MBD2 Research Articles

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

Novel insights into vancomycin-loaded calcium sulfate and negative pressure wound therapy in preventing infections in open fractures

BackgroundOpen fractures are challenging due to susceptibility to Staphylococcus aureus infections. This study examines the impact of Vancomycin-Loaded Calcium Sulfate (VLCS) and negative pressure wound therapy (NPWT) on macrophage behavior in enhancing healing and infection resistance. Both VLCS and NPWT were evaluated individually and in combination to determine their effects on macrophage polarization and infection resistance in open fractures.MethodsThrough single-cell RNA sequencing, genomic expressions in macrophages from open fracture patients treated with VLCS and NPWT were compared to a control group. The analysis focused on MBD2 gene changes related to macrophage polarization.ResultsRemarkable modifications in MBD2 expression in the treatment group indicate a shift towards M2 macrophage polarization. Additionally, the combined treatment group exhibited greater improvements in infection resistance and healing compared to the individual treatments. This shift suggests a healing-promoting atmosphere with improved infection resilience.ConclusionsVLCS and NPWT demonstrate the ability to alter macrophage behavior toward M2 polarization, which is crucial for infection prevention in open fractures. The synergistic effect of their combined use shows even greater promise in enhancing outcomes in orthopedic trauma care.

Association between the copy number variations of Methyl-CpG binding domain family and schizophrenia

Schizophrenia is recognized as one of the most severe psychiatric disorders, with its pathogenesis likely involving genetic, epigenetic, developmental, and environmental factors. Members of the Methyl-CpG Binding Domain (MBD) Family play a crucial role in the regulation of genomic DNA methylation, and studies have implicated the association between MBD family and neurodevelopmental disorders. Copy number variations (CNVs) are a significant genetic basis for human genomic variation, also playing a critical role in the genetic processes of schizophrenia. Therefore, we aimed to evaluate the susceptibility of MBD family CNVs to schizophrenia by exploring and validating them in two separate populations using CNVplex™ and qPCR methods, and to explore the relationship between MBD family CNVs and clinical phenotypes in the overall population using chi-square tests and Fisher’s exact tests. Results suggest that an increase in MBD1 gene copy number and a deficiency in MBD2 gene copy number may be associated with the risk of schizophrenia. The deficiency in MBD2 gene copy number may increase the risk of delusion of reference and delusion of persecutory in the overall sample, as well as in males. This research provides preliminary evidence supporting the association between MBD family CNVs and schizophrenia, highlighting the potential role of the MBD family in the pathogenesis of schizophrenia.

LGG-08. DIFFUSE ASTROCYTOMA IN A PATIENT WITH POSSIBLE MBD4-ASSOCIATED NEOPLASIA SYNDROME (MANS)- EXPANDING THE SPECTRUM OF MANS-ASSOCIATED NEOPLASM

Abstract BACKGROUND MBD4-associated neoplasia syndrome (MANS) is a recently identified genetic syndrome characterized by the development of colorectal polyps, acute myeloid leukemia, and uveal melanoma, with an elevated mutation burden. MANS is attributed to bi-allelic germline variations in the MBD4 gene. METHOD We present a case of a pediatric patient with diffuse astrocytoma WHO-grade II and bi-allelic germline variations in the MBD4 gene. CASE REPORT A previously healthy 10-year-old female with a significant family history of high-grade IDH wild type-glioma in the deceased older brother presented with vague symptoms and was found to have abnormal brain MRI with multiple foci of subcortical FLAIR/T2 hyperintensity involving the right frontotemporal lobe, left frontal lobe, and left thalamus. She had intact neurologic exam findings and was followed with serial brain MRIs. Over the next 2 years, she continued to be asymptomatic, but her MRI showed an increase in the size of her brain lesions. Following a stereotactic biopsy of the right temporal lesion, a diagnosis of diffuse astrocytoma (WHO-grade II) was confirmed. Molecular analysis showed point mutations in IDH1, TP53, and PTEN genes, along with LOH at 17p (including TP53 gene), and outline gene expressions for NTRK2, OLIG2, GRM3, FGF1, SOX8, MBP, MGMT genes. Given the low grade of the tumor and the absence of tumor-related symptoms, a decision was made to continue monitoring with serial MRIs. Whole exome sequencing identified germline bi-allelic missense variations in the MBD4 gene, specifically C.1405A>G and C.1573A>C. Both parents were found to be heterozygous for one of these mutations. CONCLUSION The significance of MBD4 mutation remains unknown in our patient, but bi-allelic variations coupled with the presence of a brain tumor and positive family history raise concerns about MANS in our patient. Additional studies are needed to elucidate the potential association between brain tumors and MANS.

Methyl CpG binding protein MBD2 has a regulatory role on the BRCA1 gene expression and its modulation by resveratrol in ER+, PR+ & triple-negative breast cancer cells

BackgroundResveratrol has demonstrated its ability to regulate BRCA1 gene expression in breast cancer cells, and previous studies have established the binding of MBD proteins to BRCA1 gene promoter regions. However, the molecular mechanism underlying these interactions remains to be elucidated. The aimed to evaluate the impact of MBD proteins on the regulation of BRCA1, BRCA2, and p16 genes and their consequential effects on breast cancer cells.MethodsEfficacy of resveratrol was assessed using the MTT assay. Binding interactions were investigated through EMSA, ChIP, & MeIP assay. Expression analyses of MBD genes and proteins were conducted using qRT-PCR and western blotting, respectively. Functional assays, including clonogenic, migratory, and sphere formation assays were used to assess cancer cells’ colony-forming, metastatic, and tumor-forming abilities. The cytotoxicity of resveratrol on cancer cells was also tested using an apoptosis assay.ResultsThe study determined an IC50 of 30µM for resveratrol. MBD proteins were found to bind to the BRCA1 gene promoter. Resveratrol exhibited regulatory effects on MBD gene expression, subsequently impacting BRCA1 gene expression and protein levels. Higher concentrations of resveratrol resulted in reduced colony and sphere formation, decreases migration of cancer cells, and an increases number of apoptotic cells in breast cancer cells.ImpactIdentification of MBD2-BRCA1 axis indicates their significant role in the induction of apoptosis and reduction of metastasis and proliferation in breast cancer cells. Further therapy can be designed to target these MBD proteins and resveratrol could be used along with other anticancer drugs to target breast cancer.ConclusionsIn conclusion MBD2 protein interact to the BRCA1 gene promoter, and resveratrol modulates MBD2 gene expression, which in turn regulates BRCA1 gene expression, and inhibits cell proliferation, migration, and induces apoptosis in ER+, PR+ & Triple negative breast cancer cells.

Methyl-CpG binding domain protein 2 (Mbd2) drives breast cancer progression through the modulation of epithelial-to-mesenchymal transition

Methyl-CpG-binding domain protein 2 (Mbd2), a reader of DNA methylation, has been implicated in different types of malignancies, including breast cancer. However, the exact role of Mbd2 in various stages of breast cancer growth and progression in vivo has not been determined. To test whether Mbd2 plays a causal role in mammary tumor growth and metastasis, we performed genetic knockout (KO) of Mbd2 in MMTV-PyMT transgenic mice and compared mammary tumor progression kinetics between the wild-type (PyMT-Mbd2+/+) and KO (PyMT-Mbd2−/−) groups. Our results demonstrated that deletion of Mbd2 in PyMT mice impedes primary tumor growth and lung metastasis at the experimental endpoint (postnatal week 20). Transcriptomic and proteomic analyses of primary tumors revealed that Mbd2 deletion abrogates the expression of several key determinants involved in epithelial-to-mesenchymal transition, such as neural cadherin (N-cadherin) and osteopontin. Importantly, loss of the Mbd2 gene impairs the activation of the PI3K/AKT pathway, which is required for PyMT-mediated oncogenic transformation, growth, and survival of breast tumor cells. Taken together, the results of this study provide a rationale for further development of epigenetic therapies targeting Mbd2 to inhibit the progression of breast cancer.

Abstract 4415: Utilization of MBD-seq to elucidate differentially methylated regions in cfDNA across healthy and cancer patients

Abstract 5-Methylcytosine (5-mC) is the most abundant epigenetic DNA modification in eukaryotes. The presence of 5-mC alters gene expression and genome metabolism, and its deregulation is associated with many human diseases. Cell-free DNA (cfDNA) released from apoptotic cells has recently gained attention, as mutations or epimutations (e.g. 5-mC variations) detected in cfDNA were shown to have high diagnostic potential to assess the presence, stage and outcome of several cancers. However, 5-mC detection in cfDNA remains a challenge as input material is often very scarce. MBD-Seq is a method that leverages the ability of Methyl-CpG-binding domain protein 2 (MBD2) to capture and detect highly methylated regions of DNA genome-wide. We evaluated MBD-Seq for the detection of aberrant methylation patterns in cfDNA from healthy and diseased patients. We found MBD-seq on cfDNA to be specific (over 100-fold mC:C detection ratio) and sensitive (5 ng of input cfDNA). Applying MBD-seq to samples from healthy vs colorectal and breast cancer patients showed striking differences in methylation of key tumor-associated genes. Hence, MBD-Seq can elucidate aberrant methylation patterns from cfDNA, highlighting its potential as a diagnostics tool for the detection of cancer. Citation Format: Camille Michelon, Brian Egan, Benjamin Delatte. Utilization of MBD-seq to elucidate differentially methylated regions in cfDNA across healthy and cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4415.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status

BackgroundAnalysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. ResultsMBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. Significance and noveltyReal-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

Comparison of hypoxia- and hyperoxia-induced alteration of epigene expression pattern in lungs of Pleurodeles waltl and Mus musculus.

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.

Screening and preliminary analysis of differentially expressed genes in quail embryos for feather colour based on RNA-Sequencing

The purpose of this study was to use RNA-Sequencing (RNA-Seq) to screen differentially expressed genes (DEGs) and analyse transcriptomic data from embryonic skin tissues of maroon-feather and white-feather quails. The transcriptome of embryonic skin tissues from quails was sequenced using the Illumina HiSeqTM 2000 sequencing platform. A total of 2 512 DEGs were found. Of these, 550 DEGs were up-regulated and 1 562 DEGs were down-regulated in the skin tissues of white-feather quail embryos. Five hundred and ninety-seven DEGs were enriched and annotated into 50 entries in Gene Ontology (GO) database. The total number of DEGs annotated in each entry in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was 341, enriched in 230 pathways, of which the tight junction pathway and neuroactive ligand-receptor interaction were the most significantly enriched. Candidate genes were analysed using real-time quantitative reverse transcription polymerase chain reaction (to verify the accuracy and reliability of the RNA-Seq results. The relative expression of agouti signalling protein (ASIP) and DOPA decarboxylase (DDC) in white-feather quails were increased. Relative expression of homeobox D1 (HOXD1), cathepsin D (CTSD), keratin 2 (KRT2), methyl-CpG binding domain protein 2 (MBD2), and melatonin receptor 1 (MT1) were decreased. The results of this study provide useful data on the analysis of the feather colour regulatory mechanism in white- and maroon-feather quails.

MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a TE repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

MBD3 promotes epithelial-mesenchymal transition in gastric cancer cells by upregulating ACTG1 via the PI3K/AKT pathway

BackgroundGastric cancer (GC) is a common malignancy and a leading cause of cancer-related death with high morbidity and mortality. Methyl-CpG binding domain protein 3 (MBD3), a key epigenetic regulator, is abnormally expressed in several cancers, participating in progression and metastasis. However, the role of MBD3 in GC remains unknown.MethodsMBD3 expression was assessed via public databases and validated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The prognosis of MBD3 was analysed via bioinformatics based on the TCGA dataset. The migration, invasion and proliferation of GC cells were examined by transwell, wound healing, cell counting kit (CCK)-8, colony-formation and xenograft mouse models. Epithelial-mesenchymal transition (EMT) and phosphatidylinositide 3-kinases/ protein Kinase B (PI3K/AKT) pathway markers were evaluated by Western blotting. RNA sequencing was used to identify the target of MBD3.ResultsMBD3 expression was higher in GC tissues and cells than in normal tissues and cells. Additionally, high MBD3 levels were associated with poor prognosis in GC patients. Subsequently, we proved that MBD3 enhanced the migration, invasion and proliferation abilities of GC cells. Moreover, western blot results showed that MBD3 promoted EMT and activated the PI3K/AKT pathway. RNA sequencing analysis showed that MBD3 may increase actin γ1 (ACTG1) expression to promote migration and proliferation in GC cells.ConclusionMBD3 promoted migration, invasion, proliferation and EMT by upregulating ACTG1 via PI3K/AKT signaling activation in GC cells and may be a potential diagnostic and prognostic target.

Ascorbic Acid Induced the Improved Oxygen Vacancy Defects of Bi4O5Br2 and Its Application on Photoelectrochemical Detection of DNA Demethylase MBD2 with Improved Detection Sensitivity.

Oxygen vacancy defects (OVs) are one of the main strategies for nanomaterials modification to improve the photoactivity, but current methods for fabricating OVs are usually complicated and harsh. It is important to develop simple, rapid, safe, and mild methods to fabricate OVs. By studying the effects of different weak reducing agents, the concentration of the reducing agent and the reaction time on fabrication of OVs, it is found that L-ascorbic acid (AA) gently and rapidly induces the increase of OVs in Bi4O5Br2 at room temperature. The increased OVs not only improve the adsorption of visible light, but also enhance the photocurrent response. Based on this, the preparation of OVs in Bi4O5Br2 is employed to the development of a photoelectrochemical biosensor for the detection of DNA demethylase of methyl-CpG binding domain protein 2 (MBD2). The biosensor shows a wide linear range of 0.1-400ngmL-1 and a detection limit as low as 0.03ngmL-1 (3σ). In addition, the effect of plasticizers on MBD2 activity is evaluated using this sensor. This work not only provides a novel method to prepare OVs in bismuth rich materials, but also explores a new novel evaluation tool for studying the ecotoxicological effects of contaminants.

ACD15, ACD21, and SLN regulate the accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here, we characterize two additional complex members: α-crystalline domain (ACD) containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher-order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of MBD5/6 complexes regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive a massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of the gene-silencing MBD5/6 complex and act to drive the formation of higher-order, dynamic assemblies at CG methylation (meCG) sites.

Methyl-CpG Binding Domain Protein MBD2 Is a Targetable Vulnerability in TET2 Mutant Myeloid Neoplasia

Background & Significance. Epigenetic dysregulation leading to acquired disorganization of CpG methylation (mCpG) is a hallmark of cancer in general and myeloid neoplasia (MN) in particular. TET dioxygenases (TET1, TET2 and TET3) progressively oxidize 5-methyl cytosine (mC) to facilitate CpG demethylation, a process critical to efficient transcription, lineage differentiation, and proliferation. Loss of function TET2 mutation ( TET2 MT), the predominant TET in HSPCs, isone of the most common genetic lesions in MN and often occurs early in MDS/AML as a founder lesion. It is frequently observed in clonal hematopoiesis of indeterminate potential (CHIP) 1,2, a prodromal condition linked with increased risk of MN, coronary heart disease 3, and pulmonary arterial hypertension 4. TET2 MT results in abnormal accumulation of mC in the genome resulting in loss of lineage plasticity due to transcriptional repression of differentiation-promoting genes and tumor suppressor genes (TSGs), contributing to a volatile pre-neoplastic state. Indeed, the accumulation of mC at otherwise unmethylated promoter- and enhancer-associated CpGs is a hallmark cancer 2,5. The epigenetic reader mCpG binding domain protein 2 (MBD2) specifically recognizes mCpG, thereby modulating regional gene transcription 6. This modulation occurs through the recruitment of co-repressor complexes which deliver enzymatic machinery to reorganize chromatin into compact, inaccessible, and stably silenced regions. This tightly regulated process, required for normal development and cell lineage differentiation, is hijacked by malignant cells to maintain a pro-oncogenic transcriptional program. Therefore, MBD2 inhibition may restore the normal hematopoiesis in TET2 MT HSPCs and may represent a selective therapeutic target. Methods. Genetic and epigenetic in vitro and in vivo model systems of clonal evolution including competitive repopulation assays and CRISPR-engineered cells coupled with NGS and biochemical analysis were used to establish the functional relevance of MBD2 in the clonal evolution of TET2 MT HSPCs. In addition, gene knockout or knockdown (ko/kd) were used to establish MBD2 as a novel therapeutic vulnerability in TET2 MT MN. Results. Here we report that loss of TET activity, either by TET2 MT or by neomorphic IDH1/2 MT, selectively enriches promoter/enhancer mCpG contributing to malignant evolution of TET2 MT HSPCs that creates sensitivity to MBD2 inhibition. TET2 MT creates regional accumulation of mC that recruits the MBD2-NuRd complex, resulting in silencing of TSGs as demonstrated via MBD2 chromatin immunoprecipitation assay. MBD2 ko/kd in TET2 MT cells (SIG-M5, OCIAML-5) or isogenic CRISPR-engineered leukemic cells (THP1, K562) resulted in the termination of the malignant program as reflected in a complete loss of colony forming ability in vitro. Orthotropic transplant of SIG-M5 cells (10 5 cells/mice) created leukemia in SGM3-NSGS mice with a maximum survival of 45 days, however, MBD2 ko in transplanted cells resulted in 100% disease-free survival of these mice. To further test if Mbd2 loss is sufficient to restore normal hematopoiesis and if it can abrogate the proliferative advantage of Tet2 -/- HSPCs, we developed a Tet2 -/-;Mbd2 -/- double ko (DKO) murine model by crossing Tet2 -/-with Mbd2 -/- mice. The DKO mice developed normally with restored hematopoiesis within the myeloid lineage compared to Tet2 -/-. Most importantly, the proliferative advantage of Tet2 -/-HSPCs was ablated, as reflected in the mutant fraction of total WBC and the restoration of CD11b + myeloid cells following Mbd2 loss in competitive bone marrow repopulation assays ( Fig. A).This restoration was mainly driven by the transcriptional rescue of differentiation-promoting genes which were silenced by promoter methylation in Tet2 -/- HSPCs ( Fig. B). Discussion & Conclusion. Here, for the first time, we demonstrate that MBD2 is required for maintaining the undifferentiated state of TET2 MT HSPCs with myeloid skewing. We provide several lines of evidence in human cells as well as in murine models that MBD2 is a targetable vulnerability in TET2 MT-associated leukemia. Our current findings provide a rationale for development of novel and potent non-cytotoxic therapeutic agents targeting MBD2 and their application in a biomarker-driven fashion for a large proportion of leukemias.

The Arabidopsis DNA glycosylase MBD4L repairs the nuclear genome in vivo.

DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.

Methyl-CpG binding domain (MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a β-sheet in the MBD to affect embryonic development in Bombyx mori.

Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in β1-β6 or α1 in the MBD showed that β2-β3-turns in the β-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, β4-β6 and an α-helix, play a role in stabilizing the β-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without β4-β6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact β-sheet structure in its MBD, thus ensuring silkworm embryonic development.

Metastasis-associated lung adenocarcinoma transcript 1 induces methyl-CpG-binding domain protein 4 in mice with recurrent spontaneous abortion caused by anti-phospholipid antibody positivity

IntroductionAntiphospholipid syndrome is an autoimmune disease characterized by pregnancy-related morbidity, related to persistent positivity of antiphospholipid antibodies (APL). One of the characteristics of pregnancy-related morbidity in patients with antiphospholipid syndrome is recurrent spontaneous abortion (RSA). This study aimed to examine the mechanism through which metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) regulates methyl-CpG-binding domain protein 4 (MBD4) expression in APL-positive RSA. MethodsClinical samples were subjected to microarray analysis to filter differentially expressed genes. RSA mice with APL positivity were generated, followed by adenoviral vector injection to artificially upregulate MALAT1. The effects of MALAT1 on the biological behavior of trophoblast cells were assessed. The downstream mechanism of MALAT1 was analyzed using subcellular fractionation and bioinformatics prediction, and the relationship between MALAT1 and CREB binding protein (CREBBP) or MBD4 was investigated in trophoblast cells. ResultsMALAT1 was downregulated in APL-positive RSA patients. MALAT1 was predominantly localized in the nucleus and recruited CREBBP to mediate the MBD4 transcription. In the APL-positive RSA mice overexpressing MALAT1, the expression of soluble Fms-related tyrosine kinase 1 and anticardiolipin antibody and the embryonic resorption rate were decreased, indicating that MALAT1 reduced the occurrence of RSA in mice. Moreover, MALAT1 enhanced proliferation, migration, and invasion of trophoblast cells through recruiting CREBBP to promote MBD4 expression. Silencing of CREBBP or MBD4 increased embryonic resorption rate in RSA mice overexpressing MALAT1. DiscussionMALAT1 suppresses APL-positive RSA by promoting MBD4 transcription through recruitment of CREBBP to the MBD4 promoter region.

MBD5 regulates NMDA receptor expression and seizures by inhibiting Stat1 transcription

Epilepsy is considered to result from an imbalance between excitation and inhibition of the central nervous system. Pathogenic mutations in the methyl-CpG binding domain protein 5 gene (MBD5) are known to cause epilepsy. However, the function and mechanism of MBD5 in epilepsy remain elusive. Here, we found that MBD5 was mainly localized in the pyramidal cells and granular cells of mouse hippocampus, and its expression was increased in the brain tissues of mouse models of epilepsy. Exogenous overexpression of MBD5 inhibited the transcription of the signal transducer and activator of transcription 1 gene (Stat1), resulting in increased expression of N-methyl-d-aspartate receptor (NMDAR) subunit 1 (GluN1), 2A (GluN2A) and 2B (GluN2B), leading to aggravation of the epileptic behaviour phenotype in mice. The epileptic behavioural phenotype was alleviated by overexpression of STAT1 which reduced the expression of NMDARs, and by the NMDAR antagonist memantine. These results indicate that MBD5 accumulation affects seizures through STAT1-mediated inhibition of NMDAR expression in mice. Collectively, our findings suggest that the MBD5-STAT1-NMDAR pathway may be a new pathway that regulates the epileptic behavioural phenotype and may represent a new treatment target.

MiR-146a-3p as a potential novel therapeutic by targeting MBD2 to mediate Th17 differentiation in Th17 predominant neutrophilic severe asthma

Th17 (T-helper 17) cells subtype of non-T2 (non-type 2) asthma is related to neutrophilic infiltration and resistance to inhaled corticosteroids (ICS), so is also known as severe asthma. Methyl-CpG binding domain protein 2 (MBD2) regulates the differentiation of the Th17 cells, tending to show a therapeutic target in severe asthma. miR-146a-3p is associated with anti-inflammatory characteristics and immunity. Moreover, bioinformatic analysis showed that MBD2 may be a target gene of miR-146a-3p. However, the role of miR-146a-3p in the differentiation of Th17 cells via MBD2 in severe asthma remains unknown. Here, we aimed to explore how miR-146a-3p interacts with MBD2 and affects the differentiation of Th17 cells in severe asthma. First, we recruited 30 eligible healthy people and 30 patients with severe asthma to detect the expression of miR-146a-3p in peripheral blood mononuclear cells (PBMCs) by qRT-PCR. Then, we established a HDM/LPS (house dust mite/lipopolysaccharide) exposure model of bronchial epithelial cells (BECs) to evaluate the expression of miR-146a-3p, the interaction between miR-146a-3p and MBD2 using western blot and luciferase reporter analysis and the effect of miR-146a-3p regulated Th17 cells differentiation by flow cytometry in BECs in vitro. Finally, we constructed a mouse model of Th17 predominant neutrophilic severe asthma to assess the therapeutic potential of miR-146a-3p in severe asthma and the effect of miR-146a-3p regulated Th17 cells differentiation via MBD2 in vivo. Decreased miR-146a-3p expression was noted in severe asthma patients, in the BECs and in the animal severe asthma models. Moreover, we demonstrated that miR-146a-3p suppressed Th17 cells differentiation by targeting the MBD2. miR-146a-3p overexpression significantly reduced airway hyperresponsiveness, airway inflammation and airway mucus secretion, while also inhibiting Th17 cells response in vivo, which relieved severe asthma. By targeting MBD2 to suppress Th17 cells differentiation, miR-146a-3p provides a potential novel therapeutic for Th17 predominant neutrophilic severe asthma.