Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

Left Ventricular End-Systolic Diameter May Predict Persistent Heart Failure with Reduced Ejection Fraction.

Patients with persistent heart failure (HF) with reduced ejection fraction (HFrEF) have a poorer prognosis than those with HF with improved ejection fraction (HFimpEF). However, data on the predictive value of echocardiographic parameters for persistent HFrEF are lacking. We retrospectively studied 443 patients who were diagnosed with HFrEF (EF ≤ 40%) during hospitalization and underwent echocardiography at the 1-year follow-up. We divided them into the 2 groups: HFimpEF (EF > 40%) and persistent HFrEF group at 1-year follow-up, and assessed the predictive value of echocardiographic parameters at discharge for persistent HFrEF. In total, 301/443 patients (68%) were diagnosed with persistent HFrEF and 142/443 (32%) with HFimpEF at the 1-year follow-up. Kaplan-Meier analysis revealed that the persistent HFrEF group had a poorer prognosis than the HFimpEF group (log-rank, P < 0.001). Receiver operating characteristic curve analysis revealed that left ventricular end-systolic diameter (LVESD) had the highest area under the curve (AUC) (0.70; 95% confidence interval [CI]: 0.64-0.75; cutoff value: 55 mm) among various echocardiographic parameters. LVESD was an independent predictor of persistent HFrEF at the 1-year follow-up (odds ratio: 1.07, 95%CI: 1.02-1.12) upon multivariable logistic regression analysis. The incidence of persistent HFrEF was higher in patients with an LVESD ≥ 55 mm than in those with an LVESD < 55 mm (81% versus 55%, Fisher's exact test, P < 0.001). In conclusion, an LVESD (≥ 55 mm) was associated with persistent HFrEF. Focusing on LVESD in daily practice may help clinicians with risk stratification for decision-making regarding management in patients with advanced HF refractory to guideline-directed medical therapy.

Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by the Unconventional Secretion of Cytosolic Proteins.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

Prognostic impact of guideline-directed medical therapy in patients with heart failure on regular hemodialysis.

BACKGROUND: Renin-angiotensin system inhibitor (RASi) and ß-blocker provide prognostic benefits as guideline-directed medical therapy (GDMT) in patients with heart failure and reduced ejection fraction (HFrEF). However, there is limited data for the favorable effects in such patients receiving regular hemodialysis. We aimed to evaluate the prognostic impact of RASi and ß-blocker in patients with HFrEF who receive regular hemodialysis. METHODS: In this retrospective, single-center, observational study, from 2110 consecutive patients hospitalized for HF and who survived to discharge, 97 with HFrEF who received regular hemodialysis were included for analysis. They were classified into three groups according to prescribed medication at discharge following index hospitalization: both RASi and ß-blocker (Dual-GDMT group: n = 55), either RASi or ß-blocker (Mono-GDMT group: n = 34), and neither RASi nor ß-blocker (No-GDMT group: n = 8). The primary endpoint was a composite of all-cause death and rehospitalization for heart failure. RESULTS: The mean age was 66 years and 79% of the patients were men. During the median follow-up of 501 days, the primary endpoint occurred in 43 patients (44%). Kaplan-Meier analysis revealed that the Dual-GDMT group had the lowest rates of the primary endpoint (log-rank test for trend: p < 0.001). Even after adjustment for diverse covariates (multivariate Cox regression), the Dual-GDMT (hazard ratio [HR]: 0.04, 95% confidence interval (CI): 0.005-0.32) and Mono-GDMT (HR: 0.08, 95% CI: 0.01-0.50) groups had better prognoses than the No-GDMT group. CONCLUSIONS: The prescription of RASi and/or ß-blocker was associated with a lower adverse-event rate after discharge in patients with HFrEF who were on regular hemodialysis.

Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

More than 370-Fold Increase in Antibody Affinity to Estradiol-17ß by Exploring Substitutions in the VH-CDR3.

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17ß (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.

Extracellular PKCδ signals to epidermal growth factor receptor for tumor proliferation in liver cancer cells.

Protein kinase C delta (PKCδ) is a multifunctional PKC family member and has been implicated in many types of cancers, including liver cancer. Recently, we have reported that PKCδ is secreted from liver cancer cells, and involved in cell proliferation and tumor growth. However, it remains unclear whether the extracellular PKCδ directly regulates cell surface growth factor receptors. Here, we identify epidermal growth factor receptor (EGFR) as a novel interacting protein of the cell surface PKCδ in liver cancer cells. Imaging studies showed that secreted PKCδ interacted with EGFR-expressing cells in both autocrine and paracrine manners. Biochemical analysis revealed that PKCδ bound to the extracellular domain of EGFR. We further found that a part of the amino acid sequence on the C-terminal region of PKCδ was similar to the putative EGFR binding site of EGF. In this regard, the point mutant of PKCδ in the binding site lacked the ability to bind to the extracellular domain of EGFR. Upon an extracellular PKCδ-EGFR association, ERK1/2 activation, downstream of EGFR signaling, was apparently induced in liver cancer cells. This study indicates that extracellular PKCδ behaves as a growth factor and provides a molecular basis for extracellular PKCδ-targeting therapy for liver cancer.

Prognosis and diastolic dysfunction predictors in patients with heart failure and recovered ejection fraction.

There is limited data on whether diastolic dysfunction in patients with heart failure (HF) and recovered ejection fraction (HFrecEF) is associated with worse prognosis. We retrospectively assessed 96 patients diagnosed with HFrecEF and created ROC curve of their diastolic function at the 1-year follow-up for the composite endpoint of cardiovascular death and HF readmission after the follow-up. Eligible patients were divided into two groups according to the cutoff value of E/e' ratio (12.1) with the highest AUC (0.70). Kaplan-Meier analysis showed that HFrecEF with high E/e' group had a significantly poorer prognosis than the low E/e' group (log-rank, p = 0.01). Multivariate Cox regression analysis revealed that the high E/e' group was significantly related to the composite endpoint (hazard ratio 5.45, 95% confidence interval [CI] 1.23-24.1). The independent predictors at discharge for high E/e' ratio at the 1-year follow-up were older age and female sex after adjustment for covariates (odds ratio [OR] 1.07, 95% CI 1.01-1.13 and OR 4.70, 95% CI 1.08-20.5). In conclusion, HFrecEF with high E/e' ratio might be associated with a poor prognosis. Older age and female sex were independent predictors for a sustained high E/e' ratio in patients with HFrecEF.

Ubiquitination-dependent and -independent repression of target genes by SETDB1 reveal a context-dependent role for its methyltransferase activity during adipogenesis.

The lysine methyltransferase SETDB1, an enzyme responsible for methylation of histone H3 at lysine 9, plays a key role in H3K9 tri-methylation-dependent silencing of endogenous retroviruses and developmental genes. Recent studies have shown that ubiquitination of human SETDB1 complements its catalytic activity and the silencing of endogenous retroviruses in human embryonic stem cells. However, it is not known whether SETDB1 ubiquitination is essential for its other major role in epigenetic silencing of developmental gene programs. We previously showed that SETDB1 contributes to the formation of H3K4/H3K9me3 bivalent chromatin domains that keep adipogenic Cebpa and Pparg genes in a poised state for activation and restricts the differentiation potential of pre-adipocytes. Here, we show that ubiquitin-resistant K885A mutant of SETDB1 represses adipogenic genes and inhibits pre-adipocyte differentiation similar to wild-type SETDB1. We show this was due to a compensation mechanism for H3K9me3 chromatin modifications on the Cebpa locus by other H3K9 methyltransferases Suv39H1 and Suv39H2. In contrast, the K885A mutant did not repress other SETDB1 target genes such as Tril and Gas6 suggesting SETDB1 represses its target genes by two mechanisms; one that requires its ubiquitination and another that still requires SETDB1 but not its enzyme activity.

Role of transient receptor potential vanilloid subtype 2 in lower oesophageal sphincter in rat acid reflux oesophagitis.

Gastroesophageal reflux disease (GERD) is a common gastrointestinal disorder. In the present study, we investigated TRP vanilloid subfamily member 2 (TRPV2) expression in lower oesophageal sphincter (LES) and its involvement in acid reflux oesophagitis in rats. Expression of TRPV2 and nerve growth factor mRNAs was significantly enhanced in LES of rats with reflux oesophagitis compared with normal rats. TRPV2 was mainly expressed in inhibitory motor neurons, and partly in intrinsic and extrinsic primary afferent neurons, and macrophages in LES of normal and reflux oesophagitis rats. Number of TRPV2-immunopositive nerve fibres was significantly increased, but that of nNOS-, CGRP-, and PGP9.5-nerve fibres was not changed in reflux oesophagitis compared with normal group. Probenecid produced nitric oxide production and relaxation in LES and this response was significantly enhanced in oesophagitis compared with normal group. Probenecid-induced relaxant effect was blocked by a TRPV2 inhibitor, tranilast, and a NOS inhibitor, NG-nitro-l-arginine methyl ester, in reflux oesophagitis rats. Oral administration of tranilast significantly improved body weight loss, oesophageal lesions, and epithelial thickness in oesophagitis model. These results suggest that up-regulation of TRPV2 in inhibitory motor neurons is involved in LES relaxation in oesophagitis model. TRPV2 inhibition might be beneficial for treatment of GERD.

Prognostic Impact of Early Induction of Intra-Aortic Balloon Pump Counterpulsation in High-Risk Patients With Acute Heart Failure.

BACKGROUND: Intra-aortic balloon pumping (IABP) counterpulsation provides potent supports on hemodynamic status of patients with cardiogenic shock. However, only limited numbers of patients with acute heart failure (AHF) under collapsed hemodynamic status received such benefit of IABP. We aimed to evaluate the impact of the timing of IABP induction on clinical prognosis in AHF patients at very high risk. METHODS: Of 404 consecutive AHF patients, 57 patients both with left ventricular ejection fraction (LVEF) <35% and systolic blood pressure on admission <100 mmHg were ultimately enrolled in this observational study. They were divided into 3 groups depending on IABP use; Early-IABP group (induction at ≤3 days after admission, n = 17), Late-IABP group (>3 days, n = 15) and No-IABP group (n = 25). The primary endpoint was a composite of in-hospital cardiovascular (CV) death and ventricular assisted device implantation. RESULTS: This high-risk population was typically mid-age (60 years-old), 61% male, and 75% with chronic kidney disease, and its average LVEF was 24.7%. Clinical profiles on admission were comparable among 3 subgroups, except prehospital prescription rate of loop diuretics. During hospital stay, intravenous inotropes were significantly more frequently administered in the Late-IABP group than other 2 groups. The primary endpoint was developed in 17.6% of patients in the Early-IABP group, which was significantly lower than that in the Late-IABP group (53.3%, p = 0.034) and was comparable to the No-IABP group (40.0%, p = 0.12). CONCLUSIONS: Early induction of IABP is one of the therapeutic options for improvement of in-hospital prognosis in AHF patients at very high risk.

Right atrial conduction time for predicting coexistent typical atrial flutter in patients with paroxysmal atrial fibrillation.

INTRODUCTION: Screening of coexistent typical atrial flutter (AFL) in patients with atrial fibrillation (AF) is sometimes challenging. This study investigated whether a prolonged right atrial conduction time (RACT) estimated by tissue Doppler imaging (TDI) predicts patients with concomitant AFL and AF. METHODS AND RESULTS: We retrospectively analyzed 398 patients (mean age: 61.6 years, 73.4% men) undergoing catheter ablation of paroxysmal AF. The patients were classified into two groups according to whether they had evidence of AFL (N = 122, 30.7%) determined by a clinical observation (N = 68), induction during procedures (N = 33), or AFL recurrence after procedures (N = 21) or not (N = 276, 69.3%). The preoperative RACT, defined as a longer duration between the onset of the P-wave and peak A'-wave on the right atrial lateral wall or septal wall, and total atrial conduction time (TACT), defined as the same time duration on the left atrial lateral wall, were evaluated in all patients. Patients with evidence of AFL had a significantly longer RACT than those without AFL (p < .001). A multiple logistic regression and receiver operator characteristics curve analysis revealed the ratio of the RACT and TACT (RACT/TACT) was the independent and most superior accurate cofounder for predicting evidence of AFL (area under the curve: 0.867). When adding a discriminator of an RACT/TACT ≧ 93% into the conventional screening, 98.4% of the patients with evidence of AFL were estimated to be treated during the initial procedures. CONCLUSION: The estimated RACT/TACT using the TDI may be useful for predicting patients with concomitant AFL in patients with AF.

Quantitative Volumetric Comparison of Direct Oral Anticoagulant and Vitamin K Antagonist Treatment for Pulmonary Thrombus Reduction During the Acute Phase in Symptomatic Patients.

BACKGROUND: Recent clinical trials' findings have revealed the therapeutic noninferiority of direct oral anticoagulant (DOAC) to standard therapy with vitamin K antagonist (VKA) in patients with pulmonary thromboembolism (PTE). However, few studies have quantitatively analyzed thrombus reduction in the pulmonary artery. METHODS: This observational study included 38 symptomatic PTE patients with stable hemodynamics. All patients received an intravenous heparin bolus followed by continual heparin injections immediately after the PTE diagnosis. The heparin was discontinued after edoxaban therapy began in the DOAC group (n = 22) or after the therapeutic range for the prothrombin time-international normalized ratio was achieved in the VKA group (n = 16). The thrombus volumes in the pulmonary arteries were quantitatively analyzed using contrast-enhanced computed tomography scans, and they were compared at baseline and at 2 weeks after admission. RESULTS: The pulmonary thrombus volumes declined in the VKA and DOAC groups from 7.9 to 4.2 cm3 (P = 0.048) and from 7.1 to 3.7 cm3 (P < 0.01), respectively, and the thrombus reduction rates did not differ significantly between the groups (-34% vs. -64%, respectively; P = 0.38). The fibrinogenolysis parameter changes during the14 days after admission were similar in both groups. Compared with the VKAgroup, the average hospital stay was 9days shorter in the DOAC group. There were no in-hospital deaths, and 1 case experienced major bleeding in the VKA group. CONCLUSIONS: In relation to pulmonary artery thrombus volume reduction, DOAC monotherapy for PTE may be comparable with standard therapy involving VKAs.

Controlled Radical Homopolymerization of Representative Cationically Polymerizable Vinyl Ethers.

Facile direct radical homopolymerization of vinyl ethers without a hydroxy group was achieved up to near full conversion. This polymerization was conducted in water suspension in the presence of lithium hydroxide using a thermally triggered azo-initiator of dimethyl 2,2'-azobis(2-methylpropionate). In the polymerization system, appropriate hydrogen bonding and cation-π interactions under basic conditions are keys to the successful direct radical homopolymerization. The hydrogen bonding between water and vinyl ether oxygen reduces the reactivity of the growing radical, thus suppressing unfavorable side reactions such as ß-scission. In addition, Li+ interacts with the oxygen and the vinyl group of vinyl ethers. The vinyl ether tends to be "activated" and the polymerization can be facilitated. Based on the results of free radical polymerization of vinyl ethers, controlled polymerization was also accomplished using the appropriate dithiocarbamate RAFT agent in view of the solubilities of the radical leaving group.

A case of desmoplakin mutation and delayed arrhythmogenic right ventricular cardiomyopathy/dysplasia after atrial septal defect closure.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a slow-developing cardiomyopathy characterized by ventricular arrhythmias and fibrofatty replacement of the right ventricular (RV) myocardium. Its clinical diagnosis is challenging because of its variable clinical presentation and low genetic penetrance. We describe the case of a 67-year-old man who was diagnosed as having ARVC/D with a desmoplakin mutation that appeared after occlusion of an atrial septal defect (ASD). He underwent patch closure surgery for ASD at the age of 54 years. Four years later, he underwent catheter ablation for multifocal atrial tachycardias. Because of pre-syncope and inducible sustained monomorphic ventricular tachycardia, an implantable cardioverter defibrillator was implanted. When he was admitted for worsening heart failure at the age of 61 years, the desmoplakin mutation was detected with progressive left ventricular (LV) dysfunction. Subsequently, he was diagnosed as having ARVC/D with RV dysfunction. At cardiac autopsy, characteristics of ARVC/D, including dilatation, fibrofatty changes in the right ventricle, and diffuse fibrosis in the left ventricle were detected. Along with the effect of RV dysfunction caused by ASD, the progression of LV dysfunction after ASD closure was also possibly caused by the disease progression of ARVC/D. Physicians should carefully assess the various states of ARVC/D. .

Characterization of the gastric motility response to human motilin and erythromycin in human motilin receptor-expressing transgenic mice.

Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin. The distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red. hMTLR expression was abundant in the gastric smooth muscle layer. Interestingly, higher levels of hMTLR expression were observed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice. The contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca2+-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice. Human motilin and erythromycin stimulate gastric smooth muscle contraction in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca2+. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents.

The relationship between patients' perception of type 2 diabetes and medication adherence: a cross-sectional study in Japan.

BACKGROUND: The self-management of type 2 diabetes mellitus (T2DM), which involves adherence to medical instructions on diet and nutritional advice, physical activity, medication regimen, and weight and stress management, is necessary for the treatment of T2DM.In this study, we investigated the relationship between patients' perceptions of their disease and their adherence to their medications. And we attempted to determine whether distinct subphenotypes of behavioral change of medication adherence can be discerned based on a patients' perceptions. METHOD: A cross-sectional study using a questionnaire was conducted among 157 patients with T2DM from October 2015 to September 2017. Questionnaires were administered to assess the participants' demographic and clinical characteristics, medication adherence, diabetes knowledge, and perception of being diabetic. Principal component analysis (PCA) and cluster analyses were performed to classify medication adherence patterns in the total cohort. Multiple regression analyses were performed to identify the determinant factors of medication adherence. RESULTS: PCA showed the interpretable medication adherence of patients with diabetes by using component 1 ("accessibility to medical treatment") and component 2 ("status of taking medicines"). We identified four groups that show significantly different medication adherence by using cluster analysis on the basis of the two components. Multiple regression analysis showed that body mass index (BMI), family history of diabetes, one factor of patient's perception (living an orderly life), and diabetes knowledge were found to be significant predictors of medication adherence in patients with T2DM. CONCLUSIONS: In patients with T2DM, the patient's diabetes perception of "living an orderly life" is associated with medication adherence. A poor adherence group may be able to change their adherence to diabetes treatment by developing the perception of "living an orderly life."

H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPß binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

The FBXL10/KDM2B scaffolding protein associates with novel polycomb repressive complex-1 to regulate adipogenesis.

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.