MBD2 Knockdown Research Articles

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2−/−) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2−/− mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2−/− mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

MBD2-NuRD but Not MBD3-NuRD Mediates Strong Silencing of Fetal γ-Globin Gene Expression in Human Adult Erythroid Cells

Elevated fetal hemoglobin (HbF) levels are effective in ameliorating the underlying pathophysiologic defects in β-thalassemia and sickle cell anemia. Studies aimed at a full understanding of the mechanisms that enforce silencing of HbF expression in adult erythroid cells offer the promise of targeted molecular therapy. Here we show that depletion of Methyl CpG Binding Domain Protein 2 (MBD2) or disruption of MBD2-Nucleosome Remodeling and Deacetylose Complex (NuRD) interactions results in high levels of HbF in HUDEP-2 cell progenitor derived primary erythroblasts and CD34 + progenitor derived primary adult erythroid cells.We have previously shown that depletion of MBD2 causes a 10-20 fold increase in γ-globin gene expression in adult erythroblasts of β-YAC transgenic mice. To determine the effect of total depletion of MBD2 in human erythroid cells, CRISPR/Cas9 mediated knockout was carried out in HUDEP-2 cells. The absolute level of γ-globin gene expression increased by > 100 fold and the γ/ γ+β mRNA ratio increased from 1-3% in CRISPR/Cas9 scrambled guide control cells to 50-60% in MBD2 knockout HUDEP-2 cells after erythroid differentiation, equivalent to the highest levels reported with knockout of other silencing factors. In contrast CRISPR/Cas9 mediated knockout of Methyl CpG Binding Domain Protein 3 (MBD3) had no significant effect on γ-globin gene expression levels in HUDEP-2 cells after erythroid differentiation.Partial depletion of several strong γ-globin gene silencers, including BCL11A, LRF and KLF-1 has been shown to relieve silencing in CD34+ progenitor derived primary adult erythroid cells, but differences in the differentiation states reported in individual studies make comparisons of the relative effects of these silencer factors difficult. We show here that knockdown of MBD2 by shRNA in CD34+ progenitor derived primary adult erythroid cells consistently increased γ/ γ+β mRNA rations by 7-10 fold across different stages of differentiation without affecting the level of differentiation as compared to shRNA scrambled control cells. MBD2 knockdown led to ~30-40% γ-globin mRNA levels compared to 3%-4% in scrambled shRNA control cells, a level comparable to that typically observed in sickle cell patient reticulocytes. A comparable increase in HbF levels was observed in the MBD2 shRNA knockdown CD34+ progenitor derived erythroid cells compared to scrambled shRNA controls.We have previously shown that MBD2 interacts with the CHD4 chromatin remodeling component of the NuRD complex through a coiled-coil domain, and with the histone deacetylase component of the complex via an intrinsically disordered region (IDR) within MBD2. To test the effects of disrupting these domains in a human adult erythroid cell model, MBD2 knockout HUDEP-2 cells were infected with lentiviral expression vectors containing either wild type MBD2, coiled-coil mutant MBD2, or IDR mutant MBD2. Wild type MBD2 expression resulted in significant silencing of γ-globin gene expression while neither the coiled-coil mutant nor the IDR mutant MBD2 had any silencing effect.These results establish MBD2-NuRD as a major silencer of fetal hemoglobin expression in adult human erythroid cells. Given the minimal phenotype of MBD2 knockout mice, these results suggest that the coiled-coil and IDR domains of the MBD2-NuRD complex are potential targets for therapeutic agents aimed at increasing HbF levels in patients with β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2450: Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation

Abstract Background: Epigenetic reader proteins maintain an imbalance between differentiation and self-renewal in cancer. Genetic alterations in the DNA methylation machinery (e.g., TET, DNMTs) and chromatin remodelers (e.g., KDMs, EZH1/2) are a hallmark of cancer. Methyl CpG binding domain protein 2 (MBD2) is an epigenetic reader protein which modulates regional gene transcription by recruiting co-repressor complexes to sites of CpG methylation. Our goal is to develop a novel class of anti-neoplastic epigenetic therapies targeting MBD2 to selectively reprogram tumor cells towards a terminally differentiated state and sensitize them to chemotherapy, radiation, and immunotherapy. Methods: To explore conserved mediators of MBD2 function in cancer, we used MBD2 targeting shRNA lentivirus to stably knockdown MBD2 in prostate cancer (PC3, DU145, LnCap), leukemia (SigM5, TET2WTK562 or TET2KOK562) and triple negative breast cancer (MDA-MB231-nanog-GFP) cell lines. Inhibition of MBD2 expression was confirmed by qRT-PCR and Western Blot. Proliferative potential was determined by cell counting and clonogenic potential was determined by methylcellulose-based colony forming assays. RNAseq analysis was performed on prostate cancer and leukemia cell lines. miRNA expression was analyzed using miScript miRNA PCR (Qiagen). In vivo tumor initiation capacity was analyzed using orthotropic and heterotopic xenografts in athymic NSG mice. Results: Our results show that MBD2 is required for the proliferation of triple negative breast cancer (TNBC), prostate cancer (PCa), and TET mutant leukemias (TML), in vitro. The functional mediators of MBD2's growth promoting effects were tissue/tumor context dependent. In leukemias, MBD2's growth promoting and tumor initiating effects were most pronounced in TET2 null cells (which accumulate 5mC). In TNBC, nanog-GFP reporter positive cells were more sensitive to MBD2 knockdown than reporter negative cells. In PCa and TNBC, RNAseq analysis revealed that knockdown of MBD2 led to downregulation of Myc pathway genes and increased the expression of Myc targeting microRNAs, miR33-5, miR34a, miR148a and miR363. Western blot analysis confirmed that MBD2 knockdown coordinately downregulated cMyc expression and activated p27 expression. We further demonstrated that inhibition of MBD2 diminished the tumor initiating capacity of TNBC and PCa in xenograft models and the in vivo engraftment rate of patient derived TET-/- AML. Conclusions: MBD2 knockdown diminished the proliferative capacity of PCa, TNBC and primary TET2 mutant leukemia cells in a genetic (TET2) and phenotypic (nanog+ stem/progenitor) context dependent manner. Delayed peak effect and altered differentiation markers after MBD2 inhibition suggest epigenetic reprogramming as the mechanism of growth suppression. MBD2 targets miRNA's upstream of cMyc in PCa and TNBC. MBD2 murine knockout models are developmentally normal, suggesting an acquired function in cancer with a favorable therapeutic window for targeting. Citation Format: Aysegul Balyimez, Yihong Guan, Emily Esakov, Shinjini Ganguly, Ofer Reizes, Daniel J. Lindner, Jaroslaw Maciejewski, Babal Jha, Omar Y. Mian. Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2450.

Abstract 721: CHD4 mediates genotoxic sensitivity and tumor initiation of AML cells through both MBD2-NuRD and MBD3-NuRD co-repressor complexes

Abstract Despite several advances in therapy, the 5-year survival of AML, estimated at 40-45% for the young and 10% for the elderly, remains dismal. New modalities of therapy are thus clearly needed. Research in our laboratory has identified CHD4, an ATPase chromatin re-modeling component of the NuRD co-repressor complex, as an important mediator of genotoxic sensitivity to standard chemotherapy in AML cell lines and primary AML samples. Knockdown of CHD4 also markedly reduced colonies in soft agar. Interestingly, normal hematopoietic progenitor cells did not show these phenotypic alterations. Classical NuRD complex contains either MBD2 or MBD3, and at least four other proteins. We sought to determine if the previously observed CHD4 effects are through MBD2-NuRD or MBD3-NuRD. MBD2 and MBD3 are thought to link DNA to the GATAD2/CHD4 NuRD sub-components through coiled-coil domains of both proteins interacting with those of GATAD2A/B. It was thus hypothesized that if the observed effects of CHD4 are mediated through canonical NuRD, knockdown of either MBD2 or MBD3 should be able to replicate these. In order pursue this aim, we first knocked down MBD2 in U937 cell lines and performed colony forming assays. Persistent efforts to knockdown MBD3, however, were unsuccessful. Therefore, MBD3 was knocked out using CRISPR-Cas9. Single cell cloning to generate a completely knocked out cell line, however, introduces a potential intrinsic bias in colony forming assays by selecting for cells with an inherently increased colony forming potential. A guide RNA with an ~80% overall knockout efficiency was thus selected and the assay performed on the bulk population of U937 cells prior to limiting dilution and plating. U937 cells with CHD4 knocked down were used as positive controls. Neither MBD2, nor MBD3, when knocked down by 90% or knocked out, respectively, showed any effect on colony formation in vitro using the soft agar colony forming assay. However, knocking down MBD2 in U937 cells with MBD3 knocked out, resulted a significant reduction in the colony forming potential. A similar result was obtained in the genotoxic sensitivity assay, whereby cells were treated with cytosine arabinoside (AraC) and the percentage apoptosis determined by flow cytometry through 7-AAD staining as well as caspase cleavage assays. MBD2 knockdown did not increase the genotoxic sensitivity of U937 cells, while MBD3 knockout increased it only marginally. MBD2 knockdown in MBD3 knockout U937 cell lines, however, did markedly increase the sensitivity of these cells to AraC. We conclude that in terms of mediation of AML colony forming potential and sensitivity to standard chemotherapy agents, MBD2-NuRD and MBD3-NuRD may be redundant. Future therapeutic strategies aimed at exploiting the potent effects of disrupting the NuRD complex on AML cell sensitivity to chemotherapy and tumor initiating activity need to take this into account. Citation Format: Javeria Aijaz, Justin Sperlazza, Gordon D. Ginder. CHD4 mediates genotoxic sensitivity and tumor initiation of AML cells through both MBD2-NuRD and MBD3-NuRD co-repressor complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 721.

Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.

As high fetal hemoglobin levels ameliorate the underlying pathophysiological defects in sickle cell anemia and beta (β)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemoglobin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex member MBD2 causes a 10-20-fold increase in γ-globin gene expression in adult β-globin locus yeast artificial chromosome transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in γ/γ+β mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high performance liquid chromatography. In contrast, MBD3 knockout had no appreciable effect on γ-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased γ/γ+β mRNA ratios by approximately 10-fold resulting in approximately 30-40% γ/γ+β mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiled-coil domain, and the histone deacetylase core complex via an intrinsically disordered region. Enforced expression of wild-type MBD2 in MBD2 knockout cells caused a 5-fold decrease in γ-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant MBD2 proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt complex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the MBD2 Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of MBD2 as potential therapeutic targets.

The role of IFI35 in lupus nephritis and related mechanisms

Objectives: It’s reported that multiple genes in the IFN-γ/STAT1 pathway were hypomethylated and associated with the pathogenesis of lupus nephritis (LN). Our previous study using microarray analysis suggested that interferon induced 35-kDa protein (IFI35) was hypomethylated and increased in LN. However, the role of IFI35 in LN and related mechanism remains to be elucidate.Methods: The expressions of IFNγR, STAT1, IFI35 and MBD2 in the human kidneys tissues was detected by real-time PCR and Western blot. The protein levels of IFI35 in the human kidney tissues were detected by immunohistochemistry. The methylation status of IFNγR, STAT1 and IFI35 were detected by methylation specific PCR. Cell proliferation assay was evaluated using cell counting kit 8; pcDNA-IFI35 (pcDNA-MBD2) or IFI35 RNAi (MBD2 RNAi) was used to upregulated or downregulated the expression of the IFI35 and MBD2.Results: The expressions of IFNγR, STAT1 and IFI35 in the LN kidneys were significantly higher than controls. IFI35 was expressed in mesangial cells, and positively correlated with the proliferation of mesangial cells. IFNγR, STAT1and IFI35 was hypomethylated and MBD2 was increased in LN kidneys. In vitro data confirmed those findings: after stimulating with the serum from LN patients, the proliferation of human renal mesangial cells (HRMCs) was increased. The expressions of the three members of IFNγ signal pathway were hypomethylated and upregulated. However, this effect was reversed by MBD2 knockdown. IFI35 promoted the proliferation of HRMCs and was regulated by MBD2.Conclusion: Our results demonstrated that IFI35 enhances the proliferation of mesangial cells and was regulated by MBD2 in LN.

Alteration of Scn3a expression is mediated via CpG methylation and MBD2 in mouse hippocampus during postnatal development and seizure condition

Increased expression of sodium channel SCN3A, an embryonic-expressed gene, has been identified in epileptic tissues, which is believed to contribute to the development of epilepsy. However, the regulatory mechanism of SCN3A expression under epileptic condition is still unknown. Here we showed a high level of Scn3a mRNA expression in mouse embryonic hippocampus with gradually decreasing to a low level during the postnatal development and a methylation of a specific CpG site (−39C) in the Scn3a promoter was increased in hippocampus during postnatal development, corresponding to the downregulation of Scn3a expression. Furthermore, in vitro methylation and −39C>T mutation of the Scn3a promoter decreased the reporter gene expression, suggesting an important role of the −39C site in regulating gene expression. We then demonstrated that the sequence containing −39C was a MBD2-binding motif and the CpG methylation of the promoter region increased the capability of MBD2's binding to the motif. Knockdown of MBD2 in mouse N1E-115 cells led to the −39C methylation and the downregulation of Scn3a transcription by decreasing the Scn3a promoter activity. In the hippocampus of seizure mice, the expressions of Scn3a and Mbd2 were upregulated after 10-day KA treatment. At the same time point, the −39C site was demethylated and the capability of MBD2's binding to the Scn3a promoter motif was decreased. Taken together, these findings suggest that CpG methylation and MBD2 are involved in altering Scn3a expression during postnatal development and seizure condition.

Epigenetic Regulation of Organic Anion Transporting Polypeptide 1B3 in Cancer Cell Lines

The expression of a multispecific organic anion transporter, OATP1B3/SLCO1B3, is associated with clinical prognosis and survival of cancer cells. The aims of present study were to investigate the involvement of epigenetic regulation in mRNA expression of a cancer-type variant of OATP1B3 (Ct-OATP1B3) in cancer cell lines. The membrane localization and transport functions of Ct-OATP1B3 were investigated in HEK293 cells transiently expressing Ct-OATP1B3. DNA methylation profiles around the transcriptional start site of Ct-OATP1B3 in cancer cell lines were determined. The effects of a DNA methyltransferase inhibitor and siRNA knockdown of methyl-DNA binding proteins (MBDs) on the expression of Ct-OATP1B3 mRNA were investigated. 5'-RACE identified the TSS of Ct-OATP1B3 in PK-8 cells. Ct-OATP1B3 was localized on the plasma membrane, and showed the transport activities of E217βG, fluvastatin, rifampicin, and Gd-EOB-DTPA. The CpG dinucleotides were hypomethylated in Ct-OATP1B3-positive cell lines (DLD-1, TFK-1, PK-8, and PK-45P) but were hypermethylated in Ct-OATP1B3-negative cell lines (HepG2 and Caco-2). Treatment with a DNA methyltransferase inhibitor and siRNA knockdown of MBD2 significantly increased the expression of Ct-OATP1B3 mRNA in HepG2 and Caco-2. Ct-OATP1B3 is capable of transporting its substrates into cancer cells. Its mRNA expression is regulated by DNA methylation-dependent gene silencing involving MBD2.

Methyl-CpG binding by the Mi-2/NuRD complex is required for transcriptional repression in B cells. (111.33)

Abstract We demonstrated previously that the mb-1 (Igα; Cd79a) promoter is demethylated progressively during its activation in early B cell development. Demethylation of promoter CpGs is initiated by binding of the transcription factor, Early B Cell Factor 1 (EBF1). EBF1 activates the accessibility and demethylation of mb-1 promoter DNA, which allows for binding by Pax5 prior to transcription. This process is inhibited by Mi-2/NuRD chromatin remodeling complexes. This was evidenced by knockdown of the ATPase subunit of Mi-2/NuRD, Mi-2β (CHD4), which potentiated effects of EBF1 and Pax5. Recent work in the laboratory has focused on other components of the Mi-2/NuRD complex to assess its association with methylated DNA. The Mi-2/NuRD complex contains the Methyl Binding Domain proteins 2 or 3 (MBD2 or MBD3). MBD2 has the ability to bind methylated cytosines, while MBD3 binds hydroxymethylated cytosines. Similar to depletion of Mi-2β, knockdown of MBD2 using shRNA potentiated mb-1 transcriptional activation in the presence of EBF1 and Pax5. This increased activation was similar to that obtained using 5-azacytidine, which reduces cytosine methylation by inhibiting DNA methyltransferases. In contrast, depletion of MBD3 had no effect on EBF1/Pax5-dependent promoter transcription. Our data suggest that methyl-cytosine binding by Mi-2/NuRD complexes containing MBD2 is important for transcriptional attenuation of the mb-1 gene in B cells.

Methyl-Binding Domain Protein 2–Dependent Proliferation and Survival of Breast Cancer Cells

Methyl cytosine binding domain protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where repatterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable short hairpin RNA (shRNA)-mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak antiproliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in Bagg Albino (BALB)/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by derepression of tumor suppressor genes, including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing show MBD2 binding directly to the hyper methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG island-associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and resilencing of the MBD2-dependent tumor suppressor genes. Our data suggest that uncoupling CpG methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results show a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers.

Loss of Methyl-CpG–Binding Domain Protein 2 Enhances Endothelial Angiogenesis and Protects Mice Against Hind-Limb Ischemic Injury

Despite intensive investigation, how DNA methylation influences endothelial function remains poorly understood. We used methyl-CpG-binding domain protein 2 (MBD2), an interpreter for DNA methylome-encoded information, to dissect the impact of DNA methylation on endothelial function in both physiological and pathophysiological states. Human umbilical vein endothelial cells under normal conditions express moderate levels of MBD2, but knockdown of MBD2 by siRNA significantly enhanced angiogenesis and provided protection against H(2)O(2)-induced apoptosis. Remarkably, Mbd2(-/-) mice were protected against hind-limb ischemia evidenced by the significant improvement in perfusion recovery, along with increased capillary and arteriole formation. Loss of MBD2 activated endothelial survival and proangiogenic signals downstream of vascular endothelial growth factor signaling characterized by an increase in endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor 2 expression, along with enhanced extracellular signal-regulated kinase 1/2 activation and BCL-2 expression. Mechanistic studies confirmed the methylation of CpG elements in the eNOS and vascular endothelial growth factor receptor 2 promoter. MBD2 binds to these methylated CpG elements and suppresses eNOS promoter activity. On ischemic insult, key endothelial genes such as eNOS and vascular endothelial growth factor receptor 2 undergo a DNA methylation turnover, and MBD2 interprets the changes of DNA methylation to suppress their expressions. Moreover, MBD2 modulation of eNOS expression is likely confined to endothelial cells because nonendothelial cells such as splenocytes fail to express eNOS after loss of MBD2. We provided direct evidence supporting that DNA methylation regulates endothelial function, which forms the molecular basis for understanding how environmental insults (epigenetic factor) affect the genome to modify disease susceptibility. Because MBD2 itself does not affect the methylation of DNA and is dispensable for normal physiology in mice, it could be a viable epigenetic target for modulating endothelial function in disease states.

Methyl-CpG binding protein MBD2 is implicated in methylation-mediated suppression of miR-373 in hilar cholangiocarcinoma

Aberrant expression of miRNAs is associated with particular cancers showing tissue- and clinical-feature-specificity patterns. Some miRNA genes harboring or being embedded in CpG islands undergo methylation mediated silencing. MBP, methyl CpG binding protein, suppresses transcription through binding to methylated CpG dinucleotides. Expression of miR-373 has been reported to be suppressed in malignant bile duct cell lines. Bioinformatic prediction reveals that the transcription start site (TSS) of miR-373 is implanted in a 402 bp canonical CpG island containing 26 CpG dinucleotides. In this study, we aim to determine the epigenetic regulation of miR-373 gene in hilar cholangiocarcinoma. Taqman microRNA assay shows that down-regulation of miR-373 is closely associated with poor cell differentiation, advanced clinical stage and shorter overall and disease-free survival in hilar cholangiocarcinomas. Methylation analysis shows that the promoter-associated CpG island is hypermethylated which is consistent with the inhibition of miR-373. Chromatin immunoprecipitation (ChIP) assay indicates that down-regulation of miR-373 results from the selective recruitment of MBD2 to methylated CpG islands. In contrast, MBD2 knock-down by use of a specific siRNA promoted the expression of miR-373. Reactivation of miR-373 by pharmacologic induction of 5-aza-CdR and trichostatin A (TSA) led to decreased enrichment of MBD2 at CpG island regions. Enhanced expression of exogenous MBD2 in stable QBC939 cells which stably express pGL4-m373-prom induces strengthened recruitment of MBD2. Our findings suggest that miR-373 is a methylation-mediated gene and the implication of MBD2 in methylation-mediated suppression of miR-373 plays an important role in tumourigenesis and development in hilar cholangiocarcinoma.

B1, a Novel Amonafide Analogue, Overcomes the Resistance Conferred by Bcl-2 in Human Promyelocytic Leukemia HL60 Cells

In the course of screening for novel anticancer compounds, B1 [N-(2-(dimethylamino)ethyl)-2-aminothiazonaphthalimide], a novel amonafide analogue, was generated as a new anticancer candidate. In the present study, B1 displayed stronger antitumor effects than amonafide in HL60 cells. We further examined whether B1 overcomes the resistance conferred by Bcl-2, as overcoming the resistance conferred by Bcl-2 represents an attractive therapeutic strategy against cancer. Our viability assay showed that B1 overcomes the resistance conferred by Bcl-2 in human promyelocytic leukemia HL60 cells. Various apoptosis assessment assays showed that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells by inducing apoptosis. Noticeably, we elucidated the marked downregulation of 14-3-3σ protein by B1, indicating that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells via 14-3-3σ. The analysis of chromatin immunoprecipitation assay indicated that MBD2 was associated with the methylated 14-3-3σ promoter-associated CpG island and thus interfered with transcriptional activity of the methylated promoter. Furthermore, knockdown of MBD2, using siRNA transfection, inhibited B1-induced apoptosis and overcame the resistance conferred by Bcl-2. Accordingly, these data showed the involvement of MBD2 in B1-induced apoptosis and overcoming the resistance conferred by Bcl-2, which suggested that MBD2 might guide the development of future anticancer agents.

Abstract 158: The landscape of DNA hypomethylation in liver cancer, its correlation with other cancers, and its potential role in cancer

Abstract DNA demethylation, although a common hallmark of cancer, has been relatively ignored. We used hepatic cellular carcinoma, which is one of the most common human cancers, as a model to delineate the landscape of hypomethylation in cancer, unravel the driving forces of hypomethylation and determine its potential role in cancer progression, metastasis and invasion. Using liver cancer cell lines as a model system, we tested whether the demethylation observed in liver cancer is driven by MBD2, a protein previously implicated in demethylation, which we found to be overexpressed in liver cancer. We used genome-wide promoter arrays and methylated DNA immunoprecipitation to map the sites in the genome that lost methyl marks in liver cancer patients. Affymetrix arrays were used for gene expression studies. The array data were validated by pyrosequencing and QPCR. For functional analyses, we used siRNA transfection, soft agar and invasion assays. We discovered that about 3700 transcript-encoding genes were demethylated in liver cancer samples compared to adjacent normal tissue. At least 350 of these 3700 genes were significantly induced in tumors. In the group of demethylated and induced genes, we identified a distinct subgroup of over 100 genes with CpG dense promoters that were highly methylated in normal liver, but demethylated in the tumors. Gene ontology analyses revealed that these genes are involved in cell growth, cell adhesion, signal transduction and invasion. Examination of the Ensemble methylome data suggests that these promoters are differentially methylated in a tissue specific manner but are always methylated in liver. All of the demethylated genes in liver cancer were found to be demethylated in other cancers suggesting that the demethylation of these genes is a basic requirement in cancer. The experiments in HepG2 and SkHep1 cells showed that knock-down of MBD2 with siRNA results in suppression of the majority of the hypomethylated genes and in a relevant decrease in cancer cell growth and invasion. Furthermore, the down-regulation of these genes was accompanied by an increase in promoter methylation and a decrease in MBD2 binding. It suggests that MBD2 is required for demethylation and up-regulation of these genes, which appear to be putative targets for MBD2. Our results demonstrated that DNA hypomethylation drives the coordinated expression of several gene circuitries involved in cancer growth, survival and metastasis. We showed that MBD2 is involved in driving this processes by affecting methylation and expression of important players in liver cancer growth and metastasis. Our studies established for the first time the rules governing hypomethylation in liver cancer and defined the potential functional role of hypomethylation. This study was supported by a grant from the MDEEI program of the government of Quebec and the National Cancer Institute of Canada to MS and MOST to ZGH 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 158.

Targeting a Methyl Cytosine-Binding Protein Complex to Augment Fetal/Embryonic Globin Expression.

Abstract 974The vertebrate β-type globin genes were among the first genes shown to be regulated, at least in part, by DNA methylation. The mechanism of transcriptional repression by DNA methylation is chiefly through binding of methyl cytosine binding domain (MBD) proteins and their associated co-repressor complexes. The chicken homolog to an MBD2 containing NuRD co-repressor complex (MeCPC) has previously been purified from primary erythroid cells and characterized as binding to the methylated ρ-globin promoter in erythroid cells of adult chickens in which the gene is silent [Kransdorf et al. Blood 2006; 108:2836-45]. Knockdown of MBD2 by siRNA in MEL cells stably transfected with a methylated ρ-globin gene construct leads to a greater than 10-fold increase in ρ-globin gene expression. Likewise, knockout of MBD2 results in a ∼20 fold upregulation of the human gamma globin gene in adult erythroid cells of βYAC transgenic mice [Rupon et al. PNAS 2006; 103:6617-22]. These observations suggest that disruption of the interaction of MBD2 with its co-repressor complex in adult erythropoiesis would increase fetal hemoglobin expression; a therapeutically beneficial effect for both sickle cell anemia and β-thalassemia. This possibility is further supported by the observation that DNA methylation inhibitors such as 5-azacitidine can increase the expression of γ-globin in patients. Based on these studies, we have pursued structural analysis of the interaction between MBD2 and other components from the MeCPC. We have shown that the individual coiled coil regions from MBD2 and a subunit of the NuRD complex, p66α, form a stable heterodimeric complex. Solving the structure of this coiled coil complex by NMR reveals that the interaction involves a combination of hydrophobic and ionic interactions typical of coiled coil complexes as well as a unique charge interaction involving a pair of highly conserved glutamates residues from p66α and arginine residues from MBD2. The key residues involved in binding are conserved across species, between p66α and p66β homologs, as well as between MBD2, MBD3, and the MBD3L1-L5 homologs. We have shown that the p66α coiled coil can stably bind to MBD3 in solution, indicating that similar tertiary interactions are involved in forming both MBD2 and MBD3 containing NuRD complexes. In order to explore this interaction as a potential therapeutic target, we hypothesized that over-expressing the p66α coiled coil region in tissue culture would disrupt the formation of a normal MeCPC and thereby block the function of MBD2. As predicted, expressing this region in both avian (MEL-ρ) and human (CID-βYAC) tissue culture models of globin gene regulation in adult erythroid cells induces embryonic and fetal β-type globin gene expression, respectively. Furthermore, knock-down of p66α induces fetal/embryonic globin gene expression to a similar degree as knock-down of MBD2. These studies suggest a model in which the p66α coiled peptide can bind MBD2 and block recruitment of native p66α to the NuRD complex, thereby acting in a dominant-negative manner to disrupt MBD2 function. We propose that a peptidomimetic of the p66α coiled coil region could be used therapeutically to augment fetal hemoglobin expression. Disclosures:No relevant conflicts of interest to declare.

β-Defensin-2 Promotes Resistance against Infection with P. aeruginosa

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine beta-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-gamma, MIP-2, IL-1beta, TNF-alpha, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-kappaB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-kappaB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

RNAi Targeting MBD2 Increases γ-Globin Expression in a CID-Dependent Human β-YAC Murine Fetal Bone Marrow Cell Line.

Increased fetal hemoglobin (HbF) can alleviate the symptoms and increase the life span of patients with sickle cell disease. The development of new methods to increase HbF in patients has been hampered by the lack of cell line models. Recently a new murine fetal bone marrow (FBM) cell line containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) whose growth is dependent on the chemical inducer of dimerization (CID) AP20187 (Ariad Pharmaceuticals) was described (Blau et al, J Biol Chem 280: 36642, 2005). The DNA methyltransferase (DNMTase) inhibitor 5-azacytidine increased γ-globin gene expression in this cell line suggesting that it is a good model system for studying the mechanism of DNA methylation in γ-globin gene silencing. To investigate the role of methylated DNA binding proteins in γ-globin silencing, we transduced this cell line with retrovirus vectors expressing shRNA targeting the methylated DNA binding proteins MBD2 and MBD3. Transduced pools were selected for puromycin resistance and the effect on expression of the target gene and γ-globin determined by real time PCR using the ΔΔCT method. Single cell clones were isolated by limited dilution platings for further analysis. No difference in γ-globin expression was observed between clones derived from the parental cell line and cells transduced with a nonsilencing control vector. MBD3 expression was decreased 70–85% but γ-globin expression was not affected in the selected pool or in 12 clones following transduction wth a vector targeting MBD3. In 2 selected pools transduced with an shRNA vector targeting MBD2, MBD2 expression was decreased 60 and 82% and γ-globin expression increased 3.7 fold and 4.76 fold. Mean expression of γ-globin was 3.83 fold higher in 14 clones that showed >90% decrease in MBD2 mRNA compared to 11 clonal isolates derived from cells transduced with the non-silencing control vector (p<.05). In three clones that showed the greatest induction of γ-globin, the fold change (mean +SD) of ε-, γ-, and β-globin expression was 3.98±1.75, 11.29±4.27 and 1.90±1.34, respectively, demonstrating that MBD2 knockdown increased both ε- and γ-globin expression. The level of MBD2 protein in these clones and in two additional clones that showed >90% decrease in MBD2 mRNA but no induction of γ-globin expression were compared by Western blot analysis. MBD2 was decreased >90% in all clones compared to the parental line. Decreased MBD2 levels were therefore not associated with increased γ-globin expression in all clones. We then compared shRNA-mediated MBD2 knockdown with the DNMTase inhibitor decitabine for effects on ε- and γ-globin expression. Expression of ε-globin was increased 76.0±6.1 fold, γ-globin 131±10.2 fold, and β-globin 3.6±0.38 fold following treatment with decitabine (1 × 10−6 M; 48 hours; n=3). We conclude that decreased expression of MBD2 following transduction of the CID-dependent mouse FBM βYAC cell line with an shRNA vector targeting MBD2 is associated with increased γ-globin expression, confirming previous results of analysis of βYAC-MBD2 knockout mice (Rupon et al, PNAS 103: 6617, 2006). However, the increase in γ-globin expression was approximately 10 fold greater following decitabine treatment than was achieved with shRNA-mediated MBD2 knockdown.

Functional diversification of the nematode mbd2/3 gene between Pristionchus pacificus and Caenorhabditis elegans.

BackgroundSeveral members of the Methyl-Binding Domain protein family link DNA methylation with chromatin remodeling complexes in vertebrates. Amongst the four classes of MBD proteins, MBD2/3 is the most highly conserved and widespread in metazoans. We have previously reported that an mbd2/3 like gene (mbd-2) is encoded in the genomes of the nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae. RNAi knock-down of mbd-2 in the two Caenorhabditis species results in varying percentages of lethality.ResultsHere, we report that a general feature of nematode MBD2/3 proteins seems to be the lack of a bona fide methyl-binding domain. We isolated a null allele of mbd-2 in P. pacificus and show that Ppa-mbd-2 mutants are viable, fertile and display a fully penetrant egg laying defect. This egg laying defect is partially rescued by treatment with acetylcholine or nicotine suggesting a specific function of this protein in vulval neurons. Using Yeast-two-hybrid screens, Ppa-MBD-2 was found to associate with microtubule interacting and vesicle transfer proteins.ConclusionThese results imply that MBD2/3 proteins in nematodes are more variable than their relatives in insects and vertebrates both in structure and function. Moreover, nematode MBD2/3 proteins assume functions independent of DNA methylation ranging from the indispensable to the non-essential.

MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells

AbstractThe chicken embryonic β-type globin gene, ρ, is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated ρ-globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zinc-finger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pull-down assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive ρ-globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active βA-globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent ρ-globin gene in vivo. Knock-down of MBD2 resulted in up-regulation of a methylated ρ-gene construct in mouse erythroleukemic (MEL)-ρ cells. These results represent the first purification of a MeCP1-like complex from a primary cell source and provide support for a role for MBD2 in developmental gene regulation.

Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus

The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (∼90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression.