Methyl Binding Domain Protein Research Articles

DNA hypomethylation in wood frog liver under anoxia and dehydration stresses

Wood frogs are freeze-tolerant vertebrates that can endure weeks to months frozen during the winter without breathing and with as much as 65% of total body water frozen as extracellular ice. Underlying tolerances of anoxia and of cellular dehydration support whole body freezing. One pro-survival mechanism employed by these frogs is epigenetic modifications via DNA hypomethylation processes facilitating transcriptional repression or activation. These processes involve proteins such as DNA Methyltransferases (DNMTs), Methyl Binding Domain proteins (MBDs), Ten-Eleven Translocases (TETs), and Thymine Deglycosylase (TDG). The present study evaluates the responses of these proteins to dehydration and anoxia stresses in wood frog liver. DNMT relative protein expression was reduced in liver, but nuclear DNMT activity did not change significantly under anoxia stress. By contrast, liver DNMTs and nuclear DNMT activity were upregulated under dehydration stress. These stress-specific differences were speculated to arise from Post-Translational Modifications (PTMs). DNMT3A and DNMT3B showed increased relative protein expression during recovery from dehydration and anoxia. Further, MBD1 was elevated during both conditions suggesting transcriptional repression. TET proteins showed varying responses to anoxia likely due to the absence of oxygen, a main substrate required by TETs. Similarly, TDG, an enzyme that corrects DNA damage, was downregulated under anoxia potentially due to lower levels of reactive oxygen species that damage DNA, but levels returned to normal during reperfusion of oxygen. Our results indicate differential stress-specific responses that indicate the need for more research in the DNA hypomethylation mechanisms employed by the wood frog during stress.

Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer.

Dysregulated DNA methylation in cancer is critical in the transcription machinery associated with cancer progression. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, but no treatment targeting TNBC biomarkers has yet been developed. To identify specific DNA methylation patterns in TNBC, methyl-binding domain protein 2 (MBD) sequencing data were compared in TNBC and the three other major breast cancer subtypes. Integrated analysis of DNA methylation and gene expression identified a gene set showing a correlation between DNA methylation and gene expression. ATPase Na+/K+-transporting subunit alpha 1 (ATP1A1) was found to be specifically hypomethylated in the coding sequence (CDS) region and to show increased expression in TNBC. The Cancer Genome Atlas (TCGA) database also showed that hypomethylation and high expression of ATP1A1 were strongly associated with poor survival in patients with TNBC. Furthermore, ATP1A1 knockdown significantly reduced the viability and tumor-sphere formation of TNBC cells. These results suggest that the hypomethylation and overexpression of ATP1A1 could be a prognostic marker in TNBC and that the manipulation of ATP1A1 expression could be a therapeutic target in this disease.

CHD4 Promotes Tumor Formation, Survival, and Resistance to Genotoxic Therapeutics in TP53 Mutant T-Cell Acute Lymphoblastic Leukemia Cells through the NuRD Chromatin Remodeling Complex

T-Cell acute leukemia(T-ALL) though generally responsive to current chemotherapy remains incurable in over 50% of adult patients. Importantly, the survival for patients with TP53 mutant T-ALL disease is less than 10%. We have previously shown the role of the Methyl Binding Domain protein 2-Nucleosome Remodeling and Deacetylase (MBD2-NURD) chromatin remodeling complex in triple negative breast cancer cell growth and the role of the NuRD component, Chromatin-helicase-binding protein 4(CHD4), in tumor colony formation and genotoxic resistance in acute myeloid leukemia. In the present study, we have examined the role of specific NuRD complexes and CHD4 in T-cell acute leukemia cells with mutant TP53. The prototypical NuRD complex possesses both a histone deacetylase (HDCC) sub-complex and an ATPase chromatin remodeling sub-complex containing CHD4 linked to MBD2 or MBD3 of the HDCC. CHD4 is linked to the MBD2/3 anchors of the NuRD complex, via GATAD2A or GATAD2B. Depletion of CHD4 was found to increase the rate of apoptosis in several TP53 mutant T-ALL cell lines including Jurkat, MOLT4, RPMI and CCRF by up to 5 to 10-fold. Likewise, depletion of both MBD2 and MBD3 also increased apoptosis and sensitization to the standard genotoxic chemotherapeutic agent, cytosine arabinoside (Ara-C) but had only minimal effects in normal human CD34+ hematopoietic progenitor cells. Depletion of GATAD2B and GATAD2A by shRNA also resulted in a significantly increased survival of 60 days compared to 20 days in Scramble shRNA controls, in a xenogeneic mouse model of T-ALL. Depletion of CHD4 resulted in a profound (10 to 100-fold) decrease in colony formation of the TP53 mutant T-ALL cell lines in methylcellulose. RNA seq analyses identified the apoptosis, ferroptosis and mitophagy pathways as potential mechanisms for the striking effect of NURD disruption on colony formation. In summary, CHD4, through the NuRD complex, promotes tumor cell formation, survival and therapeutic resistance of TP53 mutant T-ALL cells. Current efforts are aimed at validating these findings in primary T-ALL cells and structure-function studies to advance development of tool compounds for disrupting the NuRD complex, with the long-term goal of developing improved small molecule therapeutics for treatment resistant acute leukemia.

Streamlined construction of robust heteroprotein complexes by self-induced in-cell disulfide pairing

Biomolecules and their functional subdomains are essential building blocks in the creation of multifunctional nanocomplexes. Methyl-binding domain protein 2 (MBD2) and p66α stand out as small α-helical motifs with an ability to self-assemble into a heterodimeric coiled-coil, making them promising building units. Yet, their practical use is hindered by rapid dissociation upon dilution. In this study, novel fusion tags, MBD2 and p66α variants, were developed to covalently link during co-expression in E. coli SHuffle. Through strategic placement of cysteine at each α-helix's terminus, intracellular crosslinking occurred with high specificity and yield, facilitated by preserved α-helical interactions. This instant disulfide bonding in the oxidative cytoplasm of E. coli SHuffle efficiently overcame the need for inefficient in vitro oxidation and protein extraction prone to creating non-specific adducts and suboptimal bioprocesses. In contrast to their wild-type counterparts, the GFP-mCherry protein complex cross-linked by the fusion tags maintained the heterodimeric state even under extensive dilution. The fusion tags, when combined with the E. coli SHuffle system, allowed for the streamlined co-expression of a stable protein complex through self-induced intracellular cysteine coupling. The approach demonstrated herein holds great promise for producing multifunctional and robust heteroprotein complexes.

Selective Enrichment of Hypermethylated DNA by a Multivalent Binding Platform

AbstractThe preselection of hypermethylated DNA (hmDNA) from liquid biopsy samples is key to enable early‐stage cancer diagnostics. Due to limited selectivity of the existing preselection approaches, however, wide integration in the clinic is currently prohibited. Here, it is argued that an affinity method on a surface, such as used in affinity chromatography, can be significantly improved by employing the principles of multivalency and superselectivity. In the here proposed method, a methyl binding domain (MBD) protein immobilized at a surface is used as a receptor for (hyper)methylated DNA. By the organization of multiple MBDs on a surface, a multivalent binding platform is achieved. The MBD surface receptor density on that platform is key to increase the enrichment selectivity of hmDNA as a single MBD protein binds both methylated and non‐methylated DNA with a small difference in affinity. When the receptor density is varied, multivalent analyte binding typically responds in a non‐linear fashion, which phenomenon is called superselectivity. By careful tuning of the MBD density, it is envisaged that the selectivity for methylated over non‐methylated DNA can be optimized. Strong applicability is foreseen in a medical setting by implementing such an enrichment step in an analytical process or a lab‐on‐a‐chip device.

Optimization of the base editor BE4max in chicken somatic cells

Advanced animal reproductive and breeding biotechnology has made it possible to alter traits or create new genetic resources by the direct knock-in or knock-out of target genes. Base editing technology can achieve single-base mutations without double-stranded DNA breaks, and is a promising tool for use in the genetic modification and breeding of livestock. However, the application of base editors (BEs) in chicken has not been optimized. We evaluated the efficacy of BE4max in chicken somatic cells (DF-1). The key element of BE4max, cytosine deaminase (APOBEC), was optimized for chicken. The base editing efficiency of the optimized chBE4max editor, compared with the original BE4max editor, was improved by 10.4% ± 4.6. By inhibiting the expression of the uracil DNA glycosylase-related gene methyl binding domain protein 4 (MBD4) by siRNA in chicken DF-1 cells, the editing efficiency was enhanced by 4.43% ± 1.4 compared to the control. These results suggest that this editor may have applications in poultry breeding studies.

IDH1 mutant glioma is preferentially sensitive to the HDAC inhibitor panobinostat

IntroductionA large subset of diffusely infiltrative gliomas contains a gain-of-function mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2mut) which produces 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases, thereby inducing widespread DNA and histone methylation. Because histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain proteins, IDH1/2mut gliomas may be more dependent on HDAC activity, and therefore may be more sensitive to HDAC inhibitors.MethodsSix cultured patient-derived glioma cell lines, IDH1wt (n = 3) and IDH1mut (n = 3), were treated with an FDA-approved HDAC inhibitor, panobinostat. Cellular cytotoxicity and proliferation assays were conducted by flow cytometry. Histone modifications and cell signaling pathways were assessed using immunoblot and/or ELISA.ResultsIDH1mut gliomas exhibited marked upregulation of genes associated with the HDAC activity. Glioma cell cultures bearing IDH1mut were significantly more sensitive to the cytotoxic and antiproliferative effects of panobinostat, compared to IDH1wt glioma cells. Panobinostat caused a greater increase in acetylation of the histone residues H3K14, H3K18, and H3K27 in IDH1mut glioma cells. Another HDAC inhibitor, valproic acid, was also more effective against IDH1mut glioma cells.ConclusionThese data suggest that IDH1mut gliomas may be preferentially sensitive to HDAC inhibitors. Further, IDH1mut glioma cultures showed enhanced accumulation of acetylated histone residues in response to panobinostat treatment, suggesting a direct epigenetic mechanism for this sensitivity. This provides a rationale for further exploration of HDAC inhibitors against IDH1mut gliomas.

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with β-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat β-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with β-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34+ cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with β-thalassaemia.

Abstract 1917: The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma

Abstract Glioblastoma (GBM) is an incurable form of brain cancer with a median survival of ~15 months. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP) represents a significant clinical discovery as these patients have an enhanced survival, with a median survival of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2) which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases. This mutation occurs early in gliomagenesis and further results in aberrant DNA methylation and widespread transcriptional repression. Histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain (MBD) proteins, so we hypothesized that IDH mutant GBM exhibits enhanced reliance on HDAC activity, which is functionally significant to their cell proliferation and survival. Using a panel of 6 patient-derived cell lines grown in defined growth media, we show that G-CIMP is significantly more sensitive in vitro to the clinically approved pan histone deacetylase (HDAC) inhibitor panobinostat, with IDH mutant GBM cells exhibiting ~4 and ~10 fold lower IC/EC50 values in flow cytometric cell viability and apoptosis assays compared to IDH wild type (WT) GBM cells, respectively. For IDH mutant GBM, the average IC50 value was 13.5 nM for cell viability assays and the average EC50 value was 4.8 nM for apoptosis assays. Induction of cleaved caspase 3, a marker of apoptosis, was observed only in IDH mutant GBM cells when exposed to 10 nM panobinostat over 5 days. Analysis of proliferation via BrdU incorporation assays show that treatment with 10 nM panobinastat for 48 hours results in a 90% reduction in proliferation of IDH mutant GBM compared to only 25% reduction in IDH WT GBM cells. Molecular analysis via western blot shows that various acetylated chromatin marks, i.e. H3K14/18/27Ac, are preferentially upregulated in our IDH mutant GBM cell lines when exposed to 10 nM panobinostat for 48 hours. This was also supported by ELISA data showing that IDH mutant GBM cells have ~2 fold increase in histone H3 acetylation compared to IDH WT cells in response to 10 nM and 50 nM panobinostat. These data led us to perform a HDAC activity assay with nuclear extracts from our IDH WT and mutant GBM cells, but this assay showed no significant difference in basal HDAC activity. Overall, our results support a hypothesis where IDH mutant GBMs are more sensitive to panobinostat via preferential uptake of the drug. These data ultimately suggest that G-CIMP tumor cells display a profound sensitivity to the cytotoxic and antiproliferative effects mediated by exposure to the HDAC inhibitor panobinostat. These studies provide a strong foundation for future preclinical work evaluating the use of HDAC inhibitors as a personalized cancer therapy to treat G-CIMP. Citation Format: Thomas K. Sears, Kevin D. Woolard. The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma [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 1917.

Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation.

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector.

An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out atpH7.4in the presence of 50μM thionine and 0.5mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489μA·μM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use ofglutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Epigenetic biomarker screening by FLIM-FRET for combination therapy in ER+ breast cancer

Hormone-dependent gene expression involves dynamic and orchestrated regulation of epigenome leading to a cancerous state. Estrogen receptor (ER)-positive breast cancer rely on chromatin remodeling and association with epigenetic factors in inducing ER-dependent oncogenesis and thus cell over-proliferation. The mechanistic differences between epigenetic regulation and hormone signaling provide an avenue for combination therapy of ER-positive breast cancer. We hypothesized that epigenetic biomarkers within single nucleosome proximity of ER-dependent genes could serve as potential therapeutic targets. We described here a Fluorescence lifetime imaging-based Förster resonance energy transfer (FLIM-FRET) methodology for biomarker screening that could facilitate combination therapy based on our study. We screened 11 epigenetic-related markers which include oxidative forms of DNA methylation, histone modifications, and methyl-binding domain proteins. Among them, we identified H4K12acetylation (H4K12ac) and H3K27 acetylation (H3K27ac) as potential epigenetic therapeutic targets. When histone acetyltransferase inhibitor targeting H4K12ac and H3K27ac was combined with tamoxifen, an enhanced therapeutic outcome was observed against ER-positive breast cancer both in vitro and in vivo. Together, we demonstrate a single molecule approach as an effective screening tool for devising targeted epigenetic therapy.

Abstract C47: Obesity enhances tumor colonization and promotes expression of the spliced variant MBD2_v2 and splicing factor SRSF2 in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is a molecular subtype that accounts for approximately 15-20% of invasive breast cancer diagnoses in the United States. TNBC occurs more prevalently in premenopausal African American women (AAW), and obesity coupled with chronic inflammation is a risk factor for both incidence and recurrence. TNBC is lacking therapeutic options and persons diagnosed with TNBC have the lowest 5-year survival rates among all breast cancer patients, with AAW disproportionally bearing this burden. In order to improve TNBC patient outcomes, etiologic factors driving disparities in disease progression need to be better understood. We previously showed in TNBC cell cultures that intrinsic oncogenic redox signaling promotes self-renewal capacity of cancer stem-like cells (CSC) by maintaining expression of an epigenetic reader protein and mRNA spliced variant, methyl binding domain protein 2 variant 2 (MBD2_v2). We hypothesized that obesity-induced chronic inflammation in vivo could promote a similar cancer phenotype. Methods: To study this potential phenomenon, we developed and characterized a diet-induced obesity mouse tumor xenograft (MTX) model of TNBC in C57BL/6J Rag1 (-/-) mice, a strain that maintains a functional innate immune system, to recapitulate the proinflammatory environment induced by increased adiposity. TNBC cell lines MDA-MB-468 and MDA-MB-231 were used to generate ectopic tumor xenografts in this model. Tumor colonization and growth were measured over 150 days. RT-PCR and microarray based assays were used to detect transcriptional changes in TNBC MTXs. Results: At 5 weeks post-diet consumption, mice fed a high-fat diet displayed a 5-10% increase in body weight and increased blood glucose concentration. By the end of the study high-fat fed mice displayed a 33-43% increase in body weight and appeared to have greater central adiposity post-mortem. In overweight mice relative to healthy weight controls, TNBC cell line tumor colonization rate and MBD2_v2 expression were significantly higher. Furthermore, staining for CSC marker EpCam was greater in overweight versus control MTXs, indicating that CSC self-renewal capacity could have been enhanced. Upon global gene expression analysis of overweight MTXs, we observed a significant upregulation of stem-related factors based on fold-change expression, as well as functional enrichment for RNA splicing, including SRSF2, a splicing factor that has been shown to drive MBD2_v2 expression in pluripotent stem cells. Knockdown of SRSF2 led to decreased expression of MBD2_v2 in MDA-MB-468 cells, and decreased CSC self-renewal capacity in vitro. Conclusions: Altogether these data support MBD2_v2 regulation by redox signaling, which may be driven by a proinflammatory environment in the presence of increased adiposity. Future studies will be conducted to confirm MBD2_v2's importance in obesity-related tumor colonization in in vivo overexpression and knockdown studies, and to determine its transcriptional regulatory functions in TNBC. Citation Format: Emily A. Girsch, Gregory Dyson, Cristina Mitrea, Bin Bao, Lisa Polin, Aliccia Bollig-Fischer. Obesity enhances tumor colonization and promotes expression of the spliced variant MBD2_v2 and splicing factor SRSF2 in triple-negative breast cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr C47.

Near-Infrared Plasmonic Assemblies of Gold Nanoparticles with Multimodal Function for Targeted Cancer Theragnosis

Here we report a novel assembly structure of near-infrared plasmonic gold nanoparticles (AuNPs), possessing both photoacoustic (PA) and photothermal (PT) properties. The template for the plasmonic AuNP assembly is a bioconjugate between short double-strand DNA (sh-dsDNA) and human methyl binding domain protein 1 (MBD1). MBD1 binds to methylated cytosine-guanine dinucleotides (mCGs) within the sequence of sh-dsDNA. Hexahistidine peptides on the engineered MBD1 function as a nucleation site for AuNP synthesis, allowing the construction of hybrid conjugates, sh-dsDNA-MBD1-AuNPs (named DMAs). By varying the length of sh-dsDNA backbone and the spacer between two adjacent mCGs, we synthesized three different DMAs (DMA_5mCG, DMA_9mCG, and DMA_21mCG), among which DMA_21mCG exhibited a comparable photothermal and surprisingly a higher photoacoustic signals, compared to a plasmonic gold nanorod. Further, epidermal growth factor receptor I (EGFR)-binding peptides are genetically attached to the MBD1 of DMA_21mCG, enabling its efficient endocytosis into EGFR-overexpressing cancer cells. Notably, the denaturation of MBD1 disassembled the DMA and accordingly released the individual small AuNPs (<5 nm) that can be easily cleared from the body through renal excretion without causing accumulation/toxicity problems. This DMA-based novel approach offers a promising platform for targeted cancer theragnosis based on simultaneous PA imaging and PT therapy.

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer.

Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as a diagnostic and prognostic biological marker. Here, we describe a protocol for a versatile DNA methylation enrichment method, the Methylated CpG Island Recovery Assay (MIRA), which enables analysis of the DNA methylation status in individual genes or across the entire genome. MIRA is based on the ability of the methyl-binding domain (MBD) proteins, the MBD2B/MBD3L1 complex, to specifically bind methylated CpG dinucleotides. This easy-to-perform method can be used to analyze the methylome of bladder cancer or urothelial cells shed in the urine to elucidate the evolution of bladder carcinogenesis and/or identify epigenetic signatures of chemicals known to cause this malignancy.

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients.

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

Novel Epigenetic Controlling of Hypoxia Pathway Related to Overexpression and Promoter Hypomethylation of TET1 and TET2 in RPE Cells.

CpG methylation of DNA takes part in a specific epigenetic memory that plays crucial roles in the differentiation and abnormality of the cells. The methylation pattern aberration of genomes is affected in three ways, namely DNA methyltransferase (DNMT), ten-eleven translocation (TET), and methyl-binding domain (MBD) proteins. Of these, TET enzymes have recently been demonstrated to be master modifier enzymes in the DNA methylation process. Additionally, recent studies emphasize that not only epigenetic phenomena play a role in controlling hypoxia pathway, but the hypoxia condition also triggers hypomethylation of genomes that may help with the expression of hypoxia pathway genes. In this study, we suggested that TET1 and TET2 could play a role in the demethylation of genomes under chemical hypoxia conditions. Herein, the evaluating methylation status and mRNA expression of mentioned genes were utilized through real-time PCR and methylation-specific PCR (MSP), respectively. Our results showed that TET1 and TET2 genes were overexpressed (P < 0.05) under chemical hypoxia conditions in Retinal Pigment Epithelial (RPE) cells, whereas the promoter methylation status of them were hypomethylated in the same condition. Therefore, chemical hypoxia not only causes overexpression of TET1 and TET2 but also could gradually do promoter demethylation of same genes. This is the first study to show the relationship between epigenetics and the expression of mentioned genes related to hypoxia pathways. Furthermore, it seems that these associations in RPE cells are subjected to chemical hypoxia as a mechanism that could play a crucial role in methylation pattern changes of hypoxia-related diseases such as cancer and ischemia. J. Cell. Biochem. 118: 3193-3204, 2017. © 2017 Wiley Periodicals, Inc.

An endoparasitoid wasp influences host DNA methylation

Parasitism by endoparasitoid wasps changes the expression of various host genes, and alters host immune and developmental processes. However, it is not clearly understood how parasitism changes host gene expression in a whole genome scale. This study focused on an epigenetic control of Cotesia plutellae, an endoparasitoid wasp, against its host, Plutella xylostella. Two DNA methyltransferases (DNMT-1 and DNMT-2) are encoded in the genome of P. xylostella. In addition, methyl-binding domain proteins (MBDs) and DNA demethylation factor, ten-eleven translation protein (TET) are encoded. DNA methylation of P. xylostella genomic DNA was confirmed by restriction digestion with Gla I specific to 5-methylcytosine. DNA methylation intensity in parasitized (P) larvae was decreased compared to that in nonparasitized (NP) larvae, especially at late parasitic stage, at which expression levels of both DNMT-1 and DNMT-2 were also decreased. DNA demethylation of P. xylostella was confirmed in both NP and P larvae by restriction digestion with PvuRts1I recognizing 5-hydroxymethyl cytosine. Parasitism also suppressed expression levels of TET and MBDs. Treatment of 5-aza-2′-deoxycytidine (AZA) reduced DNA methylation intensity of NP larvae, causing suppression of hemocyte-spreading behavior and delay of immature development. RNA interference of DNMT-1 or DNMT-2 mimicked the adverse effects of AZA.

Methyl-CpG/MBD2 Interaction Requires Minimum Separation and Exhibits Minimal Sequence Specificity

Determining the pattern of methylation at CpG dinucleotides in a cell remains an essential component of epigenetic profiling. The correlations among methylation, gene expression, and accompanying disease have just begun to be explored. Many experiments for sensing methylation use a relatively inexpensive, high-throughput approach with a methyl-binding domain (MBD) protein that preferentially binds to methylated CpGs. Here, we characterize the cooperativity and sequence specificity of MBD2-DNA binding in a pulldown experiment revealing three potential biases in such experiments. The first is caused by steric clashes between two MBD2 proteins at mCpGs separated by 2 bp or less, which suggests that simultaneous binding at these sites is inhibited. This is confirmed by comparing input versus pulldown high-throughput sequencing data on M.SssI-treated samples, from which we also find that pulldown efficiency sharply increases for DNA fragments with four or more mCpGs. Analysis of these two data sets was again employed to investigate MBD2’s sequence preferences surrounding a methylated CpG (mCpG). In comparing the distributions of bases at positions with respect to an mCpG, statistically significant preferences for certain bases were found, although the corresponding biases in pulldown efficiency were all <5%. While this suggests that mCpG sequence context can mostly be ignored in MBD2 binding, the statistical certainty exhibited by our high-throughput approach bodes well for future applications.

Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana.

Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin.