Absence of Ledgf in mouse brain affects the Kmt2a/b and polycomb balance, synaptic transmission and motor function.

Tumor suppressor menin, the product of the MEN1 gene, plays a key role in controlling histone 3 lysine 4 trimethylation (H3K4me3) and gene transcription, which can regulate proliferation, apoptosis, and differentiation. However, little is known as to whether menin controls gene expression and cell proliferation and survival via regulating Polycomb group (PcG) protein complex/H3K27me3. Here we show that menin specifically represses transcription factor Paired box gene 2 (Pax2) through PcG-mediated H3K27me3 and Wilms tumor suppressor protein (WT1), a zinc finger domain-containing DNA-binding protein. Menin does not directly bind to the Pax2 locus, instead, it up-regulates WT1 expression. WT1 recruits PcG complex to the Pax2 promoter and represses expression of Pax2 through PcG-dependent H3K27me3. Moreover, WT1 also interacts with DNA methyltransferase 1 (DNMT1), and recruits DNMT1 to the Pax2 promoter, resulting in hypermethylation of CpG in the Pax2 promoter. Together, these studies have uncovered a novel epigenetic mechanism whereby menin regulates H3K27me3 and promoter DNA methylation via WT1 and suggest that WT1 protein plays an important, yet previously unappreciated role in regulating the function of the menin/PcG axis, H3K27 methylation, and DNA methylation, resulting in repression of gene transcription.

BackgroundRelapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML.ResultsAbout 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75.ConclusionOur results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.

The lens epithelium derived growth factor p75 (LEDGF/p75) is an emerging oncoprotein identified as a key player in the cellular response to oxidative stress. LEDGF/p75 is induced by oxidative stress and promotes resistance to stress-induced cell death presumably by transcriptionally activating specific stress and survival genes. LEDGF/p75 has a splice variant, p52, which induces cell death when overexpressed. We have shown elevated expression of LEDGF/p75 in advanced stage prostatic adenocarcinomas, most likely as a result of increased oxidative stress in the prostate tumor microenvironment. To understand the mechanisms by which LEDGF/p75 transcriptionally activate stress genes and promotes protection against oxidative stress-induced cell death, it was necessary to identify endogenous cellular interacting partners of this protein. Screening of a transcription factor protein array identified the Methyl CpG binding Protein 2 (MeCP2) as a possible interacting partner of LEDGF/p75. MeCP2 is mutated in patients with Rett syndrome, both activate and repress transcription and has been implicated in prostate tumor growth. This study was designed to validate MeCP2 as an interacting partner of LEDFG/p75 and to explore its influence on LEDGF/p75's function. To examine the interaction of MeCP2 and LEDGF, pull down assays were performed with purified GST-MeCP2 incubated with His-LEDGF/p75 or His-p52, and with GST-MeCP2 or GST-p75 incubated with U2OS cell lysates. The results suggested binding of MeCP2 to both LEDGF/p75 and p52. Co-immunoprecipitation assays using U2OS cell lysates revealed binding of proteins, detected by immunoblotting using specific anti-LEDGF/p75 or MeCP2 antibodies, suggesting interaction of both proteins in the cellular microenvironment. We also examined the intracellular localization of both proteins in U2OS cells by immunofluorescence microscopy. Both MeCP2 and LEDGF/p75 co-localized in the chromatin, showing a similar dense fine speckled staining pattern. To examine if MeCP2 influences LEDGF/p75's transcriptional activity, luciferase reporter assays were performed. Even though LEDGF/p75, p52 and MeCP2 transactivated the Hsp27 promoter (pr) individually, co-expression of LEDGF/p75 with MeCP2 increased Hsp27pr activity, while co-expression of LEDGF/p52 with MeCP2 decreased activity. In addition, LEDGF/p75 overexpression protected PC3 cells from tert-butyl hydrogen peroxide (TBHP) treatment; but siRNA knockdown of MeCP2 in these cells sensitized cells to TBHP treatment compared to control siRNA. In conclusion, our data suggest that LEDGF/p75 and MeCP2 are interacting partners and MeCP2 influences LEDGF/p75's transcription and survival function. Future studies will explore modulating the interaction between LEDGF/p75 and MeCP2 to sensitize prostate cancer cells to therapy-induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2945.

Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it ‘supercoiled DNA-recognition domain’ (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K9E9K9) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it.

DDB1 and CUL4 Associated Factor 7 (DCAF7) Is Essential for Hematopoiesis

The coxsackievirus and adenovirus receptor (CXADR (CAR)) is a cell adhesion molecule expressed mainly in epithelial cells. Numerous evidence indicate that CXADR has an important role in testis development and function of the blood-testis barrier (BTB) in vitro. The role of CXADR in testis physiology in vivo has, however, not been addressed. We therefore constructed a conditional CXADR knockout (cKO) mouse model in which CXADR can be depleted at any chosen timepoint by the administration of tamoxifen. We report for the first time that testicular depletion of CXADR in adult and pubertal mice does not alter BTB permeability or germ cell migration across the BTB during spermatogenesis. Adult cKO mice display normal junctional ultra-structure and localization of the junctional proteins claudin-3, occludin, junction-associated molecule-A (JAM-A), and ZO1. The BTB was intact with no leakage of biotin and lanthanum tracers into the tubular lumen. Adult CXADR cKO mice were fertile with normal sperm parameters and litter size. Breeding experiments and genotyping of the pups demonstrated that CXADR-negative sperm could fertilize WT eggs. In addition, knocking down CXADR from postnatal day 9 (P9) does not affect testicular development and BTB formation. These cKO mice were analyzed at P49 and P90 and display an intact barrier and uncompromised fertility. We conclude that CXADR possesses no direct role in testicular physiology in vivo.

ASXL2 Is a Novel Mediator of RUNX1-ETO Transcriptional Function and Collaborates with RUNX1-ETO to Promote Leukemogenesis

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2's multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of bothEZH2's canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2TAD for mediating EZH2's non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancerthat rely on the circuits wired by EZH2 and AR.

Major Histocompatibility Class II (MHC II) molecules present extracellular peptides to T helper cells and are thus responsible for initiating, regulating, and terminating immune responses to foreign agents and tumors. Expression of MHC II is induced by the inflammatory cytokine interferon gamma (IFN‐γ) which induces transcription of the Class II Transactivator (CIITA), the master regulator of MHC II genes. Transcription of CIITA through the IFN‐γ inducible CIITA promoter IV is frequently suppressed in tumor cells through specific trimethylation of histone H3 lysine 27 (H3K27me3) by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2); however the mechanisms of recruitment of PRC2 to mammalian promoters remains unknown as proteins in the PRC2 complex contain no DNA binding domains. We identify here two EZH2 binding partners which bind DNA and regulate PRC2 recruitment to CIITApIV. We demonstrate that Yin Yang 1 (YY1), the homolog of the Drosophila PRC2 recruiting protein Pleiohomoeotic (Pho), and Jumonji domain containing protein 2 (JARID2), a member of histone demethylase family, both interact with EZH2. Upon IFN‐γ stimulation, YY1 dissociates from CIITA pIV while Jarid2 CIITA pIV binding increases, suggesting novel roles for each of these proteins in regulating inducible gene promoter dynamics. Research supported by grants from the American Cancer Society, the Georgia Cancer Coalition, and the Georgia Research Alliance (to S.F. Greer).

Many promising vaccine candidates fail to elicit protective immune responses against critical human pathogens. Exposure to antigens in the gut leads to a reduction in systemic antibody responses upon subsequent vaccination due to immune tolerance, which could contribute to low efficacy of some vaccines clinically. Tolerogenic mechanisms to avoid inappropriate inflammation are partly achieved by the high abundance of regulatory T cells (Tregs) in the gut. RORγt+ FoxP3+ Tregs are a largely peripherally derived subset that depend on stimulation from microbial antigens for development and could control type-2 intestinal immune responses in some disease models. Currently, it is unclear which Treg subsets control systemic vaccine responses following oral antigen pre-exposure. By utilizing a variety of adjuvants in our tolerance model, we have determined that vaccine-specific IgG1 is suppressed following oral antigen exposure, but not IgG2c, regardless of adjuvant strength or mechanism of action. We have used a RORγt+ Treg conditional knock-out (cKO) mouse model to determine that lack of RORγt+ expression within Tregs leads to similar suppression of vaccine-specific IgG1, but not IgG2c, in groups orally exposed to vaccine antigen compared to WT controls. cKO mice exhibit higher frequencies of IL-33R+ and Gata3+ Tregs at baseline in the gut, which suggests other subsets could compensate for the loss of function of RORγt+ Tregs. Analysis of local and systemic sites by flow cytometry also suggest the T follicular-helper compartment could be altered in RORγt+ Treg cKO mice. Together, we hypothesize that RORγt+ Tregs are not solely responsible for controlling tolerance to oral antigens in a model of tolerance to a systemic vaccine. Supported by grants from NIH (R01AI127100-01, 5TL1TR002318-05)

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that plays a major role in glial cells by converting glutamate into alpha-ketoglutarate, thereby providing energy to the cell. In order to challenge GDH importance in the central nervous system (CNS), we generated CNS specific KO mice, crossing GDH-exon 7 floxed mice with Nestin-Cre mice. Homozygous CNS-specific GDH KO mice were viable and fertile. The KO mice exhibited lower body weight compared to floxed controls. Body weight reduction was not observed in new born pups, suggesting impairment in post-natal growth and energy utilization. Food intake and energy consumption measured in these mice revealed no difference between KO and controls. At the tissue level, epididymal fat was 30% larger compared to controls, while kidneys and pancreas were smaller. DEXA-scan measurements confirmed the elevated body fat composition (+23%) observed in CNS-GDH KO. Investigating whole body energy metabolism, we observed lower plasma insulin concentrations together with glucose intolerance. NMR spectroscopy performed on anesthetized animals indicated elevated levels of glutamate and glutamine in the brain of KO mice versus controls. In conclusion, the absence of GDH in the CNS modified whole body energy balance and storage. We hypothesize that impaired CNS glutamate metabolism affected energetic fluxes in the brain, with outcomes on whole body energy metabolism.

BackgroundThe RE1‐silencing transcription factor (REST) has been strongly implicated in Alzheimer’s disease (AD), healthy ageing and longevity, and there is a clear need to understand better the exact mechanisms that mediate that role. Here we study the role of REST in a series of AD‐related phenotypes in the mouse brain.MethodWe have previously generated a conditional knockout (cKO) mouse model, lacking Rest in the postnatal forebrain. Immunohistochemistry and Western blot were used to evaluate the effect of REST on neuronal, synaptic and astrocytic markers and key proteins involved in tau phosphorylation. Furthermore, RNA‐Seq (including gene ontology, gene set enrichment and protein‐protein interaction analyses) was used to study the effect of REST inactivation at the transcriptome.ResultQuantification of labelled neuronal nuclei on brain sections revealed no major neurodegeneration in Rest cKO mice. However, mild phenotypes of dendritic degeneration and as astrocytic activation were suggested. The levels of an important postsynaptic protein, PSD‐95, appeared reduced in the brain of Rest cKO mice. Interestingly, levels of proteins involved in the phosphorylation of tau, such as glycogen synthase kinase‐3beta (GSK‐3β) and p35/p25 (activators of CDK5), as well as the levels of phosphorylated tau, were found increased in the brain of Rest cKO mice. RNA‐seq results suggest that postnatal inactivation of REST down‐regulates synaptic activity and neuronal pathways, while triggering an immune response.ConclusionOur results suggest dysregulation of one of the biochemical hallmarks of Alzheimer’s disease, tau phosphorylation, and of synaptic and neuronal pathways, as well as an immune response, when REST is inactivated in the adult mouse brain.

Introduction: Dysregulated cardiomyocyte (CM) intercalated disc (ICD) function is associated with arrhythmias and heart failure development. Zonula Occludens (ZO) 1 stabilizes the ICD, and its CM-specific loss causes cardiac conduction abnormalities, including high-grade AV block. However, ZO1 loss triggers compensatory ZO2 upregulation in ventricular CMs. We therefore hypothesize that the combined loss of CM ZO1 and ZO2 causes a destabilized ICD, ventricular dysfunction and remodeling. This study aims to evaluate cardiac structure and function at baseline and under hemodynamic stress to identify ZO-related pathways. Methods: A tamoxifen-inducible MerCreMer model was used to generate a CM-specific ZO1 and ZO2 knockout (ZO1,2cKO). Cardiac assessment was performed through echocardiography, histology, and immunofluorescence for six months. ZO1,2cKO mice also underwent transverse aortic constriction (TAC) and further evaluated. Molecular studies, including western blot and qPCR, identified altered mechanisms. Results: The 6-month baseline assessment of ZO1,2cKO (n=12-20) revealed a 15% reduction in ejection fraction (EF) (±2.4%, p<0.0001), a 1mm (±0.23, p<0.0001) decrease in systolic relative wall thickness and a concurrent 10uL (±1.7, p<0.0001) increase in left ventricular volume. This was supported by a 11.5% (±4%, p=0.0337) increase in fibrosis, eccentric hypertrophy and a 30-fold upregulation of Nppa indicating cardiomyocyte stress. Confocal evaluation of ICD structure following ZO1,2cKO showed destabilized expression of ICD components including connexin 43, sodium channel protein type 5 subunit alpha. Following baseline characterization, the ZO1,2cKO mice were subjected to a 4-week hemodynamic challenge via TAC and results suggest an accelerated more prominent deterioration of function exhibiting a 20% reduction in EF with concomitant remodeling. Further bulk RNA sequencing will reveal key dysregulated pathways associated with ZO1,2cKO. In conclusion, our study highlights an underrecognized role for ZO1 and ZO2 in the stabilization of the ICD in CMs which is necessary for maintenance of normal cardiac function and structural integrity. Ongoing research aims to define their mechanistic role in the ICD and myocardium.

Tumor initiation and progression rely on intricate cellular pathways that promote proliferation while suppressing differentiation, yet the importance of pathways inhibiting differentiation in cancer remains incompletely understood. Here, we reveal a novel mechanism centered on the repression of the neuronal-specific transcription factor ARNT2 by the MYC oncogene that governs the balance between proliferation and differentiation. We found that MYC coordinates the transcriptional repression of ARNT2 through the activity of polycomb repressive complex 2 (PRC2). Notably, ARNT2, highly and specifically expressed in the central nervous system, is diminished in glioblastoma, inversely correlating with patient survival. Utilizing in vitro and in vivo models, we demonstrate that ARNT2 knockout (KO) exerts no discernible effect on the in vitro proliferation of glioblastoma cells, but significantly enhances the growth of glioblastoma cells in vivo. Conversely, ARNT2 overexpression severely dampens the growth of fully transformed glioblastoma cells subcutaneously or orthotopically xenografted in mice. Mechanistically, ARNT2 depletion diminishes differentiation and enhances stemness of glioblastoma cells. Our findings provide new insights into the complex mechanisms used by oncogenes to limit differentiation in cancer cells and define ARNT2 as a tumor suppressor in glioblastoma.

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.