Obligate Partner Research Articles

Max deletion destabilizes MYC protein and abrogates Eµ-Myc lymphomagenesis.

Although MAX is regarded as an obligate dimerization partner for MYC, its function in normal development and neoplasia is poorly defined. We show that B-cell-specific deletion of Max has a modest effect on B-cell development but completely abrogates Eµ-Myc-driven lymphomagenesis. While Max loss affects only a few hundred genes in normal B cells, it leads to the global down-regulation of Myc-activated genes in premalignant Eµ-Myc cells. We show that the balance between MYC-MAX and MNT-MAX interactions in B cells shifts in premalignant B cells toward a MYC-driven transcriptional program. Moreover, we found that MAX loss leads to a significant reduction in MYC protein levels and down-regulation of direct transcriptional targets, including regulators of MYC stability. This phenomenon is also observed in multiple cell lines treated with MYC-MAX dimerization inhibitors. Our work uncovers a layer of Myc autoregulation critical for lymphomagenesis yet partly dispensable for normal development.

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

Max-imizing the Attenuation of Myc Using Small Molecules

It has been a widely held notion within the biomedical research community that the reliable modulation of transcription factors with small molecules would represent a holy grail, given their role in directly potentiating oncogenic programs. Among the transcription factors that have been held in highest regard is Myc, since its dysregulation is among the most recurrent events in human cancer. Despite intense efforts, the ability to identify compounds that bind directly to Myc, resulting in its functional inhibition, have been met with only moderate success. However, a new approach reported by Struntz et al. (Cell Chem. Biol., 2019) focuses on a different strategy of discovering molecules that bind to Myc's obligate partner Max. Using a small-molecule microarray screen, they report the identification of KI-MS2-008, a compound that results in the stabilization of Max homodimers and the attenuation of Myc. KI-MS2-008 suppresses cancer cell grown both in vitro and within in vivo models.

Crystal Structures and Nuclear Magnetic Resonance Studies of the Apo Form of the c-MYC:MAX bHLHZip Complex Reveal a Helical Basic Region in the Absence of DNA.

The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix–loop–helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix–loop–helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.

Where Sin3a Meets STAT3: Balancing STAT3-Mediated Transcriptional Activation and Repression.

STAT3 can mediate epigenetic silencing of tumor suppressor genes (TSG). However, little is known about the molecular mechanisms involved, except that this action is mediated by DNA methylation and requires STAT3 acetylation. In this issue of Cancer Research, Gambi and colleagues confirm that oncogene-driven constitutive STAT3 acetylation is responsible for TSG silencing. Furthermore, they show that the Sin3a transcriptional repressor complex is an obligatory partner of STAT3 on the promoters of the repressed genes, shedding light on the mechanisms involved in STAT3-mediated transcriptional repression, and more importantly, identifying that the STAT3-Sin3a axis is a potential selective therapeutic target in STAT3-dependent tumors.See related article by Gambi et al., p. 3076.

Assessment of total, ligand-induced peroxisome proliferator activated receptor \u03b3 ligand activity in serum

BackgroundHumans are exposed to a complex mixture of environmental chemicals that impact bone and metabolic health, and traditional exposure assessments struggle to capture these exposure scenarios. Peroxisome proliferator activated receptor-gamma (PPARγ) is an essential regulator of metabolic and bone homeostasis, and its inappropriate activation by environmental chemicals can set the stage for adverse health effects. Here, we present the development of the Serum PPARγ Activity Assay (SPAA), a simple and cost-effective method to measure total ligand activity in small volumes of serum.MethodsFirst, we determined essential components of the bioassay. Cos-7 cells were transfected with combinations of expression vectors for human PPARγ and RXRα, the obligate DNA-binding partner of PPARγ, along with PPRE (DR1)-driven luciferase and control eGFP reporter constructs. Transfected cells were treated with rosiglitazone, a synthetic PPARγ ligand and/or LG100268, a synthetic RXR ligand, to characterize the dose response and determine the simplest and most efficacious format. Following optimization of the bioassay, we assessed the cumulative activation of PPARγ by ligands in serum from mice treated with a PPARγ ligand and commercial human serum samples.ResultsCos-7 cells endogenously express sufficient RXR to support efficacious activation of transfected PPARγ. Co-transfection of an RXR expression vector with the PPARγ expression vector did not increase PPRE transcriptional activity induced by rosiglitazone. Treatment with an RXR ligand marginally increased PPRE transcriptional activity in the presence of transfected PPARγ, and co-treatment with an RXR ligand reduced rosiglitazone-induced PPRE transcriptional activity. Therefore, the final bioassay protocol consists of transfecting Cos-7 cells with a PPARγ expression vector along with the reporter vectors, applying rosiglitazone standards and/or 10 μL of serum, and measuring luminescence and fluorescence after a 24 h incubation. Sera from mice dosed with rosiglitazone induced PPRE transcriptional activity in the SPAA in a dose-dependent and PPARγ-dependent manner. Additionally, human serum from commercial sources induced a range of PPRE transcriptional activities in a PPARγ-dependent manner, demonstrating the ability of the bioassay to detect potentially low levels of ligands.ConclusionsThe SPAA can reliably measure total PPRE transcriptional activity in small volumes of serum. This system provides a sensitive, straightforward assay that can be reproduced in any cell culture laboratory.

Hypersensitivity to Cisplatin and Gentamycin Induced Nephrotoxicity in Mice with Decreased Expression of NADPH‐Cytochrome P450 Reductase

Cytochrome P450 reductase (CPR or POR) is an obligatory redox partner for all microsomal P450 enzymes. A large, global decrease in POR expression, in a Cpr‐low mouse model, was previously found to lead to altered sex steroid hormone homeostasis, female infertility, and decreased capacity for xenobiotic metabolism. The aim of this study was to further characterize the Cpr‐low mouse model, in order to identify additional phenotypes that may reveal vulnerability in people with low POR expression. Here, we report differences between wild‐type and Cpr‐low mice in their sensitivities to drug‐induced toxicity in the renal proximal tubules. We studied two drugs, the anticancer drug cisplatin and the antibiotic drug gentamycin, which are known as renal toxicants, but are not P450 substrates. Toxicity was assessed by blood urea nitrogen (BUN) levels and histopathology of the kidney. At drug doses that do not cause renal toxicity in wild‐type mice, cisplatin caused significant increases in BUN levels over vehicle control group in both male and female Cpr‐low mice, whereas gentamycin caused significant increases in BUN levels over vehicle control group in male but not female Cpr‐low mice. Corresponding histopathological changes, including appearance of extensive proximal tubule vacuolization, were found in the kidneys of drug‐treated Cpr‐low mice. These findings suggest that POR expression level could be a risk factor for drug‐induced nephrotoxicity in patients.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ronin (Thap11) Deficiency Results in a Disease Impacting both Vitamin B12 Metabolism and Ribosome Biogenesis

Combined methylmalonic acidemia and homocystinuria (cblC type), a form of inherited intracellular vitamin B12 deficiency, is a rare metabolic and multi‐systemic disorder caused by mutations in MMACHC. Patients with cblC can have severe neurodevelopmental defects including microcephaly, hydrocephaly and seizures as well as renal, cardiac and hematological defects. Mutations in MMACHC have been the sole known causes of cblC, until recently when an X‐linked variant of the disease was described, termed cblX. This variant was found to be due to mutations in the X‐linked gene coding for the transcription cofactor HCFC1, which is known to be an obligatory partner for the transcription factor RONIN (THAP11). Intriguingly, a single patient exhibiting cblX‐like findings has been found to carry a mutation in RONIN. Patients, with both HCFC1 and RONIN mutations, were shown to have a dramatic reduction in MMACHC transcription. We have previously shown that the Hcfc1/Ronin transcriptional complex directly regulates mouse Mmachc expression. These findings suggest that cblX and the new Ronin (THAP11) disorder comprise a novel family of rare and severe cblC‐like disorders that are transcriptional in nature. As a result, we have generated a mouse model that carries the human mutation in Ronin (RoninF80L), in an effort to better understand the cellular mechanisms underlying the pathophysiology of these devastating neurodevelopmental diseases. Here we report the generation of the first mouse model, RoninF80L along with its phenotypic and molecular characterization. RoninF80L homozygous mice die soon after birth and exhibit severe brain developmental defects that recapitulate those observed in the human cblX and Ronin‐deficient patients. Moreover, consistent with a vitamin B12 deficiency, cells from RoninF80L homozygous embryos exhibit defects in cobalamin metabolism. Surprisingly however, RNA‐seq and ChIP‐seq analyses also revealed a role for ribosome biogenesis in the pathophysiology of the disease. Furthermore, we were able to show that there is also a functional deficit in ribosome biogenesis and hence protein translation. This identifies for the first time, a role for Ronin in ribosome biogenesis. Together the phenotypic and molecular data confirm that the RoninF80L mouse model will serve as a powerful tool to further uncover the pathophysiology of this complex family of diseases.Support or Funding InformationBaylor College of Medicine, Department of Molecular Physiology and Biophysics (Seed)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bidirectional Modulation of HIF‐2 Activity through Chemical Ligands

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive subunit HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, while reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found they significantly displaced pocket residue Y281. Its dramatic side-chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization. Bidirectional modulation of HIF-2 activity by antagonists or agonists through allosteric effects This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High levels of PIWI-interacting RNAs are present in the small RNA landscape of prostate epithelium from vitamin D clinical trial specimens.

BackgroundVitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates antitumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) observed in patients with high serum vitamin D. The small noncoding RNA (ncRNA) landscape includes many other RNA species that remain uncharacterized in prostate epithelium and their potential regulation by vitamin D is unknown.MethodsLaser capture microdissection (LCM) followed by small‐RNA sequencing was used to identify ncRNAs in the prostate epithelium of tissues from a vitamin D‐supplementation trial. VDR chromatin immunoprecipitation‐sequencing was performed to identify vitamin D genomic targets in primary prostate epithelial cells.ResultsIsolation of epithelium by LCM increased sample homogeneity and captured more diversity in ncRNA species compared with publicly available small‐RNA sequencing data from benign whole prostate. An abundance of PIWI‐interacting RNAs (piRNAs) was detected in normal prostate epithelium. The obligate binding partners of piRNAs, PIWI‐like (PIWIL) proteins, were also detected in prostate epithelium. High prostatic vitamin D levels were associated with increased expression of piRNAs. VDR binding sites were located near several ncRNA biogenesis genes and genes regulating translation and differentiation.ConclusionsBenign prostate epithelium expresses both piRNA and PIWIL proteins, suggesting that these small ncRNA may serve an unknown function in the prostate. Vitamin D may increase the expression of prostatic piRNAs. VDR binding sites in primary prostate epithelial cells are consistent with its reported antitumorigenic functions and a role in ncRNA biogenesis.

Bidirectional modulation of HIF-2 activity through chemical ligands.

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive subunit HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, while reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found they significantly displaced pocket residue Y281. Its dramatic side-chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization.

A revisited version of the apo structure of the ligand-binding domain of the human nuclear receptor retinoic X receptor α.

The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXRα, which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a π-bulge in helix H7, which was originally built as a regular α-helix. The presence of several CHAPS molecules, which are visible for the first time in the electron-density map and which stabilize the H1-H3 loop, which contains helix H2, are also revealed. The apo RXR structure has played an essential role in deciphering the molecular mode of action of NR ligands and is still used in numerous biophysical studies. This refined structure should be used preferentially in the future in interpreting experiments as well as for modelling and structural dynamics studies of the apo RXRα LBD.

Therapeutic Inhibition of Myc in Cancer. Structural Bases and Computer-Aided Drug Discovery Approaches.

Myc (avian myelocytomatosis viral oncogene homolog) represents one of the most sought after drug targets in cancer. Myc transcription factor is an essential regulator of cell growth, but in most cancers it is overexpressed and associated with treatment-resistance and lethal outcomes. Over 40 years of research and drug development efforts did not yield a clinically useful Myc inhibitor. Drugging the “undruggable” is problematic, as Myc inactivation may negatively impact its physiological functions. Moreover, Myc is a disordered protein that lacks effective binding pockets on its surface. It is well established that the Myc function is dependent on dimerization with its obligate partner, Max (Myc associated factor X), which together form a functional DNA-binding domain to activate genomic targets. Herein, we provide an overview of the knowledge accumulated to date on Myc regulation and function, its critical role in cancer, and summarize various strategies that are employed to tackle Myc-driven malignant transformation. We focus on important structure-function relationships of Myc with its interactome, elaborating structural determinants of Myc-Max dimer formation and DNA recognition exploited for therapeutic inhibition. Chronological development of small-molecule Myc-Max prototype inhibitors and corresponding binding sites are comprehensively reviewed and particular emphasis is placed on modern computational drug design methods. On the outlook, technological advancements may soon provide the so long-awaited Myc-Max clinical candidate.

The Degradation and Subcellular Localization of an Oncogenic protein complex in T-cell Leukemia

Background and Hypothesis: 
 LIM domain Only-2 (LMO2) is a key oncogenic driver of human T-cell acute lymphoblastic leukemia. LMO2 functions as part of a large multisubunit complex that regulates gene expression. LMO2 itself does not bind DNA but it does bind class II basic helix loop helix transcription factors, TAL1 or LYL1, which are also part of the LMO2-associated complex. We recently identified LIM domain binding protein 1 (LDB1) as an obligate LMO2 partner that is required for LMO2 protein stability in T-ALL. We believe the interaction between LDB1 and LMO2 is crucial to understanding the pathogenesis of T-ALL and is a potential therapeutic target for this aggressive leukemia. We hypothesize that in the absence of LDB1, LMO2 is rapidly degraded. Nonetheless, the mechanisms for the localization and degradation of LMO2 and its partners have not been fully explored. 
 Experimental Design or Project Methods: 
 In this study, we analyzed the first order decay of the LMO2-associated complex including LMO2, SSBP2, SSBP3, TAL1, LYL1, and LDB1. We analyzed the subcellular localization of these same proteins distinguishing between the nucleus and the cytoplasm. Our studies were enabled by cloning the HALO tag to the NH2-terminus of these proteins. Fluorescent small molecules such as HALO ligand, R110, were used for intracellular labeling followed by pulse chase analysis via flow cytometry for half-life and imaging by confocal microscopy. 
 Results: 
 We observed a hierarchy of protein stability: LDB1 had the longest half-life, followed by SSBP3, SSBP2, Tal1, LMO2 and Lyl1had the shortest half-lives. Co-expression of LDB1 conferred enhanced staility upon all protein components. All of the protein components studied showed a predominantly nuclear distribution with some in the cytoplasm. 
 Conclusion and Potential Impact: 
 The prolonged protein stability of LDB1 allows it to confer enhanced stability to LMO2 and bHLH proteins TAL1 and LYL1. Thus, targeting the assembly of the multisubunit LMO2 complex.

Conformational flexibility of histone variant CENP-ACse4 is regulated by histone H4: A mechanism to stabilize soluble Cse4

The histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly.

Myeloid Cell Hypoxia-Inducible Factors Promote Resolution of Inflammation in Experimental Colitis.

Colonic tissues in Inflammatory Bowel Disease (IBD) patients exhibit oxygen deprivation and activation of hypoxia-inducible factor 1α and 2α (HIF-1α and HIF-2α), which mediate cellular adaptation to hypoxic stress. Notably, macrophages and neutrophils accumulate preferentially in hypoxic regions of the inflamed colon, suggesting that myeloid cell functions in colitis are HIF-dependent. By depleting ARNT (the obligate heterodimeric binding partner for both HIFα subunits) in a murine model, we demonstrate here that myeloid HIF signaling promotes the resolution of acute colitis. Specifically, myeloid pan-HIF deficiency exacerbates infiltration of pro-inflammatory neutrophils and Ly6C+ monocytic cells into diseased tissue. Myeloid HIF ablation also hinders macrophage functional conversion to a protective, pro-resolving phenotype, and elevates gut serum amyloid A levels during the resolution phase of colitis. Therefore, myeloid cell HIF signaling is required for efficient resolution of inflammatory damage in colitis, implicating serum amyloid A in this process.

Loss of RNF40 Decreases NF-κB Activity in Colorectal Cancer Cells and Reduces Colitis Burden in Mice.

Inflammatory bowel diseases are linked to an increased risk of developing colorectal cancer [CRC]. Previous studies suggested that the H2B ubiquitin ligase RING finger protein-20 [RNF20] inhibited inflammatory signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB]. However, the role of RNF40, the obligate heterodimeric partner of RNF20, in the context of inflammation and CRC has not been addressed. Here, we examined the effect of RNF40 loss on CRC cells in vitro and on inflammation and inflammatory signaling in vitro and in vivo. We evaluated H2Bub1 levels in human and murine colorectal tumors by immunohistochemistry. Moreover, we correlated H2Bub1 and RNF40 levels in vivo and assessed the consequences of RNF40 depletion on cellular phenotype and gene expression in CRC cells in vitro. Finally, we examined the effect of a colon-specific loss of Rnf40 in a murine model of colitis, and assessed both local and systemic inflammation-associated consequences. In vitro studies revealed that the tumorigenic phenotype of CRC cells decreased after RNF40 depletion and displayed gene expression changes related to chromosome segregation and DNA replication, as well as decreased induction of several NF-κB-associated cytokines. This effect was associated with decreased nuclear localization of NF-κB following tumor necrosis factor alpha treatment. Consistently, the colon-specific loss of Rnf40 exerted a protective local, as well as systemic, effect following acute colitis. Our findings suggest that RNF40 plays a central role in the maintenance of tumorigenic features and inflammatory signaling by promoting nuclear NF-κB activity.

The IL-4/STAT6/PPARγ signaling axis is driving the expansion of the RXR heterodimer cistrome, providing complex ligand responsiveness in macrophages.

Retinoid X receptor (RXR) is an obligate heterodimeric partner of several nuclear receptors (NRs), and as such a central component of NR signaling regulating the immune and metabolic phenotype of macrophages. Importantly, the binding motifs of RXR heterodimers are enriched in the tissue-selective open chromatin regions of resident macrophages, suggesting roles in subtype specification. Recent genome-wide studies revealed that RXR binds to thousands of sites in the genome, but the mechanistic details how the cistrome is established and serves ligand-induced transcriptional activity remained elusive. Here we show that IL-4-mediated macrophage plasticity results in a greatly extended RXR cistrome via both direct and indirect actions of the transcription factor STAT6. Activation of STAT6 leads to chromatin remodeling and RXR recruitment to de novo enhancers. In addition, STAT6 triggers a secondary transcription factor wave, including PPARγ. PPARγ appears to be indispensable for the development of RXR-bound de novo enhancers, whose activities can be modulated by the ligands of the PPARγ:RXR heterodimer conferring ligand selective cellular responses. Collectively, these data reveal the mechanisms leading to the dynamic extension of the RXR cistrome and identify the lipid-sensing enhancer sets responsible for the appearance of ligand-preferred gene signatures in alternatively polarized macrophages.

Familial and Somatic BAP1 Mutations Inactivate ASXL1/2-Mediated Allosteric Regulation of BAP1 Deubiquitinase by Targeting Multiple Independent Domains.

Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1-ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable.Significance: Combined computational and biochemical approaches demonstrate that the BAP1-ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1-ASXL2 binding. Cancer Res; 78(5); 1200-13. ©2017 AACR.

A novel antibody-based approach to detect the functional ERCC1-202 isoform

ERCC1/XPF endonuclease plays an important role in multiple DNA repair pathways and stands as a potential prognostic and predictive biomarker for cisplatin-based chemotherapy. Four distinct ERCC1 isoforms arising from alternative splicing have been described (201, 202, 203 and 204) but only the 202 isoform is functional in DNA excision repair, when interacting with its obligate partner XPF. Currently, there is no tool to assess specifically the expression of ERCC1-202 due to high sequence homology between the four isoforms. Here, we generated monoclonal antibodies directed against the heterodimer of ERCC1 and its obligate interacting partner XPF by genetic immunization. We obtained three monoclonal antibodies (2C11, 7C3 and 10D10) recognizing specifically the heterodimer ERCC1-202/XPF as well as the ERCC1-204/XPF with no affinity to ERCC1 or XPF monomers. By combining one of these three heterodimer-specific antibodies with a commercial anti-ERCC1 antibody (clone 4F9) unable to recognize the 204 isoform in a proximity ligation assay (PLA), we managed to specifically detect the functional ERCC1-202 isoform. This methodological breakthrough can constitute a basis for the development of clinical tests to evaluate ERCC1 functional proficiency.