Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

An EMR-Based Approach to Determine Frequency, Prescribing Pattern, and Characteristics of Patients Receiving Drugs with Pharmacogenomic Guidelines.

(1) Background: This retrospective analysis utilizing electronic medical record (EMR) data from a tertiary integrated health system sought to identify patients and prescribers who would benefit from pharmacogenomic (PGx) testing based on Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. (2) Methods: EMR data from a clinical research data warehouse were analyzed from 845,518 patients that had an encounter between 2015 and 2019 at an academic medical center. Data were collected for 42 commercially available drugs with 52 evidence-based PGx guidelines from CPIC. Provider data were obtained through the EMR linked by specialty via national provider identification (NPI) number. (3) Results: A total of 845,518 patients had an encounter in the extraction period with 590,526 medication orders processed. A total of 335,849 (56.9%) patients had medication orders represented by CPIC drugs prescribed by 2803 providers, representing 239 distinct medications. (4) Conclusions: The results from this study show that over half of patients were prescribed a CPIC actionable medication from a variety of prescriber specialties. Understanding the magnitude of patients that may benefit from PGx testing, will enable the development of preemptive testing processes, physician support strategies, and pharmacist workflows to optimize outcomes should a PGx service be implemented.

KLF5 and p53 comprise an incoherent feed-forward loop directing cell-fate decisions following stress.

In response to stress, cells make a critical decision to arrest or undergo apoptosis, mediated in large part by the tumor suppressor p53. Yet the mechanisms of these cell fate decisions remain largely unknown, particularly in normal cells. Here, we define an incoherent feed-forward loop in non-transformed human squamous epithelial cells involving p53 and the zinc-finger transcription factor KLF5 that dictates responses to differing levels of cellular stress from UV irradiation or oxidative stress. In normal unstressed human squamous epithelial cells, KLF5 complexes with SIN3A and HDAC2 repress TP53, allowing cells to proliferate. With moderate stress, this complex is disrupted, and TP53 is induced; KLF5 then acts as a molecular switch for p53 function by transactivating AKT1 and AKT3, which direct cells toward survival. By contrast, severe stress results in KLF5 loss, such that AKT1 and AKT3 are not induced, and cells preferentially undergo apoptosis. Thus, in human squamous epithelial cells, KLF5 gates the response to UV or oxidative stress to determine the p53 output of growth arrest or apoptosis.

Effects of Subnormothermic Regulated Hepatic Reperfusion on Mitochondrial and Transcriptomic Profiles in a Porcine Model.

OBJECTIVE: We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. SUMMARY OF BACKGROUND DATA: Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. METHODS: Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. RESULTS: Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. CONCLUSION: Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

Microglia Influence Neurofilament Deposition in ALS iPSC-Derived Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here, we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin which exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte-conditioned medium and microglial-conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.

A GATA6-centred gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer.

OBJECTIVE: Molecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal programme. DESIGN: We combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO). RESULTS: This comprehensive human-to-mouse approach showed that GATA6 loss is necessary, but not sufficient, for the expression of ΔNp63 and the basal programme in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumours, we show that Gata6 inhibits tumour cell plasticity and immune evasion, consistent with patient-derived data, suggesting that GATA6 works as a barrier for acquiring the fully developed basal and metastatic phenotype. CONCLUSIONS: Our work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.

Impact of integrated translational research on clinical exome sequencing.

PURPOSE: Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. METHODS: From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. RESULTS: The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. CONCLUSION: Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

Inorganic arsenic promotes luminal to basal transition and metastasis of breast cancer.

Inorganic arsenic (iAs/As2 O32- ) is an environmental toxicant found in watersheds around the world including in densely populated areas. iAs is a class I carcinogen known to target the skin, lungs, bladder, and digestive organs, but its role as a primary breast carcinogen remains controversial. Here, we examined a different possibility: that exposure to iAs promotes the transition of well-differentiated epithelial breast cancer cells characterized by estrogen and progesterone receptor expression (ER+/PR+), to more basal phenotypes characterized by active proliferation, and propensity to metastasis in vivo. Our results indicate two clear phenotypic responses to low-level iAs that depend on the duration of the exposure. Short-term pulses of iAs activate ER signaling, consistent with its reported pseudo-estrogen activity, but longer-term, chronic treatments for over 6 months suppresses both ER and PR expression and signaling. In fact, washout of these chronically exposed cells for up to 1 month failed to fully reverse the transcriptional and phenotypic effects of prolonged treatments, indicating durable changes in cellular physiologic identity. RNA-seq studies found that chronic iAs drives the transition toward more basal phenotypes characterized by impaired hormone receptor signaling despite the conservation of estrogen receptor expression. Because treatments for breast cancer patients are largely designed based on the detection of hormone receptor expression, our results suggest greater scrutiny of ER+ cancers in patients exposed to iAs, because these tumors may spawn more aggressive phenotypes than unexposed ER+ tumors, in particular, basal subtypes that tend to develop therapy resistance and metastasis.

Targeting the CBP/ß-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

BACKGROUND: Although cyclic AMP-response element binding protein-binding protein (CBP)/ß-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. METHODS: To investigate the role of the CBP/ß-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/ß-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. RESULTS: We report for the first time that CBP/ß-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of "activation" markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/ß-catenin antagonism also suppresses PSC-induced migration of cancer cells. CONCLUSION: CBP/ß-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

Single-cell transcriptome reveals the novel role of T-bet in suppressing the immature NK gene signature.

The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.

Variant anatomy of the biliary system as a cause of pancreatic and peri-ampullary cancers.

BACKGROUND: The cause of most pancreatic and periampullary cancers (PAC) is unknown. Recently, anatomic variations such as pancreatobiliary maljunction have been recognized as risk factors, similar to Barrett-related gastro-esophageal cancers. METHODS: Pre-operative MRI from 860 pancreatic/biliary resections, including 322 PACs, were evaluated for low-union (cystic duct joining the common hepatic duct inside of the pancreas or within 5 mm of the pancreatic border) RESULTS: Low-union, seen <10% of the population, was present in 44% of PACs (73% distal bile duct/cholangiocarcinoma, 42% pancreatic head, and 34% ampullary). It was significantly lower(11%) in conditions without connection to the ductal system (thus not exposed to the ductal/biliary tract contents), namely mucinous cystic neoplasms and intrahepatic cholangiocarcinomas(p < 0.0001). Intra-pancreatic type low-union was seen in 16% of PACs versus 2% of controls(p < 0.0001). DISCUSSION: This study establishes an association between low-union and PACs, and points to an anatomy-induced chemical/bilious carcinogenesis. This may explain why most pancreas cancers are in the head. It is possible that the same chemical milieu, caused by conditions other than low-union/insertion, may also play a role in the remaining half of PACs. This opens various treatment opportunities including milieu modifications (chemoprevention), focused screening of at-risk patients, and early detection with possible corrective actions.

Motor Neuron Generation from iPSCs from Identical Twins Discordant for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.

Genetic variants in DGAT1 cause diverse clinical presentations of malnutrition through a specific molecular mechanism.

BACKGROUND: DGAT1, a gene encoding a protein involved in lipid metabolism, has been recently implicated in causing a rare nutritional and digestive disease presenting as Congenital Diarrheal Disorder (CDD). Genetic causes of malnutrition can be classified as metabolic disorders, caused by loss of a specific enzyme's function. However, disease driven by genetic variants in lipid metabolism genes is not well understood, and additional information is needed to better understand these effects. METHODS: We gathered a multi-institutional cohort of undiagnosed patients with a constellation of phenotypes presenting as malnutrition and metal ion dysregulation. Clinical Whole Exome Sequencing (WES) was performed on four patients and their unaffected parents. We prioritized genetic variants based on multiple criteria including population allele frequency and presumed inheritance pattern, and identified a candidate gene. Computational modeling was used to investigate if the altered amino acids are likely to result in a dysfunctional enzyme. RESULTS: We identified a multi-institutional cohort of patients presenting with malnutrition-like symptoms and likely pathogenic genomic variants within DGAT1. Multiple approaches were used to profile the effect these variants have on protein structure and function. Laboratory and nutritional intervention studies showed rapid and robust patient responses. CONCLUSIONS: This report adds on to the database for existing mutations known within DGAT1, a gene recently implicated with CDD, and also expands its clinical spectrum. Identification of these DGAT1 mutations by WES has allowed for changes in the patients' nutritional rehabilitation, reversed growth failure and enabled them to be weaned off of total parenteral nutrition (TPN).

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Molecular characterization of known and novel ACVR1 variants in phenotypes of aberrant ossification.

Diffuse idiopathic skeletal hyperostosis (DISH) is a disorder principally characterized by calcification and ossification of spinal ligaments and entheses. Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disabling disorder characterized by progressive ossification of skeletal muscle, fascia, tendons, and ligaments. These conditions manifest phenotypic overlap in the ossification of tendons and ligaments. We describe herein a patient with DISH, exhibiting heterotopic ossification of the posterior longitudinal ligament where clinical whole exome sequencing identified a variant within ACVR1, a gene implicated in FOP. This variant, p.K400E, is a novel variant, not identified previously, and occurs in a highly conserved region across orthologs. We used sequence-based predicative algorithms, molecular modeling, and molecular dynamics simulations, to test the potential for p.K400E to alter the structure and dynamics of ACVR1. We applied the same modeling and simulation methods to established FOP variants, to identify the detailed effects that they have on the ACVR1 protein, as well as to act as positive controls against which the effects of p.K400E could be evaluated. Our in silico molecular analyses support p.K400E as altering the behavior of ACVR1. In addition, functional testing to measure the effect of this variant on BMP-pSMAD 1/5/8 target genes was carried out which revealed this variant to cause increased ID1 and Msx2 expression compared with the wild-type receptor. This analysis supports the potential for the variant of uncertain significance to contribute to the patient's phenotype.

CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis.

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFß and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFß- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Aurora kinase B-phosphorylated HP1α functions in chromosomal instability.

Heterochromatin Protein 1 α (HP1α) associates with members of the chromosome passenger complex (CPC) during mitosis, at centromeres where it is required for full Aurora Kinase B (AURKB) activity. Conversely, recent reports have identified AURKB as the major kinase responsible for phosphorylation of HP1α at Serine 92 (S92) during mitosis. Thus, the current study was designed to better understand the functional role of this posttranslationally modified form of HP1α. We find that S92-phosphorylated HP1α is generated in cells at early prophase, localizes to centromeres, and associates with regulators of chromosome stability, such as Inner Centromere Protein, INCENP. In mouse embryonic fibroblasts, HP1α knockout alone or reconstituted with a non-phosphorylatable (S92A) HP1α mutant results in mitotic chromosomal instability characterized by the formation of anaphase/telophase chromatin bridges and micronuclei. These effects are rescued by exogenous expression of wild type HP1α or a phosphomimetic (S92D) variant. Thus, the results from the current study extend our knowledge of the role of HP1α in chromosomal stability during mitosis.

Disruption of FOXP3-EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation.

Background & Aims: Forkhead box protein 3 (FOXP3)+ regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function. Methods: Human FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis. T lymphocytes were isolated from mice, human blood, and lamina propria of Crohn's disease (CD) patients and non-CD controls. We performed proximity ligation or a co-immunoprecipitation assay in FOXP3-mutant+, interleukin 6 (IL6)-treated or CD-CD4+ T cells to assess FOXP3-EZH2 protein interaction. We studied IL2 promoter activity and chromatin state of the interferon γ locus via luciferase reporter and chromatin-immunoprecipitation assays, respectively, in cells expressing FOXP3 mutants. Results: EZH2 binding was abrogated by inflammatory bowel disease-associated FOXP3 cysteine 232 (C232) mutation. The C232 mutant showed impaired repression of IL2 and diminished EZH2-mediated trimethylation of histone 3 at lysine 27 on interferon γ, indicative of compromised Treg physiologic function. Generalizing this mechanism, IL6 impaired FOXP3-EZH2 interaction. IL6-induced effects were reversed by Janus kinase 1/2 inhibition. In lamina propria-derived CD4+T cells from CD patients, we observed decreased FOXP3-EZH2 interaction. Conclusions: FOXP3-C232 mutation disrupts EZH2 recruitment and gene co-repressive function. The proinflammatory cytokine IL6 abrogates FOXP3-EZH2 interaction. Studies in lesion-derived CD4+ T cells have shown that reduced FOXP3-EZH2 interaction is a molecular feature of CD patients. Destabilized FOXP3-EZH2 protein interaction via diverse mechanisms and consequent Treg abnormality may drive gastrointestinal inflammation.