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Methyl-CpG-binding domain (MBD) proteins are important DNA methylation readers that recognise methylated CpG sites and recruit histone deacetylase (HDAC) complexes and chromatin remodelling factors, leading to chromatin compaction, gene transcription, and genome integrity. Currently, MBD genes have only been identified in a few plant species and their structure and function in tea plants (Camellia sinensis) are unknown. In this study, 16 C. sinensis MBD genes (CsMBD) were identified on a genome-wide level and classified into eight classes. The CsMBD genes were mapped on nine chromosomes in tea plants, and nine pairs of CsMBD genes existed. Based on conserved domain analysis, all of the identified CsMBD proteins contained at least one MBD domain. Expression analyses showed that CsMBD genes were expressed in tissue- and organ-specific patterns. We investigated the expression patterns of CsMBD genes in response to abiotic and biotic stresses and during different plant growth and development stages. Multiple pthytohormone and stress-related cis-acting was evaluated in their promoter region, such as GGTCA, TGACG, ABRE and LTR. Specific CsMBD genes were associated with environmental stresses and developmental stages, with little overlap. Overall, our findings reveal the diverse roles of CsMBD genes under different stress and developmental conditions, highlighting candidate genes for further functional studies on tea plants.

Lung cancer is the most common cancer worldwide. Epigenetic modifications like DNA methylation play fundamental roles in the dynamic process of lung cancer. The objective of this study was to use methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) to identify novel and high-confidence DNA methylation in lung tumor. We first compared the whole-genome DNA methylation of three lung cancer cell lines, including A549, H1299, and SK-MES-1, against BEAS-2B, a lung/bronchial normal epithelial cell line. We then used pyrosequencing and OneStep qMethyl kit methods to verify the results in the cell line specimens. MBD-Seq identified 279, 8046, and 22 887 differentially methylated regions (DMRs), respectively, with 120 common DMRs among three comparison groups. Three DMRs were consistent with the MBD-Seq results by both pyrosequencing and OneStep qMethyl validations. Furthermore, OneStep qMethyl kit was also performed for functional validation of these three potential DMRs in sputum DNA from clinical participants. We successfully identified one new DMR adjacent to ATG16L2. The novel DMR might have an important function in lung carcinogenesis. Further validation of the finding in clinical specimens of lung cancer patients and functional analysis of this novel DMR in the development of lung cancer through transcriptional silencing of ATG16L2 are warranted.

Glioblastoma multiforme (GBM) heterogeneity causes a greater number of deaths than any other brain tumor, despite the availability of alkylating chemotherapy. GBM stem-like cells (GSCs) contribute to GBM complexity and chemoresistance, but it remains challenging to identify and target GSCs or factors that control their activity. Here, we identified a specific GSC subset and show that activity of these cells is positively regulated by stabilization of methyl CpG binding domain 3 (MBD3) protein. MBD3 binds to CK1A and to BTRCP E3 ubiquitin ligase, triggering MBD3 degradation, suggesting that modulating this circuit could antagonize GBM recurrence. Accordingly, xenograft mice treated with the CK1A activator pyrvinium pamoate (Pyr-Pam) showed enhanced MBD3 degradation in cells expressing high levels of O(6)-methylguanine-DNA methyltransferase (MGMT) and in GSCs, overcoming temozolomide chemoresistance. Pyr-Pam blocked recruitment of MBD3 and the repressive nucleosome remodeling and deacetylase (NuRD) complex to neurogenesis-associated gene loci and increased acetyl–histone H3 activity and GSC differentiation. We conclude that CK1A/BTRCP/MBD3/NuRD signaling modulates GSC activation and malignancy, and that targeting this signaling could suppress GSC proliferation and GBM recurrence.

Thymine:guanine base pairs are major promutagenic mismatches occurring in DNA metabolism. If left unrepaired, these mispairs can cause C to T transition mutations. In humans, T:G mismatches are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (hMBD4) and thymine-DNA glycosylase. Unlike lesion-specific DNA glycosylases, T:G-mismatch-specific DNA glycosylases specifically recognize both bases of the mismatch and remove the thymine but only from mispairs with guanine. Despite the advances in biochemical and structural characterizations of hMBD4, the catalytic mechanism of hMBD4 remains elusive. Herein, we report two structures of hMBD4 processing T:G-mismatched DNA. A high-resolution crystal structure of Asp560Asn hMBD4-T:G complex suggests that hMBD4-mediated glycosidic bond cleavage occurs via a general base catalysis mechanism assisted by Asp560. A structure of wild-type hMBD4 encountering T:G-containing DNA shows the generation of an apurinic/apyrimidinic (AP) site bearing the C1′-(S)-OH. The inversion of the stereochemistry at the C1′ of the AP-site indicates that a nucleophilic water molecule approaches from the back of the thymine substrate, suggesting a bimolecular displacement mechanism (SN2) for hMBD4-catalyzed thymine excision. The AP-site is stabilized by an extensive hydrogen bond network in the MBD4 catalytic site, highlighting the role of MBD4 in protecting the genotoxic AP-site.

The Nucleosome Remodeling and Deacetylase (NuRD) complex uniquely combines both deacetylase and remodeling enzymatic activities in a single macromolecular complex. The methyl-CpG-binding domain 2 and 3 (MBD2 and MBD3) proteins provide a critical structural link between the deacetylase and remodeling components, while MBD2 endows the complex with the ability to selectively recognize methylated DNA. Hence, NuRD combines three major arms of epigenetic gene regulation. Research over the past few decades has revealed much of the structural basis driving formation of this complex and started to uncover the functional roles of NuRD in epigenetic gene regulation. However, we have yet to fully understand the molecular and biophysical basis for methylation-dependent chromatin remodeling and transcription regulation by NuRD. In this review, we discuss the structural information currently available for the complex, the role MBD2 and MBD3 play in forming and recruiting the complex to methylated DNA, and the biological functions of NuRD.

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.

Alle Lebewesen m��ssen daf��r Sorge tragen, in ihrem umfangreichen Erbgut die gerade f��r sie ��berlebensnotwendigen Gene von denen zu unterscheiden, die nicht gebraucht werden. Insbesondere mehrzellige Organismen m��ssen diesen Prozess zellspezifisch koordinieren, trotz dessen, dass hier dieselbe Erbinformation in allen Zellen des Individuums vorliegt. Ein Mechanismus, welcher diesem Zwecke dient, ist die Modifikation von DNA-Nukleobasen, den Bausteinen des Tr��gers der Erbinformation. In S��ugetieren wie Mensch und Maus kommt hierbei der Methylierung der DNA-Nukleobase Cytosin am Kohlenstoffatom C5 des Pyrimidinrings eine besondere Rolle zu. Sie findet auf beiden Str��ngen der DNA-Doppelhelix innerhalb des kurzen Sequenzpalindroms CpG statt und tr��gt entscheidend dazu bei, dass hier ortsspezifisch andere molekulare Interaktionen f��r die Expression der Erbinformationen notwendig werden. Da das Produkt 5-Methylcytosin f��r weitere enzymatische Modifikationen wie der Oxidation zu 5-Hydroxymethylcytosin, 5-Formylcytosin oder 5-Carboxycytosin zur Verf��gung steht, k��nnen unterschiedliche Kombinationen dieser Cytosinderivate mit g��nzlich einzigartigen chemischen Eigenschaften an den komplement��ren CpG-Paaren im DNA-Doppelstrang vorliegen. Ein Aspekt, der unter dem Gesichtspunkt der epigenetischen Funktion dieser Derivate in Ermangelung technologischer Innovation sie in nat��rlichem Chromatin zu untersuchen, bislang kaum erschlossen werden konnte. Inwiefern es nun m��glich ist, solche Strang-symmetrischen oder Strang-asymmetrischen Kombinationen von Cytosinderivaten in diesen CpG-Paaren auf molekularer Ebene in der DNA-Doppelhelix zu erkennen und somit gegebenenfalls zu entschl��sseln, ist Gegenstand der vorliegenden Arbeit. Ausgehend von verschiedenen Homologen einer Proteindom��ne, welche symmetrisch methylierte CpG-Paare erkennen, den Methyl-CpG-bindenden Dom��nen (MBD), wurden aufgrund struktureller Erw��gungen und funktionaler Studien der MBD���DNA-Binding, degenerierte Proteinvariantenbibliotheken erstellt. Mithilfe eines hierf��r eigens entwickelten Hochdurchsatzverfahrens gelang es, Varianten zu identifizieren, die nahezu selektiv eine aus f��nfzehn Paarungen obiger Cytosinderivate im DNA-Doppelstrang erkennen. Neben allgemeinen Substitutionsprofilen f��r verschiedene Paarungen wurden im Speziellen mehrere MBD-Varianten entdeckt, die eine neue, nat��rlicherweise nicht vorhandene Selektivit��t f��r 5-Hydroxymethyl- und 5-Carboxymethylcytosin-haltige CpG-Paarungen aufwiesen. Aus der weiteren biochemischen und strukturellen Charakterisierung der Bindespezifit��t konnten einige Erkenntnisse ��ber die molekulare Erkennung Strang-asymmetrisch modifizierter CpG-Paarungen gewonnen werden, welche in Zukunft als Schl��ssel dienen k��nnen, die epigentische Funktion der Cytosinmodifizierung im humanen Genom mithilfe solcher speziell auf sie zugeschnittenen Sonden zu entschl��sseln.

Chronic lymphocytic leukaemia (CLL) exhibits differences between Asians and Caucasians in terms of incidence rate, age at onset, immunophenotype, and genetic profile. We performed genome-wide methylation profiling of CLL in an Asian cohort for the first time. Eight Korean patients without somatic immunoglobulin heavy chain gene hypermutations underwent methyl-CpG-binding domain sequencing (MBD-seq), as did five control subjects. Gene Ontology, pathway analysis, and network-based prioritization of differentially methylated genes were also performed. More regions were hypomethylated (2,062 windows) than were hypermethylated (777 windows). Promoters contained the highest proportion of differentially methylated regions (0.08%), while distal intergenic and intron regions contained the largest number of differentially methylated regions. Protein-coding genes were the most abundant, followed by long noncoding and short noncoding genes. The most significantly over-represented signalling pathways in the differentially methylated gene list included immune/cancer-related pathways and B-cell receptor signalling. Among the top 10 hub genes identified via network-based prioritization, four (UBC, GRB2, CREBBP, and GAB2) had no known relevance to CLL, while the other six (STAT3, PTPN6, SYK, STAT5B, XPO1, and ABL1) have previously been linked to CLL in Caucasians. As such, our analysis identified four novel candidate genes of potential significance to Asian patients with CLL.

MeCP2 is an essential transcriptional repressor that mediates transcriptional inhibition by binding to methylated DNA. The binding specificity of MeCP2 protein to methylated DNA was considered to depend on its methyl-CpG binding domain (MBD). In this study, we used atomic force microscope based single-molecular force spectroscopy to investigate the interaction of MeCP2 MBD and methylated DNA. The specific interaction forces of the MeCP2 MBD-methylated DNA complexes were measured for the first time. The dynamics was also investigated by measuring the unbinding force of the complex at different loading rates. In addition, the distribution of unbinding forces and binding probabilities of MeCP2 MBD and different DNA were studied at the same loading rate. It was found that MeCP2 MBD had weak interaction with hemi-methylated and unmethylated DNA compared to methylated DNA. This work revealed the binding characteristics of MeCP2 MBD and methylated DNA at the single-molecule level. It provides a new idea for exploring the molecular mechanism of MeCP2 in regulating methylation signals.

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.

DNA methylation is an epigenetic modification that represses gene expression. In cancer cells, alterations of the DNA methylation state in promoter regions and repetitive DNA sequences are ...

Embryonic stem cells (ESCs) exhibit two salient features beneficial for regenerative medicine: unlimited self-renewal and pluripotency. Methyl-CpG-binding domain protein 3 (Mbd3), a scaffolding component of the nucleosome remodeling deacetylase complex, is a specific regulator of pluripotency, as ESCs lacking Mbd3 are defective for lineage commitment potential but retain normal self-renewal properties. However, functional similarities and dissimilarities among the three Mbd3 isoforms (a, b, and c) have not been intensively explored. Herein, we demonstrated that Mbd3c, which lacks an entire portion of the MBD domain, exerted equivalent activity for counteracting the defective lineage commitment potential of Mbd3-knockout ESCs. Our analyses also revealed that the coiled-coil domain common to all three MBD3 isoforms, but not the MBD domain, plays a crucial role in this activity. Mechanistically, our data demonstrate that the activity of the coiled-coil domain is exerted, at least in part, through recruitment of polycomb repressive complex 2 to a subset of genes linked to development and organogenesis, thus establishing stable transcriptional repression.

Global DNA methylation levels have been considered as biomarkers for cancer diagnostics because transposable elements that constitute approximately 45% of the human genome are hypomethylated in cancer cells. We have previously reported a homogeneous assay for measuring methylated CpG content of genomic DNA based on bioluminescence resonance energy transfer (BRET) using methyl-CpG-binding domain (MBD)-fused luciferase (MBD-luciferase). In this study, a homogeneous assay for measuring unmethylated CpG content of genomic DNA in the same platform was developed using CXXC domain-fused luciferase (CXXC-luciferase) that specifically recognizes unmethylated CpG. In this assay, CXXC-luciferase recognizes unmethylated CpG on genomic DNA, whereby BRET between luciferase and the fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the genomic DNA concentration (R2 = 0.99) and unmethylated CpG content determined by the bisulfite method (R2 = 0.97). There was a significant negative correlation between the BRET signal of the CXXC-luciferase-based assay and that of the MBD-luciferase-based assay (R2 = 0.92). Moreover, we demonstrated that the global DNA methylation level determined using the bisulfite method was dependent on the ratio of the BRET signal in the MBD-luciferase-based assay to the total BRET signal in the MBD-luciferase- and CXXC-luciferase-based assays (R2 = 0.99, relative standard deviation < 2.2%, and analysis speed < 35 min). These results demonstrated that global DNA methylation levels can be quantified by calculating the BRET signal ratio without any calibration curve.

Cytosine modifications diversify and structure the genome thereby controlling proper development and differentiation. Here, we focus on the interplay of the 5-methylcytosine reader Mbd1 and modifier Tet1 by analyzing their dynamic subcellular localization and the formation of the Tet oxidation product 5-hydroxymethylcytosine in mammalian cells. Our results demonstrate that Mbd1 enhances Tet1-mediated 5-methylcytosine oxidation. We show that this is due to enhancing the localization of Tet1, but not of Tet2 and Tet3 at heterochromatic DNA. We find that the recruitment of Tet1 and concomitantly its catalytic activity eventually leads to the displacement of Mbd1 from methylated DNA. Finally, we demonstrate that increased Tet1 heterochromatin localization and 5-methylcytosine oxidation are dependent on the CXXC3 domain of Mbd1, which recognizes unmethylated CpG dinucleotides. The Mbd1 CXXC3 domain deletion isoform, which retains only binding to methylated CpGs, on the other hand, blocks Tet1-mediated 5-methylcytosine to 5-hydroxymethylcytosine conversion, indicating opposite biological effects of Mbd1 isoforms. Our study provides new insights on how cytosine modifications, their modifiers and readers cross-regulate themselves.

DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

Methyl-CpG binding domain proteins (MBD) can specifically bind to methylated CpG sites and play important roles in epigenetic gene regulation. Here, we identified and functionally characterized the MBD protein in Tribolium castaneum. T. castaneum genome encodes only one MBD protein: TcMBD2/3. RNA interference targeting this gene at different developmental stages caused lethal phenotypes including metamorphosis deficiency in larvae and pupae, gastrointestinal system problems and fecundity deficiency in adult. Moreover, Tcmbd2/3 knockdown adult showed progressive reduced locomoter activity, a typical neurodegeneration phenotype. This is a common feature of DNA methylation in mammals and has not been found in other insects. However, band shift assays demonstrated that TcMBD2/3 could not bind to methylated DNA, indicating the essential roles of TcMBD2/3 is independent of DNA methylation. Our study provides Tcmbd2/3 plays important roles in T. castaneum and gives new insights into the potential mechanism of action of MBD proteins in insect.

Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the Arabidopsis SWR1-C has not been thoroughly characterized. Here, we aim to identify interacting partners of a conserved Arabidopsis SWR1 subunit ACTIN-RELATED PROTEIN 6 (ARP6). We isolate nine predicted components and identify additional interactors implicated in histone acetylation and chromatin biology. One of the interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacts with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 is required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 is preferentially bound to nucleosome-depleted regions at the 5’ ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes., The SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation, but its composition remains largely uncharacterized in plants. Here, the authors report that methyl-CpG-binding domain 9 (MBD9) is a SWR1-C interacting protein required for histone H2A.Z deposition in Arabidopsis.

Abnormal DNA methylations such as hypermethylation on tumor suppressor genes and global hypomethylation have been recognized as hallmarks of cancer. Previously, we reported a bioluminescence resonance energy transfer (BRET)-based global DNA methylation level assay using a methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc) and unmethylated CpG-binding domain-fused firefly luciferase (CXXC-Fluc). The BRET signal between MBD-Fluc and BOBO-3 DNA intercalating dye depends on the methylated CpG contents, whereas the BRET signal between CXXC-Fluc and BOBO-3 depends on the unmethylated CpG contents. Therefore, the global DNA methylation level can be quantified using the BRET assay. However, these assays must be performed separately, because the same luciferase fuses to both MBD and CXXC. In this study, we developed a one-step quantification assay of global DNA methylation based on a multicolor BRET assay using MBD-Fluc and CXXC-fused Oplophorus luciferase (CXXC-Oluc). We demonstrated that MBD-Fluc and CXXC-Oluc simultaneously excite BOBO-3 and BOBO-1 DNA intercalating dyes on genomic DNA, respectively. Moreover, the BRET signals produced from MBD-Fluc and CXXC-Oluc depended on the methylation status of the CpG contents. These results demonstrate that global DNA methylation can be quantified by this multicolor BRET assay in a single tube. Graphical abstract ᅟ.

DNA methyltransferase (DNMT) inhibitors are epigenetic drugs used to treat myelodysplastic syndrome. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects; however, the relationships between these characteristics have not been established yet due to the lack of a method to induce only DNA demethylation. Herein, we show that a fusion protein comprised of the methyl‐CpG‐binding domain (MBD) and the catalytic domain of Ten‐eleven translocation protein 1 (TET1‐CD) globally demethylates and upregulates a number of methylated genes. These upregulated genes frequently contained CpG islands (CGIs) within ± 1000 bp of the transcription start site (TSS). Interestingly, 65% of the genes upregulated fivefold or more by MBD‐TET1‐CDwt were also reactivated after treatment with a DNMT inhibitor, 5‐azacytidine (Aza‐CR), suggesting that gene reactivation by both methods primarily shares the same mechanism, DNA demethylation. In order to examine whether DNA demethylation affects the growth of cancer cells, we have established a tetracycline inducible system that can regulate the expression of MBD‐TET1‐CDwt in a prostate cancer cell line, LNCaP. The induction of MBD‐TET1‐CDwt demethylated and upregulated glutathione S‐transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with the reactivation of methylated genes, induction of MBD‐TET1‐CDwt extensively suppressed the growth of LNCaP cells through G1/S arrest. These results clearly indicate that TET oxidase activity recruited at methyl‐CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation and allows us to analyze the effect of only global DNA demethylation in a wide variety of cancer cells.

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature.

In tomato (Solanum lycopersicum), high pigment mutations (hp-1 and hp-2) were mapped to genes encoding UV-damaged DNA binding protein 1 (DDB1) and de-etiolated-1 (DET1), respectively. Here we characterized a tomato methyl-CpG-binding domain protein SlMBD5 identified by yeast two-hybrid screening using SlDDB1 as a bait. Yeast two-hybrid assay demonstrated that the physical interaction of SlMBD5 with SlDDB1 is mediated by the C-termini of SlMBD5 and the β-propeller-C (BPC) of SlDDB1. Co-immunoprecipitation analyses revealed that SlMBD5 associates with SlDDB1-interacting partners including SlDET1, SlCUL4, SlRBX1a and SlRBX1b in vivo. SlMBD5 was shown to target to nucleus and dimerizes via its MBD motif. Electrophoresis mobility shift analysis suggested that the MBD of SlMBD5 specifically binds to methylated CpG dinucleotides but not to methylated CpHpG or CpHpH dinucleotides. SlMBD5 expressed in protoplast is capable of activating transcription of CG islands, whereas CUL4/DDB1 antagonizes this effect. Overexpressing SlMBD5 resulted in diverse developmental alterations including darker green fruits with increased plastid level and elevated pigmentation, as well as enhanced expression of SlGLK2, a key regulator of plastid biogenesis. Taken together, we hypothesize that the physical interaction of SlMBD5 with the CUL4-DDB1-DET1 complex component may affect its binding activity to methylated DNA and subsequently attenuate its transcription activation of downstream genes.

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition., Author summary The histone H2A variant, H2A.Z, is found in all known eukaryotes and plays important roles in transcriptional regulation. H2A.Z is selectively incorporated into nucleosomes within many genes by the activity of a conserved ATP-dependent chromatin remodeling complex in yeast, insects, and mammals. Whether this complex exists in the same form in plants, and how the complex is targeted to specific genomic locations have remained open questions. In this study we demonstrate that plants do indeed utilize a complex analogous to those of fungi and animals to deposit H2A.Z, and we also identify several new proteins that interact with this complex. We found that one such interactor, Methyl-CpG-BINDING DOMAIN 9 (MBD9), is required for H2A.Z incorporation at thousands of genomic sites that share a distinct histone modification profile. The histone binding properties of MBD9 suggest that it may guide H2A.Z deposition to specific sites by interacting with modified nucleosomes and with the H2A.Z deposition complex. We hypothesize that this represents a general paradigm for the targeting of H2A.Z to specific sites.

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.

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate formation in mice. This TCDD treatment, which may be considered an environmental factor in cleft palate formation, is associated with alterations in DNA methylation. However, the underlying molecular mechanisms of DNA methylation produced by TCDD in mouse embryos are poorly understood. DNA methyltransferases (DNMTs) and methyl‑CpG binding domain proteins (MBDs) are thought to be closely associated with the actions of DNA methylation. Therefore, the present study tested the hypothesis that this cleft palate inducing effect of TCDD will alter the expression levels of DNMTs and various MBDs in palate tissue of fetal mice. Pregnant C57BL/6J mice were treated with either TCDD (64 µg/kg) or corn oil (control) at embryonic day 10.5 (E10.5) and fetal palates were harvested for structural and molecular analyses at E13.5, E14.5, E15.5 and E17.5. Expression levels of DNMTs and MBDs were assayed using reverse transcription‑quantitative polymerase chain reaction and western blotting. The incidence of cleft palates in the TCDD group was 98.24%, whereas no cases of cleft palate were observed in the control group. Expression levels of DNMTs and MBDs were significantly increased in the TCDD group compared with the control. The results demonstrate clear alterations in DNMTs and MBDs, as induced by TCDD, and suggest that such alterations are important in cleft palate formation in fetal mice.

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

Although methyl CpG binding domain protein-2 (MeCP2) is commonly understood to function as a silencing factor at methylated DNA sequences, recent studies also show that MeCP2 can bind unmethylated sequences and coordinate gene activation. MeCP2 displays broad binding patterns throughout the genome, with high expression levels similar to histone H1 in neurons. Despite its significant presence in the brain, only subtle gene expression changes occur in the absence of MeCP2. This may reflect a more complex regulatory mechanism of MeCP2 to complement chromatin binding. Using an RNA immunoprecipitation of native chromatin technique, we identify MeCP2 interacting microRNAs in mouse primary cortical neurons. In addition, comparison with mRNA sequencing data from Mecp2-null mice suggests that differentially expressed genes may indeed be targeted by MeCP2-interacting microRNAs. These findings highlight the MeCP2 interaction with microRNAs that may modulate its binding with chromatin and regulate gene expression.

Detailed biological knowledge about the potential importance of the methylome is typically lacking for common diseases. Therefore, methylome-wide association studies (MWAS) are critical to detect disease relevant methylation sites. Methyl-CpG-binding domain sequencing (MBD-seq) offers potential advantages compared to antibody-based enrichment, but performance depends critically on using an optimal protocol. Using an optimized protocol, MBD-seq can approximate the sensitivity/specificity obtained with whole-genome bisulfite sequencing, but at a fraction of the costs and time to complete the project. Thus, MBD-seq offers a comprehensive first pass at the CpG methylome and is economically feasible with the samples sizes required for MWAS.

DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.

UNLABELLED In 2009, esophageal cancer was recorded as the fifth most commonly diagnosed cancer and the fourth leading cause of cancer-related death in China. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of esophageal cancers. Genetic factors might play an important role in the carcinogenesis of ESCC. We conducted a hospital-based case-control study to evaluate the association between methyl-CpG binding domain 4 (MBD4) rs3138373 A>G, rs2005618 T>C, and rs3138355 G>A tag single nucleotide polymorphisms and the risk of developing ESCC. A total of 629 ESCC patients and 686 controls were recruited. Genotypes were determined using the ligation detection reaction method. When the MBD4 rs3138355 GG homozygous genotype was used as the reference group, the GA, AA, and GA/AA genotypes were not associated with ESCC risk. In the recessive model, when the MBD4 rs3138355 GG/GA genotypes were used as the reference group, the AA homozygous genotype was associated with a 28% decreased risk for ESCC (AA vs. GG/GA adjusted odds ratio=0.72, 95% confidence interval=0.53-0.99, P=0.040). The MBD4 rs3138373 A>G and rs2005618 T>C single nucleotide polymorphisms were not associated with ESCC risk. The MBD4 rs3138355 G>A polymorphism was associated with a significantly decreased risk of ESCC among male and older patients. The MBD4 rs3138355 GG genotype was associated with a decreased risk of ESCC among male patients and the elderly. Additional, larger studies are required to confirm these current findings.

DNA methylation is an important form of epigenetic regulation in both normal development and cancer. Methyl-CpG-binding domain protein 1 (MBD1) is highly related to DNA methylation. Its MBD domain recognizes and binds to methylated CpGs. This binding allows it to trigger methylation of H3K9 and results in transcriptional repression. The CXXC3 domain of MBD1 makes it a unique member of the MBD family due to its affinity to unmethylated DNA. MBD1 acts as an epigenetic regulator via different mechanisms, such as the formation of the MCAF1/MBD1/SETDB1 complex or the MBD1-HDAC3 complex. As methylation status always changes along with carcinogenesis or neurogenesis, MBD1 with its interacting partners, including proteins and non-coding RNAs, participates in normal or pathological processes and functions in different regulatory systems. Because of the important role of MBD1 in epigenetic regulation, it is a good candidate as a therapeutic target for diseases.

Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (Arabidopsis thaliana) line expressing the Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) and Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenes, we isolated an mbd7 (for methyl-CpG-binding domain protein7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis.

DNA methylation is one of the major epigenetic marks in the mammalian genome to define chromatin higher-order structure, and plays essential roles in various developmental processes. In the mammalian genome, DNA methylation mainly occurs at the 5th position of cytosine bases in a palindromic 5'-CG-3'dinucleotide sequence. Methyl CpG binding domain (MBD) proteins recognize symmetrically methylated CpG sites (5mCG/5mCG) through a conserved MBD, and recruit transcriptional repressors or chromatin modifiers. One of the MBD proteins, MBD4, uniquely contains a C-terminal glycosylation domain together with an N-terminal MBD, and functions as a mismatch DNA repair enzyme specific for T/G or U/G mismatch bases generated by spontaneous deamination of 5-methylcytosine. The base excision activity of MBD4 is also implicated in active DNA demethylation initiated by the conversion of 5-methylcytosine to thymine by deaminases. Unlike other MBD proteins, MBD4 recognizes not only 5mCG/5mCG but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine (5mCG/TG). In addition, our biochemical data demonstrate that MBD also binds to intermediates in DNA demethylation pathways, such as 5-hydroxymethyl-cytosine (hmC), 5-carboxyl-cytosine and 5-hydroxy-uracil. The crystal structures of MBDMBD4 in complex with 5mCG/TG, 5mCG/5mCG or 5mCG/hmCG provide new structural insights into the versatility of base recognition by MBD4. A DNA interface of MBD4 has flexible structural features, in which an extensive hydration water network supports the versatile base specificity of MBD4. The versatile base recognition by MBDMBD4 implies multi-functional roles of MBD4 in the regulation of dynamic DNA methylation patterns.

Human telomerase reverse transcriptase (hTERT) is expressed in most cancer cells. Paradoxically, its promoter is embedded in a hypermethylated CpG island. A short region escapes to this alteration, allowing a basal level of transcription. However, the methylation of adjacent regions may play a role in the maintenance of low hTERT expression. It is now well established that methyl-CpG binding domain proteins mediate the transcriptional silencing of hypermethylated genes. The potential involvement of these proteins in the control of hTERT expression was firstly investigated in HeLa cells. Chromatin immunoprecipitation assays showed that only methyl-CpG-binding domain protein 2 (MBD2) associated the hypermethylated hTERT promoter. In MBD2 knockdown HeLa cells, constitutively depleted in MBD2, neither methyl CpG binding protein 2 (MeCP2) nor MBD1 acted as substitutes for MBD2. MBD2 depletion by transient or constitutive RNA interference led to an upregulation of hTERT transcription that can be downregulated by expressing mouse Mbd2 protein. Our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT in HeLa cells. This specific transcriptional repression was also observed in breast, liver and neuroblastoma cancer cell lines. Thus, MBD2 seems to be a general repressor of hTERT in hTERT-methylated telomerase-positive cells.

Reproductive isolation and speciation are driven by the convergence of environmental and genetic variation. The integration of these variation sources is thought to occur through epigenetic marks including DNA methylation. Proteins containing a methyl-CpG-binding domain (MBD) bind methylated DNA and interpret epigenetic marks, providing a dynamic yet evolutionarily adapted cellular output. Here, we report the Drosophila MBD-containing proteins, dMBD-R2 and dMBD2/3, contribute to reproductive isolation and survival behavioral strategies. Drosophila melanogaster males with a reduction in dMBD-R2 specifically in octopamine (OA) neurons exhibit courtship toward divergent interspecies D. virilis and D. yakuba females and a decrease in conspecific mating success. Conspecific male-male courtship is increased between dMBD-R2-deficient males while aggression is reduced. These changes in adaptive behavior are separable as males with a hypermethylated OA neuronal genome exhibited a decrease in aggression without altering male-male courtship. These results suggest Drosophila MBD-containing proteins are required within the OA neural circuitry to inhibit interspecies and conspecific male-male courtship and indicate that the genetically hard-wired neural mechanisms enforcing behavioral reproductive isolation include the interpretation of the epigenome.

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.

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing. DOI: http://dx.doi.org/10.7554/eLife.19893.001

Methyl-CpG binding protein 2 (MeCP2) has recently been characterized as an oncogene frequently amplified in several types of cancer. However, its precise role in gastric cancer (GC) and the molecular mechanism of MeCP2 regulation are still largely unknown. Here we report that MeCP2 is highly expressed in primary GC tissues and the expression level is correlated with the clinicopathologic features of GC. In our experiments, knockdown of MeCP2 inhibited tumor growth. Molecular mechanism of MeCP2 regulation was investigated using an integrated approach with combination of microarray analysis and chromatin immunoprecipitation sequencing (ChIP-Seq). The results suggest that MeCP2 binds to the methylated CpG islands of FOXF1 and MYOD1 promoters and inhibits their expression at the transcription level. Furthermore, we show that MeCP2 promotes GC cell proliferation via FOXF1-mediated Wnt5a/β-Catenin signaling pathway and suppresses apoptosis through MYOD1-mediated Caspase-3 signaling pathway. Due to its high expression level in GC and its critical function in driving GC progression, MeCP2 represents a promising therapeutic target for GC treatment., Highlights • MeCP2 inhibits FOXF1 and MYOD1 transcription by binding to their promoter regions. • MeCP2 promotes GC cell proliferation via FOXF1-mediated Wnt/β-Catenin signaling pathway. • MeCP2 suppresses GC cell apoptosis through MYOD1-mediated Caspase-3 signaling pathway. Gastric cancer is the fourth most common malignant cancer and the third most frequent cause of cancer-related deaths worldwide. The molecular mechanism underlying gastric carcinogenesis and progression is still unknown. Methyl-CpG binding protein 2 (MeCP2) has recently been characterized as an oncogene frequently amplified in several types of cancer. However, its precise role and the molecular mechanism of MeCP2 regulation in gastric cancer are largely unknown. Our results show that MeCP2 promotes gastric cancer cell proliferation via FOXF1-mediated Wnt5a/β-Catenin signaling pathway and suppresses cell apoptosis through MYOD1-mediated Caspase-3 signaling pathway. MeCP2 represents a promising therapeutic target for gastric cancer treatment.

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.