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Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences

January 2013
Nucleic Acids Research 41(5)

January 2013
41(5)

Source
PubMed

License
CC BY-NC 3.0

Authors:

The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2–NuRD and MBD3–NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2–NuRD, in contrast to MBD3–NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes.

LacI-MBD2 or LacI-MBD3 assemble MBD2–NuRD or MBD3–NuRD on the LacO array. (A) F42B8 (U2OS) cells were transfected with GFP-LacI-MBD2b or GFP-LacI-MBD3. GFP or Cherry signals were directly determined, and indirect immunofluorescence was carried out with antibodies against Mi2 (αMi-2), RbAp46 (αRbAp46) and HDAC1 (αHDAC1). Merged colour images show DAPI staining in addition. (B) Schematic representation and summary of the assembly of the different NuRD components. Green arrows indicate recruitment of the components pointed out by the arrowhead, whereas the grey arrows indicate no assembly.

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In contrast to MBD3 and to other NuRD components, LacI-MBD2a or LacI-MBD2b dramatically compact the euchromatic LacO array. RREB1 cells were transfected with GFP-LacI, GFP-LacI-MBD2a/b, GFP-LacI-MBD3 and GFP-LacI-HDAC1 as indicated. Samples were visualized for DNA (blue) and GFP (green). The arrows point to the compact array or to the expanded array (dotted circle).

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In contrast to GFP-LacI-MBD3, GFP-LacI-MBD2a or GFP-LacI-MBD2b repress the euchromatic LacO promoter. (A) Illustration of the reporter-gene including seven LacI-binding sites (LacO-repeats). Reporter construct was used to generate stable reporter cell clones in HeLa cells. (B) Western Blot analysis of the indicated GFP-LacI-fusion proteins expressed in the stable A10 clone. GAPDH antibody was used as loading control. (C) The A10 clone was transiently transfected with either GFP-LacI, GFP-LacI-VP16, GFP-LacI-MBD2a, GFP-LacI-MBD2b or GFP-LacI-MBD3. Gene activity is expressed as fold change relative to the untransfected A10 clone. (D) Binding analysis by ChIP-qPCR was performed after transfection of the indicated constructs using a GFP antibody and expressed relative to untransfected cells. (E, F) GFP-LacI-MBD2a and GFP-LacI-MBD2b change the H3K9me3/H3K9ac ratio after binding to the reporter. ChIP-qPCR was performed with antibodies recognizing H3K9ac (E) and H3K9me3 (F) and expressed relative to input. Error bars indicate the standard error of three individual replicates.

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MBD2 and MBD3 bind similar sequence classes. (A) MBD2 and MBD3 distribution of significantly bound regions in comparison with the distribution of genomic features (TSS, TSS upstream, TES, exon, intron, intergenic). (B) Peaks of MBD2 and MBD3 binding are enriched at exons and TSS, but not at intergenic regions and introns. Enrichments are calculated relative to the frequency of these features in the genome. (C) MBD2 and MBD3 bind to TSS. Plotted is the average profile of V5-MBD2, V5-MBD3 or V5 read coverage in a 6000 bp window around the TSS of all RefSeq genes. (D) MBD3 sites are often co-occupied by MBD2. Plots illustrate average V5 binding (left panel), V5-MBD2 (center panel) and V5-MBD3 binding (right panel) over MBD2 peaks (orange) and MBD3 peaks (blue).

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MBD2 sites are highly enriched for methylated DNA, whereas MBD3 sites are devoid of DNA methylation. (A) Venn diagram shows overlap of MBD2- and MBD3-binding regions with CpG islands. (B) Average relative (percentage) CpG methylation level within CpG islands associated with MBD2, MBD3 or of all CpG islands [island definition from (21)]. MBD2-associated islands show high methylation, whereas MBD3-associated islands are devoid of methylation as compared with the average island methylation [Wilcoxon signed-rank test: P = 0 (MBD2) and P = 3.5 × 10−57(MBD3)].

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Supplementary Data

Data

January 2013

Katharina Günther · Mareike Rust · Joerg Leers · Thomas Boettger · Rainer Renkawitz

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

Preprint

Full-text available

Apr 2024

Chromatin organization controls DNA’s accessibility to regulatory factors to influence gene expression. Heterochromatin, or transcriptionally silent chromatin enriched in methylated DNA and methylated histone tails, self-assembles through multivalent interactions with its associated proteins into a condensed, but dynamic state. Liquid-liquid phase separation (LLPS) of key heterochromatin regulators, such as heterochromatin protein 1 (HP1), plays an essential role in heterochromatin assembly and function. Methyl-CpG-binding protein 2 (MeCP2), the most studied member of the methyl-CpG-binding domain (MBD) family of proteins, has been recently shown to undergo LLPS in the absence and presence of methylated DNA. These studies provide a new mechanistic framework for understanding the role of methylated DNA and its readers in heterochromatin formation. However, the details of the molecular interactions by which other MBD family members undergo LLPS to mediate genome organization and transcriptional regulation are not fully understood. Here, we focus on two MBD proteins, MBD2 and MBD3, that have distinct but interdependent roles in gene regulation. Using an integrated computational and experimental approach, we uncover the homotypic and heterotypic interactions governing MBD2 and MBD3 phase separation and DNA’s influence on this process. We show that despite sharing the highest sequence identity and structural homology among all the MBD protein family members, MBD2 and MBD3 exhibit differing residue patterns resulting in distinct phase separation mechanisms. Understanding the molecular underpinnings of MBD protein condensation offers insights into the higher-order, LLPS-mediated organization of heterochromatin.

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

Article

Full-text available

May 2024
BMC CANCER

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

Dissecting the roles of MBD2 isoforms and domains in regulating NuRD complex function during cellular differentiation

Article

Full-text available

Jun 2023

The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.

MBD2a-NuRD binds to the methylated γ-globin gene promoter and uniquely forms a complex required for silencing of HbF expression

Article

Jun 2023
P NATL ACAD SCI USA

During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.

The NuRD Complex in Neurodevelopment and Disease: A Case of Sliding Doors

Article

Full-text available

Apr 2023

The Nucleosome Remodelling and Deacetylase (NuRD) complex represents one of the major chromatin remodelling complexes in mammalian cells, uniquely coupling the ability to “open” the chromatin by inducing nucleosome sliding with histone deacetylase activity. At the core of the NuRD complex are a family of ATPases named CHDs that utilise the energy produced by the hydrolysis of the ATP to induce chromatin structural changes. Recent studies have highlighted the prominent role played by the NuRD in regulating gene expression during brain development and in maintaining neuronal circuitry in the adult cerebellum. Importantly, components of the NuRD complex have been found to carry mutations that profoundly affect neurological and cognitive development in humans. Here, we discuss recent literature concerning the molecular structure of NuRD complexes and how the subunit composition and numerous permutations greatly determine their functions in the nervous system. We will also discuss the role of the CHD family members in an array of neurodevelopmental disorders. Special emphasis will be given to the mechanisms that regulate the NuRD complex composition and assembly in the cortex and how subtle mutations may result in profound defects of brain development and the adult nervous system.

CARM1 hypermethylates the NuRD chromatin remodeling complex to promote cell cycle gene expression and breast cancer development

Article

Apr 2024

Protein arginine methyltransferase CARM1 has been shown to methylate a large number of non-histone proteins, and play important roles in gene transcriptional activation, cell cycle progress, and tumorigenesis. However, the critical substrates through which CARM1 exerts its functions remain to be fully characterized. Here, we reported that CARM1 directly interacts with the GATAD2A/2B subunit in the nucleosome remodeling and deacetylase (NuRD) complex, expanding the activities of NuRD to include protein arginine methylation. CARM1 and NuRD bind and activate a large cohort of genes with implications in cell cycle control to facilitate the G1 to S phase transition. This gene activation process requires CARM1 to hypermethylate GATAD2A/2B at a cluster of arginines, which is critical for the recruitment of the NuRD complex. The clinical significance of this gene activation mechanism is underscored by the high expression of CARM1 and NuRD in breast cancers, and the fact that knockdown CARM1 and NuRD inhibits cancer cell growth in vitro and tumorigenesis in vivo. Targeting CARM1-mediated GATAD2A/2B methylation with CARM1 specific inhibitors potently inhibit breast cancer cell growth in vitro and tumorigenesis in vivo. These findings reveal a gene activation program that requires arginine methylation established by CARM1 on a key chromatin remodeler, and targeting such methylation might represent a promising therapeutic avenue in the clinic.

The Nucleosome Remodelling and Deacetylation complex coordinates the transcriptional response to lineage commitment in pluripotent cells

Article

Full-text available

Dec 2023
Biol Open

As cells exit the pluripotent state and begin to commit to a specific lineage they must activate genes appropriate for that lineage while silencing genes associated with pluripotency and preventing activation of lineage-inappropriate genes. The Nucleosome Remodelling and Deacetylation (NuRD) complex is essential for pluripotent cells to successfully undergo lineage commitment. NuRD controls nucleosome density at regulatory sequences to facilitate transcriptional responses, and also has been shown to prevent unscheduled transcription (transcriptional noise) in undifferentiated pluripotent cells. How these activities combine to ensure cells engage a gene expression program suitable for successful lineage commitment has not been determined. Here we show that NuRD is not required to silence all genes. Rather it restricts expression of genes primed for activation upon exit from the pluripotent state, but maintains them in a transcriptionally permissive state in self-renewing conditions which facilitates their subsequent activation upon exit from naïve pluripotency. We further show that NuRD coordinates gene expression changes, which acts to maintain a barrier between different stable states. Thus NuRD-mediated chromatin remodelling serves multiple functions, including reducing transcriptional noise, priming genes for activation and coordinating the transcriptional response to facilitate lineage commitment.

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

Preprint

Full-text available

Nov 2023

Background It has been shown that Resveratrol regulate BRCA1 gene expression in breast cancer cells and that MBD proteins bind to the BRCA1 gene promoter regions, the molecular link or mechanism has yet to be established. In This study aimed to evaluate the effect of MBD proteins in the regulation of BRCA1, BRCA2, and p16, genes as well as their impacts on breast cancer cells. Methods Efficacy of resveratrol was calculated by MTT assay. The binding assay was performed by EMSA, ChIP, & MeIP assay. MBD genes & proteins expression were analyzed by qRT-PCR & western blotting. Clonogenic, migratory, and sphere formation assays were used to assess cancer cells' colony-forming, metastatic, and tumor-forming abilities. The cytotoxicity of resveratrol on cancer cells was also tested using an apoptosis assay. Results This study found that IC50 of resveratrol was 30µM. MBD proteins bind to the BRCA1 gene promoter. Resveratrol regulates MBD genes expression, which in turn adversely influences BRCA1 gene expression and protein expression. A high concentration of Resveratrol reduced the colony & sphere formation as well as migration of cancer cells. It also increases no. of apoptotic cells in breast cancer cells. Conclusions In conclusion MBD2 protein interact to the BRCA1 gene promoter, and resveratrol modulates MBD2 gene expression, which in turn regulates BRCA1 gene expression, and inhibits cell proliferation, migration, and induces apoptosis in ER+, PR+ & Triple negative breast cancer cells. Impact Identification of MBD2-BRCA1 axis indicates their significant role in the induction of apoptosis and reduction of metastasis and proliferation in Breast cancer cells. Further therapy can be designed to target these MBD proteins and resveratrol could be used along with other anticancer drugs to target breast cancer.

Rise of the Chromodomain Helicase DNA‐Binding (CHD) Chromatin Remodelling Machines

Chapter

Mar 2023

Andrej Alendar

Vital processes such as transcription, repair, replication and recombination continuously shape cellular chromatin landscape. The chromodomain helicase DNA‐binding (CHD) family of enzymes regulate chromatin dynamics by utilising energy from ATP hydrolysis to alter nucleosomal position, structure and composition, thereby modulating accessibility of the underlying DNA sequence. The CHD enzymes are highly conserved in evolution, and genetic studies in fungi, plants and animals demonstrate their critical roles in regulation of cellular identity and fate and organismal development. Advances in genomic studies over the past two decades led to the identification of frequent DNA copy‐number alterations, mutations and aberrant expression of CHDs in human malignancies and various disorders, highlighting their importance as relevant biomarkers or targets for therapeutic intervention. Key Concepts CHD family of enzymes (CHD1‐9) are evolutionary conserved ATP‐dependent chromatin remodelers that mobilise nucleosomes to regulate DNA‐templated processes, such as transcription, replication and repair. They are critical for normal organismal development and are frequently mutated in human disorders and cancers. Chromodomain helicase DNA‐binding proteins (CHD1‐9) are evolutionary conserved family of ATP‐dependent chromatin remodelers that mobilise nucleosomes to regulate DNA‐templated processes such as transcription, replication and DNA repair. CHD enzymes share a common domain structure: two N‐terminal chromodomains, a central ATPase/helicase domain, and a C‐terminal DNA‐binding domain. CHD proteins play crucial roles in diverse cellular processes, including embryonic development, stem cell maintenance and differentiation, and tissue‐specific gene expression. Dysregulation of CHD proteins has been associated with various human diseases, including cancer and neurodevelopmental disorders. CHD proteins are attractive targets for the development of new therapies for diseases associated with chromatin dysfunction. Understanding the molecular mechanisms underlying the function of CHD proteins may pave the way for the development of new drugs that target these proteins and improve the treatment of a variety of human diseases.

Synonymous alterations of cancer-associated Trp53 CpG mutational hotspots cause fatal developmental jaw malocclusions but no tumors in knock-in mice

Article

Full-text available

Apr 2023
PLOS ONE

Intragenic CpG dinucleotides are tightly conserved in evolution yet are also vulnerable to methylation-dependent mutation, raising the question as to why these functionally critical sites have not been deselected by more stable coding sequences. We previously showed in cell lines that altered exonic CpG methylation can modify promoter start sites, and hence protein isoform expression, for the human TP53 tumor suppressor gene. Here we extend this work to the in vivo setting by testing whether synonymous germline modifications of exonic CpG sites affect murine development, fertility, longevity, or cancer incidence. We substituted the DNA-binding exons 5-8 of Trp53, the mouse ortholog of human TP53, with variant-CpG (either CpG-depleted or -enriched) sequences predicted to encode the normal p53 amino acid sequence; a control construct was also created in which all non-CpG sites were synonymously substituted. Homozygous Trp53-null mice were the only genotype to develop tumors. Mice with variant-CpG Trp53 sequences remained tumor-free, but were uniquely prone to dental anomalies causing jaw malocclusion (p < .0001). Since the latter phenotype also characterises murine Rett syndrome due to dysfunction of the trans-repressive MeCP2 methyl-CpG-binding protein, we hypothesise that CpG sites may exert non-coding phenotypic effects via pre-translational cis-interactions of 5-methylcytosine with methyl-binding proteins which regulate mRNA transcript initiation, expression or splicing, although direct effects on mRNA structure or translation are also possible.

In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition

Article

Full-text available

Dec 1996

Carmen C Robinett

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

Ultrafast and memory-efficient alignment of short reads to the human genome

Article

Full-text available

Jan 2009

Bowtie 1 is a fast and memory-efficient program for aligning short reads to mammalian genomes. Burrows-Wheeler indexing allows Bowtie to align more than 25 million 35-bp reads per CPU hour to the human genome in a memory footprint of as little as 1.1 gigabytes. Bowtie extends previous Burrows-Wheeler techniques with a quality-aware search algorithm that permits mismatches. Multiple processor cores can be used simultaneously to achieve greater alignment speed. Bowtie is free, open source software available for download from http://bowtie.cbcb.umd.edu . The Burrows-Wheeler Transformation of a text T, BWT(T), is constructed as shown to the right. The Burrows- Wheeler Matrix of T is the matrix whose rows are all distinct cyclic rotations of T$ sorted lexicographically ($ is "less than" all other characters). BWT(T) is the sequence of characters in the last column of this matrix.

NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment

Article

May 2012

Transcriptional heterogeneity within embryonic stem cell (ESC) populations has been suggested as a mechanism by which a seemingly homogeneous cell population can initiate differentiation into an array of different cell types. Chromatin remodeling proteins have been shown to control transcriptional variability in yeast and to be important for mammalian ESC lineage commitment. Here we show that the Nucleosome Remodeling and Deacetylation (NuRD) complex, which is required for ESC lineage commitment, modulates both transcriptional heterogeneity and the dynamic range of a set of pluripotency genes in ESCs. In self-renewing conditions, the influence of NuRD at these genes is balanced by the opposing action of self-renewal factors. Upon loss of self-renewal factors, the action of NuRD is sufficient to silence transcription of these pluripotency genes, allowing cells to exit self-renewal. We propose that modulation of transcription levels by NuRD is key to maintaining the differentiation responsiveness of pluripotent cells.

Ultrafast and memory-efficient alignment of short DNA sequences to the human genome

Article