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Biological systems to sense and respond to metabolic perturbations are critical for the maintenance of cellular homeostasis. Here we describe a hepatic system in this context orchestrated by the transcriptional corepressor C-terminal binding protein 2 (CtBP2) that harbors metabolite-sensing capabilities. The repressor activity of CtBP2 is reciprocally regulated by NADH and acyl-CoAs. CtBP2 represses Forkhead box O1 (FoxO1)-mediated hepatic gluconeogenesis directly as well as Sterol Regulatory Element-Binding Protein 1 (SREBP1)-mediated lipogenesis indirectly. The activity of CtBP2 is markedly defective in obese liver reflecting the metabolic perturbations. Thus, liver-specific CtBP2 deletion promotes hepatic gluconeogenesis and accelerates the progression of steatohepatitis. Conversely, activation of CtBP2 ameliorates diabetes and hepatic steatosis in obesity. The structure-function relationships revealed in this study identify a critical structural domain called Rossmann fold, a metabolite-sensing pocket, that is susceptible to metabolic liabilities and potentially targetable for developing therapeutic approaches., Sensing of nutrient status coordinates the regulation of liver glucose and lipid metabolism, and is important for metabolic homeostasis. Here the authors report that transcriptional the corepressor CtBP2 can sense nutrient status and coordinate repression of liver glucose and lipid metabolism via Fox01 and SREBP1, respectively.

Despite the importance of nitric oxide signaling in multiple biological processes, its role in tissue regeneration remains largely unexplored. Here, we provide evidence that inducible nitric oxide synthase (iNos) translocates to the nucleus during zebrafish tailfin regeneration and is associated with alterations in the nuclear S-nitrosylated proteome. iNos inhibitors or nitric oxide scavengers reduce protein S-nitrosylation and impair tailfin regeneration. Liquid chromatography/tandem mass spectrometry reveals an increase of up to 11-fold in the number of S-nitrosylated proteins during regeneration. Among these, Kdm1a, a well-known epigenetic modifier, is S-nitrosylated on Cys334. This alters Kdm1a binding to the CoRest complex, thus impairing its H3K4 demethylase activity, which is a response specific to the endothelial compartment. Rescue experiments show S-nitrosylation is essential for tailfin regeneration, and we identify downstream endothelial targets of Kdm1a S-nitrosylation. In this work, we define S-nitrosylation as an essential post-translational modification in tissue regeneration., The role of the post-translational modifications in tissue regeneration is still not clearly understood. Here, the authors show that many nuclear proteins change S-nitrosylation state in the regenerating zebrafish tailfin, highlighting the importance of Kdm1a S-nitrosylation in the repair process.

DAXX and ATRX are tumor suppressor proteins that form a histone H3.3 chaperone complex and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT). Here, we show that DAXX and ATRX knock-out (KO) U87-T cells that have acquired ALT-like features have defects in p53 chromatin binding and DNA damage response. RNA-seq analysis revealed that p53 pathway is among the most perturbed. ChIP-seq and ATAC-seq revealed a genome-wide reduction in p53 DNA-binding and corresponding loss of chromatin accessibility at many p53 response elements across the genome. Both DAXX and ATRX null cells showed a depletion of histone H3.3 and accumulation of γH2AX at many p53 sites, including subtelomeres. These findings indicate that loss of DAXX or ATRX can compromise p53 chromatin binding and p53 DNA damage response in ALT-like cells, providing a link between histone composition, chromatin accessibility and tumor suppressor function of p53.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice., Molecular diversity of proprioceptive neuron types (Ia, Ib and II PNs) is unclear. Here, the authors characterized the functional organization and development of eight subtypes of PNs in mice. Importantly, Ia subtypes are plastic, suggesting a role in adaptive proprioception during motor behavior.

Circadian pace is modulated by light intensity, known as the Aschoff’s rule, with largely unrevealed mechanisms. Here we report that photoreceptor CRY2 mediates blue light input to the circadian clock by directly interacting with clock core component PRR9 in blue light dependent manner. This physical interaction dually blocks the accessibility of PRR9 protein to its co-repressor TPL/TPRs and the resulting kinase PPKs. Notably, phosphorylation of PRR9 by PPKs is critical for its DNA binding and repressive activity, hence to ensure proper circadian speed. Given the labile nature of CRY2 in strong blue light, our findings provide a mechanistic explanation for Aschoff’s rule in plants, i.e., blue light triggers CRY2 turnover in proportional to its intensity, which accordingly releasing PRR9 to fine tune circadian speed. Our findings not only reveal a network mediating light input into the circadian clock, but also unmask a mechanism by which the Arabidopsis circadian clock senses light intensity.

The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor., The repressive states of peroxisome proliferator-activated receptor γ (PPARγ) are ill-defined, despite nuclear receptors being a major drug target. Here authors demonstrate multiple structurally distinct repressive states, providing a structural rationale for ligand bias in a nuclear receptor.

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units., The BEN-solo proteins—including Insensitive (Insv), Elba1 and Elba2—function in both transcriptional repression and chromatin insulation. Here, the authors investigate the role of these proteins in Drosophila embryos, finding that ELBA and Insv function as general insulators and partition active chromatin to ensure proper gene activation in Drosophila.

SPINDOC is tightly associated with the histone H3K4me3 effector protein SPIN1. To gain a better understanding of the biological roles of SPINDOC, we identified its interacting proteins. Unexpectedly, SPINDOC forms two mutually exclusive protein complexes, one with SPIN1 and the other with PARP1. Consistent with its ability to directly interact with PARP1, SPINDOC expression is induced by DNA damage, likely by KLF4, and recruited to DNA lesions with dynamics that follows PARP1. In SPINDOC knockout cells, the levels of PARylation are reduced, in both the absence and presence of DNA damage. The SPINDOC/PARP1 interaction promotes the clearance of PARP1 from damaged DNA, and also impacts the expression of known transcriptional targets of PARP1. To address the in vivo roles of SPINDOC in PARP1 regulation, we generate SPINDOC knockout mice, which are viable, but slightly smaller than their wildtype counterparts. The KO mice display reduced levels of PARylation and, like PARP1 KO mice, are hypersensitive to IR-induced DNA damage. The findings identify a SPIN1-independent role for SPINDOC in the regulation of PARP1-mediated PARylation and the DNA damage response., SPINDOC is known to interact with Spindlin1 (SPIN1), a histone code effector protein. Here, the authors show that SPINDOC is distributed between two distinct protein complexes, one comprising SPIN1 and the other one with PARP1. Their results suggest a role for SPINDOC in the regulation of PARP1- mediated PARylation and the DNA damage response.

The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding., The ligand-activated transcription factor farnesoid X receptor (FXR) acts as a cellular sensor for bile acids and is of interest as a drug target. Here the authors employ X-ray crystallography and NMR to characterize the molecular determinants of FXR agonists, antagonists and a partial agonist that drive FXR activation and antagonism.

Osteoarthritis (OA) has been recognized as the most common chronic age-related disease. Cartilage degeneration influences OA therapy. Here we report that hematopoietic pre-B cell leukemia transcription factor-interacting protein (HPIP) is essential for OA development. Elevated HPIP levels are found in OA patients. Col2a1-CreERT2/HPIPf/f mice exhibit obvious skeletal abnormalities compared with their HPIPf/f littermates. HPIP deficiency in mice protects against developing OA. Moreover, intra-articular injection of adeno-associated virus carrying HPIP-specific short hairpin RNA in vivo attenuates OA histological signs. Notably, in vitro RNA-sequencing and chromatin immunoprecipitation sequencing profiles identify that HPIP modulates OA cartilage degeneration through transcriptional activation of Wnt target genes. Mechanistically, HPIP promotes the transcription of Wnt targets by interacting with lymphoid enhancer binding factor 1 (LEF1). Furthermore, HPIP potentiates the transcriptional activity of LEF1 and acetylates histone H3 lysine 56 in the promoters of Wnt targets, suggesting that HPIP is an attractive target in OA regulatory network., Osteoarthritis (OA) is associated with cartilage degeneration, and no effective therapy exists. Here, the authors show that the HPIP protein modulates OA progression by regulating Wnt signaling, and that its knockdown in joints via AAV-mediated gene silencing attentuates pathology.

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

18 p.-8 fig., Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration., This work was supported by National Institutes of Health grants AG054108, AG021904, and AG017617 (to AMC) and AG038072 (to AMC, FM, and EG), the JPB Foundation (to AMC), the Michael J. Fox Foundation (to AMC and EG), the Rainwater Charitable Foundation (to AMC and EG) and the Backus Foundation (to AMC). Chemical synthesis, NMR and MS data were in part supported by NIH awards P30 CA013330 and 1S10OD016305. Research in PB and RGS lab is funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 to PB and RTI2018-098990-J-I00 to RGS. CAR was supported by T32 GM007491 and MM by T32 AG023475.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development., TUTase mediated uridylation of mRNA promotes degradation. Here, Scheer, de Almeida et al. show that Arabidopsis TUTase URT1 interacts directly with the translation inhibitor and decay factor DECAPPING5 and suppresses siRNA biogenesis by preventing accumulation of deadenylated mRNAs

Recurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-‘reading’ bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy., The fusion gene ZMYND11-MBTD1 (ZM) is present in a subgroup of patients with acute myeloid leukaemia (AML). Here, the authors show that ZM expression induces AML in a murine model though activating the NuA4/TIP60 histone acetyltransferase complex.

Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1-mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we perform a genome-wide single guide RNA (sgRNA) screen for genes involved in ERV silencing and identify the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out (ko) cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We find that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analyses reveal that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observe strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions., Endogenous retroviruses (ERVs) compose a significant portion of mammalian genomes; however, how ERVs are regulated is not well known. Here the authors performed a genome-wide sgRNA screen to identify Morc3 as a mediator of ERV silencing. They show Morc3 associates with the H3.3 chaperone Daxx, and that loss of Morc3 leads to reduced H3.3 at ERVs.

Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume. Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations—supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations., Structural studies of nuclear receptor transcription factors revealed that nearly all nuclear receptors share a conserved helix 12 dependent transcriptional activation mechanism. Here the authors present two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation, where helix 12 is located within the orthosteric ligand-binding pocket instead, and discuss mechanistic implications.

Emergence of an aggressive androgen receptor (AR)-independent neuroendocrine prostate cancer (NEPC) after androgen-deprivation therapy (ADT) is well-known. Nevertheless, the majority of advanced-stage prostate cancer patients, including those with SPINK1-positive subtype, are treated with AR-antagonists. Here, we show AR and its corepressor, REST, function as transcriptional-repressors of SPINK1, and AR-antagonists alleviate this repression leading to SPINK1 upregulation. Increased SOX2 expression during NE-transdifferentiation transactivates SPINK1, a critical-player for maintenance of NE-phenotype. SPINK1 elicits epithelial-mesenchymal-transition, stemness and cellular-plasticity. Conversely, pharmacological Casein Kinase-1 inhibition stabilizes REST, which in cooperation with AR causes SPINK1 transcriptional-repression and impedes SPINK1-mediated oncogenesis. Elevated levels of SPINK1 and NEPC markers are observed in the tumors of AR-antagonists treated mice, and in a subset of NEPC patients, implicating a plausible role of SPINK1 in treatment-related NEPC. Collectively, our findings provide an explanation for the paradoxical clinical-outcomes after ADT, possibly due to SPINK1 upregulation, and offers a strategy for adjuvant therapies., SPINK1 upregulation is found in ~10–25% of prostate cancers. Here, they show that whilst SPINK1 is transcriptionally repressed by androgen receptor and its co-repressor REST, it is upregulated after androgen-deprivation therapy, which leads to neuroendocrine differentiation of prostate cancer cells.

Impairment of intrinsic plasticity is involved in a range of neurological disorders such as epilepsy. However, how intrinsic excitability is regulated is still not fully understood. Here we report that the epigenetic factor Chromodomain Y-like (CDYL) protein is a critical regulator of the initiation and maintenance of intrinsic neuroplasticity by regulating voltage-gated ion channels in mouse brains. CDYL binds to a regulatory element in the intron region of SCN8A and mainly recruits H3K27me3 activity for transcriptional repression of the gene. Knockdown of CDYL in hippocampal neurons results in augmented Nav1.6 currents, lower neuronal threshold, and increased seizure susceptibility, whereas transgenic mice over-expressing CDYL exhibit higher neuronal threshold and are less prone to epileptogenesis. Finally, examination of human brain tissues reveals decreased CDYL and increased SCN8A in the temporal lobe epilepsy group. Together, our findings indicate CDYL is a critical player for experience-dependent gene regulation in controlling intrinsic excitability., Alterations in intrinsic plasticity are important in epilepsy. Here the authors show that the epigenetic factor CDYL regulates the gene expression of the voltage gated sodium channel, Nav1.6, which contributes to seizures in a rat model of epilepsy.

In response to extracellular signals, many signalling proteins associated with the plasma membrane are sorted into endosomes. This involves endosomal fusion, which depends on the complexes HOPS and CORVET. Whether and how their subunits themselves modulate signal transduction is unknown. We show that Vps11 and Vps18 (Vps11/18), two common subunits of the HOPS/CORVET complexes, are E3 ubiquitin ligases. Upon overexpression of Vps11/Vps18, we find perturbations of ubiquitination in signal transduction pathways. We specifically demonstrate that Vps11/18 regulate several signalling factors and pathways, including Wnt, estrogen receptor α (ERα), and NFκB. For ERα, we demonstrate that the Vps11/18-mediated ubiquitination of the scaffold protein PELP1 impairs the activation of ERα by c-Src. Thus, proteins involved in membrane traffic, in addition to performing their well-described role in endosomal fusion, fine-tune signalling in several different ways, including through ubiquitination., Endosomal fusion depends on the HOPS and CORVET complexes but whether and how their subunits modulate signal transduction is not fully understood. Here, the authors show that the HOPS/CORVET subunits Vps11 and Vps18 are E3 ubiquitin ligases that are involved in the regulation of ERα signalling.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis., Non-small cell lung cancer (NSCLC) is often associated with metastasis to the lungs. Here, the authors perform independent screens and identify NuRD as a co-repressor of ZEB1, and demonstrate TBC1D2b as a downstream target of ZEB1/NuRD complex regulating NSCLC metastasis.

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor (PPAR) gamma converts a transcriptionally neutral antagonist (GW9662) into a repressive inverse agonist (T0070907) relative to basal cellular activity. X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for corepressor-selective inverse agonism. NMR spectroscopy reveals that PPARγ exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPARγ for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide routes for developing corepressor-selective repressive PPARγ ligands., Peroxisome proliferator-activated receptor gamma (PPARγ) is a target for insulin sensitizing drugs. Here the authors combine NMR, X-ray crystallography and MD simulations and report a structural mechanism for eliciting PPARγ inverse agonism, where coactivator binding is inhibited and corepressor binding promoted, which causes PPARγ repression.

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer., Cigarette smoke induces microRNA dysregulation in cancers. Here, the authors show that cigarette smoke promotes the maturation of oncogenic primary miR-25 due to METTL3 hypomethylation, and mature miR-25 suppresses PH domain leucine-rich repeat protein phosphatase 2, resulting in oncogenic AKT activation in pancreatic cancer.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value, In pancreatic neuroendocrine tumors (PanNETs) ATRX, DAXX, and MEN1 are commonly mutated (A-D-M mutant PanNETs). Here, the authors find in a cohort of PanNETS 58% are A-D-M mutant PanNETs, with a worse clinical outcome and differences in gene expression and methylation compared to A-D-M wild type cases- these gene expression differences suggest that A-D-M mutant PanNETs potentially originate from a cell type similar to alpha cells.

Autophagy cargo recognition and clearance are essential for intracellular protein quality control. SQSTM1/p62 sequesters intracellular aberrant proteins and mediates cargo delivery for their selective autophagic degradation. The formation of p62 non-membrane-bound liquid compartments is critical for its function as a cargo receptor. The regulation of p62 phase separation/condensation has yet been poorly characterised. Using an unbiased yeast two-hybrid screening and complementary approaches, we found that DAXX physically interacts with p62. Cytoplasmic DAXX promotes p62 puncta formation. We further elucidate that DAXX drives p62 liquid phase condensation by inducing p62 oligomerisation. This effect promotes p62 recruitment of Keap1 and subsequent Nrf2-mediated stress response. The present study suggests a mechanism of p62 phase condensation by a protein interaction, and indicates that DAXX regulates redox homoeostasis, providing a mechanistic insight into the prosurvival function of DAXX., The autophagy protein p62 undergoes liquid-liquid phase separation but how this is regulated is unclear. Here, the authors report that the histone chaperone DAXX interacts with p62 in the cytoplasm to drive its phase separation.

The ATRX–DAXX histone chaperone complex incorporates the histone variant H3.3 at heterochromatic regions in a replication-independent manner. Here, we present a high-resolution x-ray crystal structure of an interaction surface between ATRX and DAXX. We use single amino acid substitutions in DAXX that abrogate formation of the complex to explore ATRX-dependent and ATRX-independent functions of DAXX. We find that the repression of specific murine endogenous retroviruses is dependent on DAXX, but not on ATRX. In support, we reveal the existence of two biochemically distinct DAXX-containing complexes: the ATRX–DAXX complex involved in gene repression and telomere chromatin structure, and a DAXX–SETDB1–KAP1–HDAC1 complex that represses endogenous retroviruses independently of ATRX and H3.3 incorporation into chromatin. We find that histone H3.3 stabilizes DAXX protein levels and can affect DAXX-regulated gene expression without incorporation into nucleosomes. Our study demonstrates a nucleosome-independent function for the H3.3 histone variant., The ATRX-DAXX histone chaperone complex incorporates H3.3 in heterochromatin in a replication-independent manner. Here, the authors present a high-resolution x-ray crystal structure of an interaction surface between ATRX and DAXX, and characterize ATRX-dependent and-independent functions of DAXX.

EGFR is required for animal development, and dysregulation of EGFR is critically implicated in malignant transformation. However, the molecular mechanism underlying the regulation of EGFR expression remains poorly explored. Here we report that the zinc-finger protein ZNF516 is a transcription repressor. ZNF516 is physically associated with the CtBP/LSD1/CoREST complex and transcriptionally represses a cohort of genes including EGFR that are critically involved in cell proliferation and motility. We demonstrate that the ZNF516–CtBP/LSD1/CoREST complex inhibits the proliferation and invasion of breast cancer cells in vitro and suppresses breast cancer growth and metastasis in vivo. Significantly, low expression of ZNF516 is positively associated with advanced pathological staging and poor survival of breast carcinomas. Our data indicate that ZNF516 is a transcription repressor and a potential suppressor of EGFR, adding to the understanding of EGFR-related breast carcinogenesis and supporting the pursuit of ZNF516 as a potential therapeutic target for breast cancer., EGFR is a well-known oncogene; however, the mechanisms regulating its expression are still unclear. Here, analysing genome-wide chromatin associations, the authors show that in breast cancer cells ZNF516 represses EGFR transcription through the interaction with the CtBP/LSD1/CoREST complex.

Chromosome alignment is required for accurate chromosome segregation. Chromosome misalignment can result in genomic instability and tumorigenesis. Here, we show that NF-κB activating protein (NKAP) is critical for chromosome alignment through anchoring CENP-E to kinetochores. NKAP knockdown causes chromosome misalignment and prometaphase arrest in human cells. NKAP dynamically localizes to kinetochores, and is required for CENP-E kinetochore localization. NKAP is SUMOylated predominantly in mitosis and the SUMOylation is needed for NKAP to bind CENP-E. A SUMOylation-deficient mutant of NKAP cannot support the localization of CENP-E on kinetochores or proper chromosome alignment. Moreover, Bub3 recruits NKAP to stabilize the binding of CENP-E to BubR1 at kinetochores. Importantly, loss of NKAP expression causes aneuploidy in cultured cells, and is observed in human soft tissue sarcomas. These findings indicate that NKAP is a novel and key regulator of mitosis, and its dysregulation might contribute to tumorigenesis by causing chromosomal instability., The kinetochore-bound motor CENP-E plays a critical role in chromosome alignment. Here, the authors show that NF-κB activating protein (NKAP) dynamically localises to kinetochores, is SUMOylated during mitosis, and this modification is required for NKAP to bind CENP-E and localise CENP-E to the kinetochore.

The histone H3.3 chaperone DAXX is implicated in formation of heterochromatin and transcription silencing, especially for newly infecting DNA virus genomes entering the nucleus. Epstein-Barr virus (EBV) can efficiently establish stable latent infection as a chromatinized episome in the nucleus of infected cells. The EBV tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency. Here we report the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4. BNRF1 DID contacts DAXX HBD and histones through non-conserved loops. The BNRF1-DAXX interface is responsible for BNRF1 localization to PML-nuclear bodies typically associated with host-antiviral resistance and transcriptional repression. Paradoxically, the interface is also required for selective transcription activation of viral latent cycle genes required for driving B-cell proliferation. These findings reveal molecular details of virus reprogramming of an antiviral histone chaperone to promote viral latency and cellular immortalization., The Epstein-Barr virus tegument protein BNRF1 is required for the establishment of selective viral gene expression during latency and interacts with the histone chaperone DAXX. Here the authors provide structural insight into how BNRF1 hijacks the DAXX-histone H3.3-H4 complex.

Epigenetic regulatory complexes play key roles in the modulation of transcriptional regulation underlying neural stem cell (NSC) proliferation and progeny specification. How specific cofactors guide histone demethylase LSD1/KDM1A complex to regulate distinct NSC-related gene activation and repression in cortical neurogenesis remains unclear. Here we demonstrate that Rcor2, a co-repressor of LSD1, is mainly expressed in the central nervous system (CNS) and plays a key role in epigenetic regulation of cortical development. Depletion of Rcor2 results in reduced NPC proliferation, neuron population, neocortex thickness and brain size. We find that Rcor2 directly targets Dlx2 and Shh, and represses their expressions in developing neocortex. In addition, inhibition of Shh signals rescues the neurogenesis defects caused by Rcor2 depletion both in vivo and in vitro. Hence, our findings suggest that co-repressor Rcor2 is critical for cortical development by repressing Shh signalling pathway in dorsal telencephalon., Epigenetic regulation plays a key role in cortical development. Here the authors show that Rcor2, a co-repressor of the histone demethylase LSD1/KDM1A complex, regulates neural progenitor cell proliferation and cortical neurogenesis by repressing sonic hedgehog signaling.

Maternal effect genes play critical roles in early embryogenesis of model organisms where they have been intensively investigated. However, their molecular function in mammals remains largely unknown. Recently, we identified a subcortical maternal complex (SCMC) that contains four proteins encoded by maternal effect genes (Mater, Filia, Floped and Tle6). Here we report that TLE6, similar to FLOPED and MATER, stabilizes the SCMC and is necessary for cleavage beyond the two-cell stage of development. We document that the SCMC is required for formation of the cytoplasmic F-actin meshwork that controls the central position of the spindle and ensures symmetric division of mouse zygotes. We further demonstrate that the SCMC controls formation of the actin cytoskeleton specifically via Cofilin, a key regulator of F-actin assembly. Our results provide molecular insight into the physiological function of TLE6, its interaction with the SCMC and their roles in the symmetric division of the zygote in early mouse development.

Upon stimulation by Wnt ligands, the canonical Wnt/beta-catenin signalling pathway results in the stabilization of beta-catenin and its translocation into the nucleus to form transcriptionally active complexes with sequence-specific DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family proteins. In the absence of nuclear beta-catenin, TCF proteins act as transcriptional repressors by binding to Groucho/Transducin-Like Enhancer of split (TLE) proteins that function as co-repressors by interacting with histone deacetylases whose activity leads to the generation of transcriptionally silent chromatin. Here we show that the transcription factor Ladybird homeobox 2 (Lbx2) positively controls the Wnt/beta-catenin signalling pathway in the posterior lateral and ventral mesoderm of the zebrafish embryo at the gastrula stage, by directly interfering with the binding of Groucho/TLE to TCF, thereby preventing formation of transcription repressor complexes. These findings reveal a novel level of regulation of the canonical Wnt/beta-catenin signalling pathway occurring in the nucleus and involving tissue-specific derepression of TCF by Lbx2.

Glioblastoma (GBM) is the most common and deadly malignant brain cancer, with a median survival of less than two years. GBM displays a cellular complexity that includes brain tumour-initiating cells (BTICs), which are considered as potential key targets for GBM therapies. Here we show that the transcription factors FOXG1 and Groucho/TLE are expressed in poorly differentiated astroglial cells in human GBM specimens and in primary cultures of GBM-derived BTICs, where they form a complex. FOXG1 knockdown in BTICs causes downregulation of neural stem/progenitor and proliferation markers, increased replicative senescence, upregulation of astroglial differentiation genes, and decreased BTIC-initiated tumour growth upon intracranial transplantation into host mice. These effects are phenocopied by Groucho/TLE knockdown or dominant-inhibition of the FOXG1:Groucho/TLE complex. These results provide evidence that transcriptional programs regulated by FOXG1 and Groucho/TLE are important for BTIC-initiated brain tumour growth, implicating FOXG1 and Groucho/TLE in GBM tumorigenesis.

The growth of advanced prostate cancer depends on androgen receptor signalling, however treatment options are limited. Here we report the disruption of specific protein-protein interactions involving LXXLL motifs in androgen receptor-coregulator proteins such as PELP1 using a novel, small molecule peptidomimetic (D2). D2 is stable, non-toxic and efficiently taken up by prostate cancer cells. Importantly, D2 blocks androgen-induced nuclear uptake and genomic activity of the androgen receptor. Furthermore, D2 abrogates androgen-induced proliferation of prostate cancer cells in vitro with an IC50 of 40 nM, and inhibits tumour growth in a mouse xenograft model. D2 also disrupts androgen receptor-coregulator interactions in ex vivo cultures of primary human prostate tumours. These findings provide evidence that targeting androgen receptor-coregulator interactions using peptidomimetics may be a viable therapeutic approach for patients with advanced prostate cancer.