Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells
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PROVIDER: PRJNA501901 | ENA |
REPOSITORIES: ENA
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Project description:As high fetal hemoglobin (HbF) levels ameliorate the underlying pathophysiologic defects in sickle cell anemia and β-thalassemia, understanding the mechanisms that enforce silencing of HbF postnatally offers the promise of effective molecular therapy. Depletion of Methyl cytosine Binding Domain protein 2 (MBD2) causes a 10-20 fold increase in γ-globin gene expression in adult β-YAC transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, CRISPR-Cas9 mediated knockout (KO) was carried out in Human Umbilical cord Derived Erythroid Progenitor-2 (HUDEP-2) erythroid cells resulting in γ/γ+β mRNA levels of ~50% and ~40% HbF by HPLC. In contrast, MBD3 KO had no appreciable effect on γ-globin mRNA. Knockdown (Kd) of MBD2 in primary adult erythroid cells consistently increased γ/γ+β mRNA ratios by ~10-fold resulting in ~30-40% γ/γ+β mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the CHD4 chromatin remodeling component of NuRD through a coiled-coil (CC) domain, and the histone deacetylase (HDCC) component via an intrinsically disordered region (IDR). Enforced expression of wild-type MBD2 in MBD2KO HUDEP-2 cells caused a 5-fold decrease in γ-globin mRNA while neither the CC mutant nor the IDR mutant MBD2 proteins had an inhibitory effect. Consistently, co-immunoprecipitation assays showed that the CC and IDR domain mutations disrupt NuRD complex formation by dissociating the CHD4 and HDCC domains respectively. These results establish MBD2-NuRD as a major silencer of HbF in human erythroid cells and point to the CC and IDR domains of MBD2-NuRD as potential therapeutic targets.
2020-01-27 | GSE121992 | GEO
Project description:To obtain a comprehensive and quantitative view on MBD2 vs MBD3-NuRD complex stoichiometry, we performed biotin co-immunoprecipitations in Mbd3 KO ES cells expressing either biotin-tagged MBD2a or MBD3a and identified known NuRD complex members using label-free mass spectrometry (Supplementary Fig. 8b-c). We then calculated the intensity-based absolute quantification (iBAQ) values of the most predominant and statistically significant MBD-interacting proteins in both cell lines, which can be used to estimate the relative abundance. While we observe very similar complex composition between MBD2a-NuRD and MBD3a-NuRD, peptides shared between SALL1-4 proteins show a preferred interaction with the MBD2a-NuRD complex.
2023-06-30 | PXD042407 | Pride
Project description:The heterogeneous collection of 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 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. This suggests a functional connection between MBD2 binding to chromatin and splicing.
2013-02-08 | GSE41003 | GEO
Project description:The heterogeneous collection of 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 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. This suggests a functional connection between MBD2 binding to chromatin and splicing. 9 Total samples were analysed. Three individual replicates for HeLa cells treated with siRNA against MBD2, MBD3 or scrambled siRNA were performed. We calculated the fold change of gene expression of cells treated with MBD2- or MBD3-siRNA over cells treated with scrambled siRNA.
2013-02-08 | E-GEOD-41003 | biostudies-arrayexpress
Project description:The heterogeneous collection of NuRD complexes can be grouped into the MBD2 or MBD3 containing complexes MBD2-NuRD and MBD3-NuRD. 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 a strong enrichment for MBD2 at methylated CpG sequences, whereas CpGs bound by MBD3 are devoid of methylation. Gene activity of MBD2 bound genes is four fold lower as compared to genes bound by MBD3. 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. This suggests a functional connection between MBD2 binding to chromatin and splicing.
2013-02-08 | GSE41006 | GEO
Project description:The heterogeneous collection of NuRD complexes can be grouped into the MBD2 or MBD3 containing complexes MBD2-NuRD and MBD3-NuRD. 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 a strong enrichment for MBD2 at methylated CpG sequences, whereas CpGs bound by MBD3 are devoid of methylation. Gene activity of MBD2 bound genes is four fold lower as compared to genes bound by MBD3. 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. This suggests a functional connection between MBD2 binding to chromatin and splicing. V5 ChIP followed by high throughput sequencing in HeLa cells transiently transfected with either V5-MBD2b, V5-MBD3 or V5.
2013-02-08 | E-GEOD-41006 | biostudies-arrayexpress
Project description: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.
2023-05-23 | GSE199541 | GEO
Project description:Cytosine methylation on DNA is an important epigenetic and regulatory mark. Chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins modulate chromatin structure and transcription at methylated loci. Two MBD proteins, Mbd2 and Mbd3, are mutually exclusive members of the NuRD chromatin remodeling complex, and have been shown to bind methylated or hydroxymethylated DNA, respectively. However, a recent study called both results into question, showing that chromatin binding by these proteins is partially (Mbd2) or completely (Mbd3) independent of DNA methylation/hydroxymethylation. Here we re-analyze these data and observe discrepancies with both conclusions. Furthermore, we describe multiple new datasets that demonstrate the dependence of endogenous Mbd2 and Mbd3 on DNA methylation. Interestingly, we find that Mbd2 and Mbd3 are also dependent on one another for binding, likely due to the fact that both are required for normal levels of DNA methylation/hydroxymethylation. These findings describe a regulatory loop controlling the DNA methylation machinery and its readers.
2016-11-15 | GSE79769 | GEO
Project description:Cytosine methylation on DNA is an important epigenetic and regulatory mark. Chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins modulate chromatin structure and transcription at methylated loci. Two MBD proteins, Mbd2 and Mbd3, are mutually exclusive members of the NuRD chromatin remodeling complex, and have been shown to bind methylated or hydroxymethylated DNA, respectively. However, a recent study called both results into question, showing that chromatin binding by these proteins is partially (Mbd2) or completely (Mbd3) independent of DNA methylation/hydroxymethylation. Here we re-analyze these data and observe discrepancies with both conclusions. Furthermore, we describe multiple new datasets that demonstrate the dependence of endogenous Mbd2 and Mbd3 on DNA methylation. Interestingly, we find that Mbd2 and Mbd3 are also dependent on one another for binding, likely due to the fact that both are required for normal levels of DNA methylation/hydroxymethylation. These findings describe a regulatory loop controlling the DNA methylation machinery and its readers.
2016-11-15 | GSE86965 | GEO
Project description:Cytosine methylation on DNA is an important epigenetic and regulatory mark. Chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins modulate chromatin structure and transcription at methylated loci. Two MBD proteins, Mbd2 and Mbd3, are mutually exclusive members of the NuRD chromatin remodeling complex, and have been shown to bind methylated or hydroxymethylated DNA, respectively. However, a recent study called both results into question, showing that chromatin binding by these proteins is partially (Mbd2) or completely (Mbd3) independent of DNA methylation/hydroxymethylation. Here we re-analyze these data and observe discrepancies with both conclusions. Furthermore, we describe multiple new datasets that demonstrate the dependence of endogenous Mbd2 and Mbd3 on DNA methylation. Interestingly, we find that Mbd2 and Mbd3 are also dependent on one another for binding, likely due to the fact that both are required for normal levels of DNA methylation/hydroxymethylation. These findings describe a regulatory loop controlling the DNA methylation machinery and its readers.
2016-11-15 | GSE79770 | GEO