatherogenic actions, the mechanisms remain poorly understood. Here we report the identification of a molecular pathway by which PPAR-gamma represses the transcriptional activation of inflammatory response genes in mouse macrophages. The initial step of this pathway involves ligand-dependent SUMOylation of the PPAR-gamma ligand-binding domain, which targets PPAR-gamma to nuclear receptor corepressor (NCoR)-histone deacetylase-3 (HDAC3) complexes on inflammatory gene promoters. This in turn prevents recruitment of the ubiquitylation/19S proteosome machinery that normally mediates the signal-dependent removal of corepressor complexes required for gene activation. As a result, NCoR complexes are not cleared from the promoter and target genes are maintained in a repressed state. This mechanism provides an explanation for how an agonist-bound nuclear receptor can be converted from an activator of transcription to a promoter-specific repressor of NF-kappaB target genes that regulate immunity and homeostasis.

Nuclear receptors regulate many biologically important processes in development and homeostasis by their bimodal function as repressors and activators of gene transcription. A finely tuned modulation of the transcriptional activities of nuclear receptors is crucial for determining highly specific and diversified programmes of gene expression. Recent studies have provided insights into the molecular mechanisms that are required to switch between repression and activation functions, the combinatorial roles of the multiple cofactor complexes that are required for mediating transcriptional regulation, and the central question of how several different signalling pathways can be integrated at the nuclear level to achieve specific profiles of gene expression.

Acetylation and deacetylation of nucleosomal histones have profound effects on gene transcription in all eukaryotes. In humans, three highly homologous class I and four class II histone deacetylase (HDAC) enzymes have been identified to date. The class I deacetylases HDAC1 and HDAC2 are components of multisubunit complexes, one of which could associate with the nuclear hormone receptor corepressor, N-CoR. N-CoR also interacts with class II deacetylases HDAC4, HDAC5, and HDAC7. In comparison with HDAC1 and HDAC2, HDAC3 remains relatively uncharacterized, and very few proteins have been shown to interact with HDAC3. Using an affinity purification approach, we isolated an enzymatically active HDAC3 complex that contained members of the nuclear receptor corepressor family. Deletion analysis of N-CoR revealed that HDAC3 binds multiple N-CoR regions in vitro and that all of these regions are required for maximal binding in vivo . The N-CoR domains that interact with HDAC3 are distinct from those that bind other HDACs. Transient overexpression of HDAC3 and microinjection of Abs against HDAC3 showed that a component of transcriptional repression mediated by N-CoR depends on HDAC3. Interestingly, data suggest that interaction with a region of N-CoR augments the deacetylase activity of HDAC3. These results provide a possible molecular mechanism for HDAC3 regulation and argue that N-CoR is a platform in which distinct domains can interact with most of the known HDACs.

Checkpoint kinase 2 (Chk2) is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

Activation of toll-like receptors (TLRs) leads to derepression and subsequent activation of inflammatory response genes that play essential roles in innate and acquired immunity. Derepression requires signal-dependent turnover of the nuclear receptor corepressor NCoR from target promoters, but the mechanisms remain poorly understood. Here, we report that TLR4 uses NFkappaB to deliver IKKepsilon to target promoters that contain "integrated circuits" of kappaB and AP-1 sites, resulting in local phosphorylation of c-Jun and subsequent NCoR clearance. In contrast, TLR2 signaling leads to rapid activation of CaMKII and phosphorylation of the TBLR1 component of NCoR complexes, bypassing the requirement for c-Jun phosphorylation and enabling NCoR clearance from promoters lacking integrated kappaB elements. Intriguingly, the IKKvarepsilon-dependent clearance pathway is sensitive to transrepression by liver X receptors, while the CaMKII-dependent pathway is not. These findings reveal mechanisms for integration of TLR, calcium, and nuclear receptor signaling pathways that underlie pathogen-specific responses and disease-specific programs of inflammation.

The peroxisome proliferator-activated receptor gamma (PPARgamma) regulates adipocyte differentiation and glucose homeostasis and is the molecular target of thiazolidinediones (TZDs) that act as insulin-sensitizers in patients with type 2 diabetes. PPARgamma is also expressed in macrophages and negatively regulates the programme of macrophage activation by repressing a subset of AP1 and NF-kappaB-dependent genes. Recent genetic, molecular and biochemical studies support the idea that PPARgamma inhibits inflammatory gene expression in activated macrophages by a NCoR/sumoylation-dependent pathway. Sumoylation of PPARgamma targets it to NCoR corepressor complexes that are bound to inflammatory response gene promoters and prevents their signal-dependent clearance that is normally a prerequisite for transcriptional activation. As a consequence, genes remain in a repressed state. Because the ligand-induced allosteric changes that promote entry of PPARgamma into this transrepression pathway are distinct from those that mediate interactions with conventional coactivators, these findings may facilitate the development of novel PPARgamma ligands that retain antidiabetic activities but have reduced side effects.

A key strategy to achieve regulated gene expression in higher eukaryotes is to prevent illegitimate signal-independent activation by imposing robust control on the dismissal of corepressors. Here, we report that many signaling pathways, including Notch, NF-kappaB, and nuclear receptor ligands, are subjected to a dual-repression "checkpoint" based on distinct corepressor complexes. Gene activation requires the release of both CtBP1/2- and NCoR/SMRT-dependent repression, through the coordinate action of two highly related exchange factors, the transducer beta-like proteins TBL1 and TBLR1, that license ubiquitylation and degradation of CtBP1/2 and NCoR/SMRT, respectively. Intriguingly, their function and differential specificity reside in only five specific Ser/Thr phosphorylation site differences, regulated by direct phosphorylation at the level of the promoter, as exemplified by the role of PKCdelta in TBLR1-dependent dismissal of NCoR. Thus, our data reveal a strategy of dual-factor repression checkpoints, in which dedicated exchange factors serve as sensors for signal-specific dismissal of distinct corepressors, with specificity imposed by upstream signaling pathways.

The peroxisome proliferator-activated receptor gamma (PPARgamma) regulates adipocyte differentiation and glucose homeostasis and is the molecular target of thiazolidinediones (TZDs) that act as insulin-sensitizers in patients with type 2 diabetes. PPARgamma is also expressed in macrophages and negatively regulates the programme of macrophage activation by repressing a subset of AP1 and NF-kappaB-dependent genes. Recent genetic, molecular and biochemical studies support the idea that PPARgamma inhibits inflammatory gene expression in activated macrophages by a NCoR/sumoylation-dependent pathway. Sumoylation of PPARgamma targets it to NCoR corepressor complexes that are bound to inflammatory response gene promoters and prevents their signal-dependent clearance that is normally a prerequisite for transcriptional activation. As a consequence, genes remain in a repressed state. Because the ligand-induced allosteric changes that promote entry of PPARgamma into this transrepression pathway are distinct from those that mediate interactions with conventional coactivators, these findings may facilitate the development of novel PPARgamma ligands that retain antidiabetic activities but have reduced side effects.

Multiple enzymatic activities are required for transcriptional initiation. The enzyme DNA topoisomerase II associates with gene promoter regions and can generate breaks in double-stranded DNA (dsDNA). Therefore, it is of interest to know whether this enzyme is critical for regulated gene activation. We report that the signal-dependent activation of gene transcription by nuclear receptors and other classes of DNA binding transcription factors, including activating protein 1, requires DNA topoisomerase IIβ-dependent, transient, site-specific dsDNA break formation. Subsequent to the break, poly(adenosine diphosphate–ribose) polymerase–1 enzymatic activity is induced, which is required for a nucleosome-specific histone H1–high-mobility group B exchange event and for local changes of chromatin architecture. Our data mechanistically link DNA topoisomerase IIβ–dependent dsDNA breaks and the components of the DNA damage and repair machinery in regulated gene transcription.

The nuclear receptor corepressor (NCoR) and the related factor known as silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) are essential components of multiprotein complexes that mediate active repression by unliganded nuclear receptors. Recent studies suggest that NCoR and SMRT can interact with and exert repressive effects on several other classes of DNA-binding transcription factors, but the physiological importance of these interactions has not been established. Here, investigation of endogenous transcriptional programs regulated by NCoR in macrophages reveals that NCoR acts as a transcriptional checkpoint for activator protein (AP)-1-dependent gene networks that regulate diverse biological processes including inflammation, cell migration, and collagen catabolism, with loss of NCoR, resulting in derepression of AP-1 target genes. The NCoR corepressor complex imposes an active block of exchange of c-Jun for c-Jun/c-Fos heterodimers, with targeted deletion of the c-Jun locus, resulting in loss of NCoR complexes from AP-1 target genes under basal conditions. The checkpoint function of NCoR is relieved by signal-dependent phosphorylation of c-Jun, which directs removal of NCoR/HDAC3/TBL1/TBLR1 complexes through recruitment of a specific ubiquitylation complex, as a prerequisite to the default binding of c-Jun/c-Fos heterodimers and transcriptional activation. The requirement for a checkpoint function to achieve the appropriate dynamic range of transcriptional responses to inflammatory signals is likely to be used by other signal-dependent transcription factors that regulate diverse homeostatic and developmental processes.

The mechanisms that control the precisely regulated switch from gene repression to gene activation represent a central question in mammalian development. Here, we report that transcriptional activation mediated by liganded nuclear receptors unexpectedly requires the actions of two highly related F box/WD-40-containing factors, TBL1 and TBLR1, initially identified as components of an N-CoR corepressor complex. TBL1/TBLR1 serve as specific adaptors for the recruitment of the ubiquitin conjugating/19S proteasome complex, with TBLR1 selectively serving to mediate a required exchange of the nuclear receptor corepressors, N-CoR and SMRT, for coactivators upon ligand binding. Tbl1 gene deletion in embryonic stem cells severely impairs PPARγ-induced adipogenic differentiation, indicating that TBL1 function is also biologically indispensable for specific nuclear receptor-mediated gene activation events. The role of TBLR1 and TBL1 in cofactor exchange appears to also operate for c-Jun and NFκB and is therefore likely to be prototypic of similar mechanisms for other signal-dependent transcription factors.

Mammalian organogenesis requires the expansion of pluripotent precursor cells before the subsequent determination of specific cell types, but the tissue-specific molecular mechanisms that regulate the initial expansion of primordial cells remain poorly defined. We have genetically established that Six6 homeodomain factor, acting as a strong tissue-specific repressor, regulates early progenitor cell proliferation during mammalian retinogenesis and pituitary development. Six6, in association with Dach corepressors, regulates proliferation by directly repressing cyclin-dependent kinase inhibitors, including the p27Kip1 promoter. These data reveal a molecular mechanism by which a tissue-specific transcriptional repressor-corepressor complex can provide an organ-specific strategy for physiological expansion of precursor populations.

Transcription of the ERBB2 oncogene is repressed by oestrogen in human breast cancer cells. We show that a 218 bp fragment of the human ERBB2 gene promoter is responsive to oestrogen in transient transfection in ZR75.1 and SKBR.3 cells when the oestrogen receptor is expressed. Deletion analysis of this fragment shows that a sequence located at the 5' end, which is known to mediate ERBB2 overexpression in breast cancer, is also responsible for the oestrogen response. This sequence binds AP-2 transcription factors and appears functionally identical to an element of the oestrogen-dependent enhancer described in the first intron of human ERBB2. We observed that oestrogen treatment down-regulates expression of AP-2 proteins but does not affect the DNA binding activity of AP-2. Constitutive expression of AP-2beta or AP-2gamma, but not AP-2alpha, abrogates the estrogenic repression. Our results demonstrate that AP-2 transcription factors are implicated in the oestrogenic regulation of ERBB2 gene expression and suggest a complex interplay involving the different AP-2 isoforms and other unidentified factors.

Retinoic acid and thyroid hormone receptors can act alternatively as ligand-independent repressors or ligand-dependent activators, based on an exchange of N-CoR or SMRT-containing corepressor complexes for coactivator complexes in response to ligands. We provide evidence that the molecular basis of N-CoR recruitment is similar to that of coactivator recruitment, involving cooperative binding of two helical interaction motifs within the N-CoR carboxyl terminus to both subunits of a RAR-RXR heterodimer. The N-CoR and SMRT nuclear receptor interaction motifs exhibit a consensus sequence of LXX I/H I XXX I/L, representing an extended helix compared to the coactivator LXXLL helix, which is able to interact with specific residues in the same receptor pocket required for coactivator binding. We propose a model in which discrimination of the different lengths of the coactivator and corepressor interaction helices by the nuclear receptor AF2 motif provides the molecular basis for the exchange of coactivators for corepressors, with ligand-dependent formation of the charge clamp that stabilizes LXXLL binding sterically inhibiting interaction of the extended corepressor helix.

CREB-binding proteins (CBP) and p300 are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including CREB, nuclear receptors, and STATs. CBP and p300 function in part by mediating the assembly of multiprotein complexes that contain additional cofactors such as p300/CBP interacting protein (p/CIP), a member of the p160/SRC family of coactivators, and the p300/CBP associated factor p/CAF. In addition to serving as molecular scaffolds, CBP and p300 each possess intrinsic acetyltransferase activities that are required for their function as coactivators. Here we report that the adenovirus E1A protein inhibits the acetyltransferase activity of CBP on binding to the C/H3 domain, whereas binding of CREB, or a CREB/E1A fusion protein to the KIX domain, fails to inhibit CBP acetyltransferase activity. Surprisingly, p/CIP can either inhibit or stimulate CBP acetyltransferase activity depending on the specific substrate evaluated and the functional domains present in the p/CIP protein. While the CBP interaction domain of p/CIP inhibits acetylation of histones H3, H4, or high mobility group by CBP, it enhances acetylation of other substrates, such as Pit-1. These observations suggest that the acetyltransferase activities of CBP/p300 and p/CAF can be differentially modulated by factors binding to distinct regions of CBP/p300. Because these interactions are likely to result in differential effects on the coactivator functions of CBP/p300 for different classes of transcription factors, regulation of CBP/p300 acetyltransferase activity may represent a mechanism for integration of diverse signaling pathways.

SummaryTimely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however, the regulatory strategies underlying each factor’s effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of peroxisome proliferator-activator receptor γ (PPARγ) in adipocytes requires G protein suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARγ-mediated regulation of a specific transcriptional program, including the lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.