MBD2 Research Articles

PiR-31470 epigenetically suppresses the expression of glutathione S-transferase pi 1 in prostate cancer via DNA methylation

Inactivation of glutathione S-transferase pi 1 (GSTP1) via hypermethylation is an early and common event in prostate carcinogenesis. Functional inactivation of GSTP1 increases the susceptibility to oxidative stress and enhance progression risk of the prostatic carcinoma. In this study, we hypothesized that the Piwi-interacting RNA (piRNA) could be a sequence-recognition and guidance molecule for induction of promoter methylation of GSTP1 facilitating prostate carcinogenesis. We found that piR-31470 was highly expressed in prostate cancer cells, and piR-31470 could bind to piwi-like RNA-mediated gene silencing 4 (PIWIL4) to form the PIWIL4/piR-31470 complex. This complex could bind to the nascent RNA transcripts of GSTP1, and recruit DNA methyltransferase 1, DNA methyltransferase 3 alpha and methyl-CpG binding domain protein 2 to initiate and maintain the hypermethylation and inactivation of GSTP1. Our data demonstrated that the overexpression of piR-31470 inhibited the levels of GSTP1 and increased vulnerability to oxidative stress and DNA damage in human prostate epithelial RWPE1 cells. In conclusion, this study characterized the roles of the PIWIL4/piR-31470 complex in the regulation of the transcription of GSTP1 by methylating the CpG island of GSTP1. This discovery may provide a novel therapeutic strategy by targeting piRNAs for the epigenetic treatment of prostate cancer.

Abstract C44: MBD3 stabilizes MYC, leading to metastatic outgrowth of pancreatic cancer in the liver

Abstract Introduction: Pancreatic cancer remains a challenging disease, with an overall cumulative 5-year survival rate less than 5%. Metastases are responsible for over 90% of cancer-related deaths. Despite significant progress made in cancer drug development, our understanding of the mechanisms driving metastatic outgrowth remains limited. Systematic dissection of the molecular mechanisms underlying metastatic progression is necessary for the development of new therapeutic strategies. Method: To identify the important genes driving metastatic outgrowth, we performed a novel in vivo cDNA screening using a high metastatic-capacity line (M-4964Liv) and a low metastatic-capacity line (D-4964Liv), which we derived from in vivo passaging of a cell line from a KPC mouse. MBD3 regulated MYC stability was examined by cycloheximide chase assay and further confirmed by polymerase chain reaction and Western blotting. Phenotypic effect of MBD3 was evaluated by sphere assay in 3D and intrasplenic model of liver metastasis in vivo. Results: Methyl-CpG binding domain protein 3 (MBD3) was identified in our screen. We initially evaluated MBD3 with pancreatosphere formation assays. In doing so, we showed that MBD3 overexpression leads to significant augmentation of sphere formation, while knockdown abrogates sphere-forming capacity. We subsequently validated MBD3 in vivo with liver metastases assays, through both overexpression and knockdown, as being a facilitator of metastatic outgrowth. In addition, we observed high expression of MBD3 across a panel of pancreatic cancer cell lines that readily form liver metastases upon splenic injection. To understand how MBD3 may lead to metastases, we used a cycloheximide chase assay and determined that MBD3 overexpression increases MYC stability. With knockout of MBD3 using CRISPR/Cas9, we demonstrated that MBD3 mediates MYC stabilization by increasing the expression of phospho-MYC at Ser-62 (stable form of MYC). In the absence of MBD3, we observed an increase in phospho-MYC at Thr-58 expression (unstable form of MYC). Consequently, we observed a decrease in expression of CDK7, a downstream target of MYC, after MBD3 knockdown. CDK7 is a known master regulator of cyclin-dependent kinases, and loss of CDK7 drives cells to quiescence. Intriguingly, downregulation of MYC expression, through destabilization with MBD3 knockdown, led to activation of negative feedback pathway as observed with an increase in phospho-GSK3β at Ser-9, which inactivates GSK3β and thereby decreases MYC ubiquitination. Conclusion: MBD3 leads to stabilization of MYC in pancreatic cancer cell lines, which results in an increase in the ability to form liver metastases. Further dissection of this previously undescribed MBD-mediated mechanism may lead to new therapeutic targeting of MYC for patients with pancreatic cancer. Citation Format: Alok Ranjan, Surajit Sinha, Reed Ayabe, Michael Wach, Samantha Ruff, Shreya Gupta, James McDonald, Kirsten Remmert, Imani Alexander, Jonathan Hernandez. MBD3 stabilizes MYC, leading to metastatic outgrowth of pancreatic cancer in the liver [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C44.

MUC1-C Activates the NuRD Complex to Drive Dedifferentiation of Triple-Negative Breast Cancer Cells.

The NuRD chromatin remodeling and deacetylation complex, which includes MTA1, MBD3, CHD4, and HDAC1 among other components, is of importance for development and cancer progression. The oncogenic mucin 1 (MUC1) C-terminal subunit (MUC1-C) protein activates EZH2 and BMI1 in the epigenetic reprogramming of triple-negative breast cancer (TNBC). However, there is no known link between MUC1-C and chromatin remodeling complexes. Here, we showed that MUC1-C binds directly to the MYC HLH-LZ domain and identified a previously unrecognized MUC1-C→MYC pathway that regulates the NuRD complex. MUC1-C/MYC complexes selectively activated the MTA1 and MBD3 genes and posttranscriptionally induced CHD4 expression in basal- but not luminal-type BC cells. In turn, MUC1-C formed complexes with these NuRD components on the ESR1 promoter. Downregulating MUC1-C decreased MTA1/MBD3/CHD4/HDAC1 occupancy and increased H3K27 acetylation on the ESR1 promoter, with induction of ESR1 expression and downstream estrogen response pathways. Targeting MUC1-C and these NuRD components also induced expression of FOXA1, GATA3, and other markers associated with the luminal phenotype. These findings support a model in which MUC1-C activates the NuRD complex to drive dedifferentiation and reprogramming of TNBC cells. SIGNIFICANCE: MUC1-C directly interacts with MYC to activate the NuRD complex, mediating regulation of the estrogen receptor in triple-negative breast cancer cells.

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

A model of pulldown alignments from SssI-treated DNA improves DNA methylation prediction

BackgroundProtein pulldown using Methyl-CpG binding domain (MBD) proteins followed by high-throughput sequencing is a common method to determine DNA methylation. Algorithms have been developed to estimate absolute methylation level from read coverage generated by affinity enrichment-based techniques, but the most accurate one for MBD-seq data requires additional data from an SssI-treated Control experiment.ResultsUsing our previous characterizations of Methyl-CpG/MBD2 binding in the context of an MBD pulldown experiment, we build a model of expected MBD pulldown reads as drawn from SssI-treated DNA. We use the program BayMeth to evaluate the effectiveness of this model by substituting calculated SssI Control data for the observed SssI Control data. By comparing methylation predictions against those from an RRBS data set, we find that BayMeth run with our modeled SssI Control data performs better than BayMeth run with observed SssI Control data, on both 100 bp and 10 bp windows. Adapting the model to an external data set solely by changing the average fragment length, our calculated data still informs the BayMeth program to a similar level as observed data in predicting methylation state on a pulldown data set with matching WGBS estimates.ConclusionIn both internal and external MBD pulldown data sets tested in this study, BayMeth used with our modeled pulldown coverage performs better than BayMeth run without the inclusion of any estimate of SssI Control pulldown, and is comparable to – and in some cases better than – using observed SssI Control data with the BayMeth program. Thus, our MBD pulldown alignment model can improve methylation predictions without the need to perform additional control experiments.

Increased HERV-E clone 4\u20131 expression contributes to DNA hypomethylation and IL-17 release from CD4+ T cells via miR-302d/MBD2 in systemic lupus erythematosus

BackgroundIncreased human endogenous retroviruses E clone 4–1 (HERV-E clone 4–1) mRNA expression is observed in systemic lupus erythematosus (SLE) patients and associates with the disease activity. In this study, we want to further investigate the mechanism of HERV-E clone 4–1 mRNA upregulation and its roles in SLE progression.MethodsCD4+ T cells were isolated from venous blood of SLE patients or healthy controls and qRT-PCR was used to detect HERV-E clone 4–1 mRNA expression. We then investigated the regulation of Nuclear factor of activated T cells 1 (NFAT1) and Estrogen receptor-α (ER-α) on HERV-E clone 4–1 transcription and the functions of HERV-E clone 4–1 3′ long terminal repeat (LTR) on DNA hypomethylation and IL-17 release.ResultsWe found HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients and positively correlated with SLE disease activity. This is associated with the activation of Ca2+/calcineurin (CaN)/NFAT1 and E2/ER-α signaling pathway and DNA hypomethylation of HERV-E clone 4–1 5’LTR. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE through miR-302d/Methyl-CpG binding domain protein 2 (MBD2)/DNA hypomethylation and IL-17 signaling via its 3’LTR.ConclusionsHERV-E clone 4–1 mRNA upregulation is due to the abnormal inflammation/immune/methylation status of SLE and it could act as a potential biomarker for diagnosis of SLE. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE via its 3’LTR and the signaling pathways it involved in may be potential therapeutic targets of SLE.

Overexpression of MBD3 Improves Reprogramming of Cloned Pig Embryos.

Methyl-CpG-binding domain protein 3 (MBD3) is a core component of the nucleosome remodeling and deacetylase (NuRD) complex, which is crucial for pluripotent stem cell differentiation and embryonic development. MBD3 was shown to play important roles in transcription factor-induced somatic cell reprogramming. Expression level of MBD3 was demonstrated to be higher in somatic cell nuclear transfer-generated cloned pig embryos than in fertilization-derived porcine embryos. However, the functions of MBD3 in nuclear transfer-mediated somatic cell reprogramming are unknown. In this study, MBD3 was overexpressed in cloned pig embryos, and the effects of MBD3 overexpression on gene transcription, DNA methylation, and in vitro developmental competence of cloned pig embryos were analyzed. Results indicated that overexpression of MBD3 in cloned pig embryos not only increased blastocyst rate and number of cells per blastocyst but also upregulated mRNA expression levels and decreased the DNA methylation of NANOG, OCT4, and LINE1 genes to the levels close to those in in vivo fertilization-produced pig embryos. These findings suggest that overexpression of MBD3 improves reprogramming of cloned pig embryos.

Abstract 3680: SRSF2-dependent MBD2v2 expression is induced by obesity and promotes tumor-initiating triple negative breast cancer stem cells

Abstract Obesity is a risk factor for triple negative breast cancer (TNBC) incidence and poor outcomes, the underlying molecular biology of which remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl-CpG-binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD2v2, is dependent on reactive oxygen species (ROS) and is crucial for maintenance and expansion of cancer stem cell-like cells (CSCs). The relevance of CSCs is that they are a subpopulation of cancer cells recognized as the source of malignant tumor initiation, and give rise to drug resistance and metastatic recurrence. Because obesity is coupled with inflammation and ROS, we hypothesized that obesity could fuel an increase in MBD2v2 expression to promote the tumor-initiating CSC phenotype in TNBC cells in vivo. In this study we sought to characterize the role of obesity in regulating MBD2v2 expression in TNBC tumors, and better understand the mechanism regulating MBD2v2 expression in TNBC cells. Analysis of TNBC patient datasets revealed associations between high tumor MBD2v2 expression and high relapse rates and body mass index (BMI). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD2v2 expression is governed by ROS-dependent expression of the serine and arginine-rich splicing factor 2 (SRSF2). Analysis of TNBC patient datasets also revealed an association between high tumor SRSF2 expression and high relapse rates and BMI. We employed a diet-induced obesity (DIO) mouse model to investigate if obesity influenced MBD2v2 expression and increased tumor initiation capacity of inoculated TNBC cell lines. MBD2v2 and SRSF2 levels were increased in TNBC cell line-derived tumors, which formed more frequently in DIO mice, relative to tumors in lean control mice. Stable MBD2v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo. SRSF2 knockdown resulted in decreased MBD2v2 expression, decreased CSCs in TNBC cell cultures and hindered tumor formation DIO mice. The data provide concurring evidence that SRSF2-regulated MBD2v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights in support of the epidemiological evidence that obesity is a risk factor for TNBC. The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity-linked cancers, which reinforces the relevance of this study. Citation Format: Emily A. Teslow, Cristina Mitrea, Bin Bao, Ramzi M. Mohammad, Lisa A. Polin, Gregory Dyson, Kristen S. Purrington, Aliccia Bollig-Fischer. SRSF2-dependent MBD2v2 expression is induced by obesity and promotes tumor-initiating triple negative breast cancer stem cells [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 3680.

Capture of a functionally active methyl-CpG binding domain by an arthropod retrotransposon family.

The repressive capacity of cytosine DNA methylation is mediated by recruitment of silencing complexes by methyl-CpG binding domain (MBD) proteins. Despite MBD proteins being associated with silencing, we discovered that a family of arthropod Copia retrotransposons have incorporated a host-derived MBD. We functionally show how retrotransposon-encoded MBDs preferentially bind to CpG-dense methylated regions, which correspond to transposable element regions of the host genome, in the myriapod Strigamia maritima. Consistently, young MBD-encoding Copia retrotransposons (CopiaMBD) accumulate in regions with higher CpG densities than other LTR-retrotransposons also present in the genome. This would suggest that retrotransposons use MBDs to integrate into heterochromatic regions in Strigamia, avoiding potentially harmful insertions into host genes. In contrast, CopiaMBD insertions in the spider Stegodyphus dumicola genome disproportionately accumulate in methylated gene bodies compared with other spider LTR-retrotransposons. Given that transposons are not actively targeted by DNA methylation in the spider genome, this distribution bias would also support a role for MBDs in the integration process. Together, these data show that retrotransposons can co-opt host-derived epigenome readers, potentially harnessing the host epigenome landscape to advantageously tune the retrotransposition process.

Structural analyses reveal that MBD3 is a methylated CG binder.

The MBD3, a methyl-CpG-binding domain (MBD)-containing protein, is a core subunit of the Mi-2/NuRD complex. Recent reports show that MBD3 recognizes both methylated CG (mCG)- and hydroxymethylated CG (hmCG)-containing DNA, with a preference for hmCG. However, whether the MBD3-MBD indeed has methyl-CG-binding ability is controversial. In this study, we provided the structural basis to support the ability of MBD3-MBD to bind mCG-containing DNA. We found that the MBD3-MBD bound to mCG-containing DNA through two conserved arginine fingers, and preferentially bound to mCG over hmCG, similar to other methyl-CpG-binding MBD proteins. Compared to its closest homolog MBD2, the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for a weaker mCG DNA binding ability. Based on the complex structure of MBD3-MBD with a nonpalindromic AmCGC DNA, we suggest that all the mCG-binding MBD domains can recognize mCG-containing DNA without orientation selectivity, consistent with our observations that the sequences outside the mCG dinucleotide do not affect mCG DNA binding significantly. DNA cytosine methylation is evolutionarily conserved in most metazoans, and most invertebrates have only one MBD gene, MBD2/3. We also looked into the mCG DNA binding ability of some invertebrates MBD2/3 and found that the conserved arginine fingers and a conserved structural fold are required for methylated DNA binding by MBD2/3-MBDs in invertebrates. Hence, our results demonstrate that mCG-binding arginine fingers embedded into a conserved structural fold are essential structural features for MBD2/3s binding to methylated DNA among metazoans.

Obesity-induced MBD2_v2 expression promotes tumor-initiating triple-negative breast cancer stem cells.

Obesity is a risk factor for triple‐negative breast cancer (TNBC) incidence and poor outcomes, but the underlying molecular biology remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl‐CpG‐binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD variant 2 (MBD2_v2), is dependent on reactive oxygen species (ROS) and is crucial for maintenance and expansion of cancer stem cell‐like cells (CSCs). Because obesity is coupled with inflammation and ROS, we hypothesized that obesity can fuel an increase in MBD2_v2 expression to promote the tumor‐initiating CSC phenotype in TNBC cells in vivo. Analysis of TNBC patient datasets revealed associations between high tumor MBD2_v2 expression and high relapse rates and high body mass index (BMI). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD2_v2 expression is governed by ROS‐dependent expression of serine‐ and arginine‐rich splicing factor 2 (SRSF2). We employed a diet‐induced obesity (DIO) mouse model that mimics human obesity to investigate whether obesity causes increased MBD2_v2 expression and increased tumor initiation capacity in inoculated TNBC cell lines. MBD2_v2 and SRSF2 levels were increased in TNBC cell line‐derived tumors that formed more frequently in DIO mice relative to tumors in lean control mice. Stable MBD2_v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo. SRSF2 knockdown resulted in decreased MBD2_v2 expression, decreased CSCs in TNBC cell cultures, and hindered tumor formation in vivo. This report describes evidence to support the conclusion that MBD2_v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights into support of the epidemiological evidence that obesity is a risk factor for TNBC. The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity‐linked cancers, which reinforces the relevance of this report.

Methyl-CpG Binding Domain Protein 2 Inhibits the Malignant Characteristic of Lung Adenocarcinoma through the Epigenetic Modulation of 10 to 11 Translocation 1 and miR-200s

It has been reported that disorders of epigenetic modulation play a critical role in carcinogenesis. Methyl-CpG binding domain protein 2 (MBD2) is known to act as an epigenetic modulator in various types of tumors; however, the role of MBD2 in lung adenocarcinoma (LUAD) remains unclear. Herein, we demonstrated the down-regulation of MBD2 in LUAD compared with adjacent nontumor tissues. The down-regulation of MBD2 in LUAD was correlated with metastasis and poor survival. In addition, MBD2 inhibited tumor metastasis by maintaining the expression of the miR-200s, which suppressed the invasive properties of tumors. Also, MBD2 positively correlated with 5-hydroxymethylcytosine content in the promoter of miR-200s. The conventional view is that MBD2 acts as a transcriptional suppressor. However, the data revealed that MBD2 may act as a transcriptional activator by recruiting 10 to 11 translocation 1 (TET1) and forming a chromatin-remodeling complex. The MBD2-TET1 complex locates to the TET1 promoter and removes the methyl residues in this region, thereby activating TET1 transcription. TET1 also acted as a tumor suppressor in LUAD. Taken together, the data demonstrate the correlation between MBD2, miR-200s, and TET1, and tumor suppressive effect of MBD2 through up-regulation of TET1 and the miR-200s.

Mbd3 Promotes Reprogramming of Primary Human Fibroblasts.

Mbd3 (Methyl-CpG binding domain protein), a core member of NuRD (nucleosome remodelling and deacetylation) is essential for embryogenesis. However, its role in reprogramming of somatic cells into induced pluripotent stem cells (iPSC) remains controversial. Some reports suggest that Mbd3 inhibits pluripotency, whilst others show that it greatly enhances reprogramming efficiency. Our study is the first to assess the role of Mbd3 on reprogramming of primary human fibroblasts using Yamanaka episomal plasmids (Reprogramming factors (RF) under feeder-free conditions. We showed that shRNA-mediated partial depletion of Mbd3 resulted in >5-fold reduction in the efficiency of reprogramming of primary human fibroblasts. Furthermore, iPSC that emerged after knock-down of Mbd3 were incapable of trilineage differentiation even though they expressed all markers of pluripotency. In contrast, over-expression of the Mbd3b isoform along with the Yamanaka episomal plasmids increased the number of fibroblast derived iPSC colonies by at least two-fold. The resulting colonies were capable of trilineage differentiation. Our results, therefore, suggest that Mbd3 appears to play an important role in reprogramming of primary human fibroblasts, which provides further insight into the biology of reprogramming but also has direct implication for translation of iPSC to clinic.

Zinc finger E-box-binding homeobox 1 mediates aerobic glycolysis via suppression of sirtuin 3 in pancreatic cancer.

AIMTo uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODSEndogenous zinc finger E-box binding homeobox-1 (ZEB1) was silenced using a lentivirus-mediated method, and the impact of ZEB1 and methyl-CpG binding domain protein 1 (MBD1) on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3 (SIRT3) expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatin-immunoprecipitation assays.RESULTSZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1.CONCLUSIONZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.

Poly(ADP-ribose) polymerase-1 promotes expression of miR-155 by the up-regulation of methyl-CpG binding domain protein 2 in TK6 cells exposed to hydroquinone

Hydroquinone (HQ), one of the major metabolites of benzene, can induce aberrant gene expression. MiR-155, a tumor activator, participates in various biological processes, including DNA damage response. However, the molecular mechanism of aberrant miR-155 expression is still not completely elucidated. Here, we investigated the mechanism of abnormal expression of miR-155 induced by poly(ADP-ribose)polymerase-1 (PARP-1) expression in HQ-treated TK6 lymphoblastoid cells. We examined the expression of genes related to abnormal expression of miR-155 to explore the reason for this phenomenon. The results of the present study showed that miR-155 was significantly increased and reactive oxygen species (ROS) were decreased in cells treated with HQ for 72 h compared with PBS-treated cells. Meanwhile, E4F1, PARP-1 and PARP-1 related co-regulators (NF-κB, HDAC1, and HDAC2), acetylated histone H3 (H3Ac) were increased in a concentration-dependent manner. Experiments for treatment with 5-AzaC (DNMTs inhibitor), TSA (HDACs inhibitor), DOX (to activate PARP-1) or MG132 (proteasome inhibitor) revealed that the MBDs and PARP-1 was positively associated with miR-155 expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of miR-155, H3Ac and MBD2 protein were decreased, compared with negative control. In conclusion, PARP-1 activates expression of miR-155 via acetylation by regulating MBD2 in TK6 cells exposed to HQ.

A Whole Methylome Study of Ethanol Exposure in Brain and Blood: An Exploration of the Utility of Peripheral Blood as Proxy Tissue for Brain in Alcohol Methylation Studies.

Recent reviews have highlighted the potential use of blood-based methylation biomarkers as diagnostic and prognostic tools of current and future alcohol use and addiction. Due to the substantial overlap that often exists between methylation patterns across different tissues, including blood and brain, blood-based methylation may track methylation changes in brain; however, little work has explored the overlap in alcohol-related methylation in these tissues. To study the effects of alcohol on the brain methylome and identify possible biomarkers of these changes in blood, we performed a methylome-wide association study in brain and blood from 40 male DBA/2J mice that received either an acute ethanol (EtOH) or saline intraperitoneal injection. To investigate all 22 million CpGs in the mouse genome, we enriched for the methylated genomic fraction using methyl-CpG binding domain (MBD) protein capture followed by next-generation sequencing (MBD-seq). We performed association tests in blood and brain separately followed by enrichment testing to determine whether there was overlapping alcohol-related methylation in the 2 tissues. The top result for brain was a CpG located in an intron of Ttc39b (p=5.65×10-08 ), and for blood, the top result was located in Espnl (p=5.11×10-08 ). Analyses implicated pathways involved in inflammation and neuronal differentiation, such as CXCR4, IL-7, and Wnt signaling. Enrichment tests indicated significant overlap among the top results in brain and blood. Pathway analyses of the overlapping genes converge on MAPKinase signaling (p=5.6×10-05 ) which plays a central role in acute and chronic responses to alcohol and glutamate receptor pathways, which can regulate neuroplastic changes underlying addictive behavior. Overall, we have shown some methylation changes in brain and blood after acute EtOH administration and that the changes in blood partly mirror the changes in brain suggesting the potential for DNA methylation in blood to be biomarkers of alcohol use.

Genome-wide analysis of genes encoding MBD domain-containing proteins from tomato suggest their role in fruit development and abiotic stress responses.

In tomato, DNA methylation has an inhibitory effect on fruit ripening. The inhibition of DNA methyltransferase by 5-azacytidine results in premature fruit ripening. Methyl CpG binding domain (MBD) proteins are the readers of DNA methylation marks and help in the recruitment of chromatin-modifying enzymes which affect gene expression. Therefore, we investigate their contribution during fruit development. In this study, we identified and analyzed 18 putative genes of Solanum lycopersicum and Solanum pimpinellifolium encoding MBD proteins. We also identified tomato MBD syntelogs in Capsicum annum and Solanum tuberosum. Sixty-three MBD genes identified from four different species of solanaceae were classified into three groups. An analysis of the conserved domains in these proteins identified additional domains along with MBD motif. The transcript profiling of tomato MBDs in wild-type and two non-ripening mutants, rin and Nr, indicated constructive information regarding their involvement during fruit development. When we performed a stage-specific expression analysis during fruit ripening, a gradual decrease in transcript accumulation in the wild-type fruit was detected. However, a very low expression was observed in the ripening mutants. Furthermore, many ethylene-responsive cis-elements were found in SlMBD gene promoters, and some of them were induced in the presence of exogenous ethylene. Further, we detected the possible role of these MBDs in abiotic stresses. We found that few genes were differentially expressed under various abiotic stress conditions. Our results provide an evidence of the involvement of the tomato MBDs in fruit ripening and abiotic stress responses, which would be helpful in further studies on these genes in tomato fruit ripening.

MBD1 Contributes to the Genesis of Acute Pain and Neuropathic Pain by Epigenetic Silencing of Oprm1 and Kcna2 Genes in Primary Sensory Neurons.

The transmission of normal sensory and/or acute noxious information requires intact expression of pain-associated genes within the pain pathways of nervous system. Expressional changes of these genes after peripheral nerve injury are also critical for neuropathic pain induction and maintenance. Methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, regulates gene transcriptional activity. We report here that MBD1 in the primary sensory neurons of DRG is critical for the genesis of acute pain and neuropathic pain as DRG MBD1-deficient mice exhibit the reduced responses to acute mechanical, heat, cold, and capsaicin stimuli and the blunted nerve injury-induced pain hypersensitivities. Furthermore, DRG overexpression of MBD1 leads to spontaneous pain and evoked pain hypersensitivities in the WT mice and restores acute pain sensitivities in the MBD1-deficient mice. Mechanistically, MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 is likely a key player under the conditions of acute pain and neuropathic pain.SIGNIFICANCE STATEMENT In the present study, we revealed that the mice with deficiency of methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, in the DRG displayed the reduced responses to acute noxious stimuli and the blunted neuropathic pain. We also showed that DRG overexpression of MBD1 produced the hypersensitivities to noxious stimuli in the WT mice and rescued acute pain sensitivities in the MBD1-deficient mice. We have also provided the evidence that MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 may participate in the genesis of acute pain and neuropathic pain likely through regulating DNMT3a-controlled Oprm1 and Kcna2 gene expression in the DRG neurons.

Backbone and side-chain resonance assignments of the methyl-CpG-binding domain of MBD6 from Arabidopsis thaliana.

Epigenetic regulation is essential to various biological phenomena such as cell differentiation and cancer. DNA methylation is one of the most important epigenetic signals, as it is directly involved in gene silencing of transposable elements, genomic imprinting, and chromosome X inactivation. To mediate these processes, methyl-CpG-binding domain (MBD) proteins recognize specific signals encoded in the form of DNA methylation patterns. AtMBD6, one of the 12 MBD proteins in Arabidopsis thaliana, shares a high sequential homology in the MBD domain with mammalian MBD proteins, but a detailed characterization of its structural and functional properties remains elusive. Here, we report the 1H, 13C, and 15N resonance assignments of the isolated MBD domain of AtMBD6. Analysis of the chemical shift data implied that the MBD domain of AtMBD6 has a secondary structure similar to that of mammalian MeCP2, while the β-strands β1 and β3 of AtMBD6 were found to be longer than those of MeCP2. The structural differences provide insight into the different recognition mechanisms of methylated DNA by plant and mammalian MBDs. The assignments reported here will aid further analyses such as titration experiments and three-dimensional structure determination using NMR to yield a detailed characterization of the interaction between AtMBD6 and methylated DNAs.

Effects of maternal feed intake restriction during pregnancy on the expression of growth regulation, imprinting and epigenetic transcription-related genes in foetal goats

Maternal nutrition during gestation is a leading factor of modifying the foetal epigenome and phenotype for mammals. Imprinting genes have important roles in regulating foetal growth, programming and development. There, however, are limited data available on the effects of feed intake restriction on the expression of imprinting genes in pregnant goats. The present study, therefore, was conducted to assess the effects of maternal feed intake restriction on the relative abundance of mRNA for growth imprinting, DNA methyltransferase (DNMT) and epigenetic transcription-related genes in the liver and heart of foetal goats during gestation. A total of 24 Liuyang black goats (2.0±0.3 yr) with similar body weight (BW, 31.22±8.09 kg) and parity (2) were allocated equally to either a control group (CG) or a restriction group (RG) during both early (from 26 to 65 days) and late (from 96 to 135 days) gestation. All goats were fed a mixed diet and had free access to fresh water. The feed of the RG was 40% less than that of the CG. The early and late gestation goats were weighed, bled and slaughtered on days 65 and 135 of gestation, respectively. In early gestation, the foetal weight, body length, the weight of foetal heart and liver were greater (P < 0.05) in the RG. The CpG methylation of genomic DNA in the foetal heart was less (P = 0.0001) in the RG. The relative abundance of mRNA of methyl-CpG-binding domain protein 2 (MBD2) and methyl-CpG-binding domain protein 3 (MBD3) genes in the foetal liver were greater (P < 0.05) in the RG. During the late gestation, the foetal weight, heart weight and liver weight were less (P < 0.05) in the RG. The relative abundance of mRNA for the MBD2 gene (P = 0.043) in the foetal heart, and the ten-eleven translocation protein 1 (TET1) gene (P < 0.05) in both the foetal heart and liver were greater in the RG. These results indicate feed intake restriction during gestation influenced foetal development and regulated the relative abundance of mRNA for epigenetic transcription-related genes.