Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal β-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiledcoil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.epigenetics | gene regulation D NA methylation involves the enzymatic addition of a methyl group at the C5 position of symmetrically opposed cytosine bases in a double-stranded cytosine-guanosine sequence (CpG). Regions of increased CpG content (CpG islands) often are found associated with promoters and, when methylated, silence expression of the associated gene (1, 2). Although most CpG islands are largely unmethylated in normal adult tissues, a subset of CpG islands is methylated in specific tissue subtypes, stages of differentiation, and development. Importantly, hypermethylation and silencing of tumor suppressor genes represents a pro-oncogenic change found in a wide range of malignancies (3). These observations have raised interest in DNA methylation as both an important genetic regulatory mechanism and a potential therapeutic target for either re-expression of developmentally silenced genes or reversing tumor suppressor gene silencing in cancer (4, 5).The methyl cytosine binding proteins include a family that specifically recognizes the methylated CpG sequence through an ∼60 amino acid methyl-binding domain (MBD). There are five members of the MBD family in mammals: methyl CpG-binding protein 2 (MeCP2), the first to be identified (6), and MBD1 through MBD4 (7). We and others have isolated and characterized an MBD2-containing nucleosome remodeling and deacetylation (NuRD) complex (referred to as "MBD2-NuRD") that binds methylated DNA and regulates transcription of the associated gene (8-10). The MBD2-NuRD complex comprises at least one homolog of six core proteins: MBD2, retinoblastoma protein-associated protein (RbAp46 or -48) Mi-2(α or β), p66(α or β), histone deacetylase (HDAC1 or 2), and metastasis associated (MTA1 or -2) (Fig. 1A). However, the specific interactions involved in the formation of the MBD2-NuRD complex have not been delineated clearly; information that is key to understanding (i) ...

An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD, play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. We observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine chemical inducer of dimerization hematopoietic cells and in CD34(+) progenitor-derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, <50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34(+) progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.

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Background: Trimethylamine N-oxide (TMAO) is reported to promote the pathogenesis of atherosclerosis and be associated with cardiovascular events risk. It is unknown whether plasma TMAO is associated with plaque morphology in patients with acute myocardial infarction. We investigated the relationship between the culprit plaque morphology and plasma TMAO concentration in patients with ST-segment–elevation myocardial infarction. Methods and Results: A prospective series of 211 patients with ST-segment–elevation myocardial infarction who underwent preintervention optical coherence tomography examination for the culprit lesion were enrolled; 77 and 69 patients were categorized as plaque rupture and plaque erosion, respectively. Plasma TMAO levels, detected using stable isotope dilution liquid chromatography tandem mass spectrometry, were significantly higher in patients with plaque rupture than in those with plaque erosion (3.33 μM; interquartile range: 2.48–4.57 versus 1.21 μM; interquartile range: 0.86–1.91; P <0.001). After adjustments for traditional risk factors, elevated TMAO levels remained independently correlated with plaque rupture (adjusted odds ratio: 4.06, 95% CI, 2.38–6.91; P <0.001). The area under the receiver operating characteristic curve for plaque rupture versus plaque erosion was 0.89. At a cutoff level of 1.95 μM, TMAO had a sensitivity of 88.3% and specificity of 76.8% in discriminating plaque rupture from plaque erosion. Conclusions: High levels of plasma TMAO independently correlated with plaque rupture in patients with ST-segment–elevation myocardial infarction. Moreover, TMAO might be a useful biomarker for plaque rupture to improve risk stratification and management in patients with ST-segment–elevation myocardial infarction. Clinical Trial Registration: URL: https://www.clinicaltrials.gov . Unique identifiers: NCT03593928.

Key Points CHD4 depletion sensitizes AML cells but not normal CD34+ progenitors to genotoxic agents by relaxing chromatin and impairing DSB repair. CHD4 depletion modulates expression of AML cell genes that regulate tumor formation in vivo and colony formation in vitro.

The methylation pattern of a 248-base pair proximal transcribed region (248) of the avian embryonic -globin gene was found to correlate inversely with stagespecific expression in avian erythroid cells. In vitro methylation of the 248 segment alone (in the absence of promoter methylation) resulted in a 5-fold inhibition of transcription in a transient transfection assay in primary erythroid cells in which the transfected gene is packaged into nucleosomal chromatin. This effect was observed if the 248 segment was positioned adjacent to the promoter but not when it was located 2.7 kilobases downstream. Fully methylated but not unmethylated 248 formed a novel cell type-specific methyl cytosinebinding protein complex (MeCPC) that contained methyl binding domain protein-2 (MBD-2) and histone deacetylase 1 proteins but differed from MeCP-1. The histone deacetylase inhibitor trichostatin A failed to relieve methylation-mediated repression of transcription from the -gene promoter, supporting the notion of the dominance of methylation over histone deacetylation in silencing through CpG-rich sequences at this locus. These data demonstrate that site-specific methylation of a vertebrate gene 5-transcribed region alone at the exact CpGs that are methylated in vivo can suppress transcription in homologous primary cells and facilitate binding to a cell type-specific MeCPC.

The chicken embryonic ␤-type globin gene, , is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated -globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zincfinger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pulldown assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive -globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active ␤ A -globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent -globin IntroductionMethylation of the 5-position of cytosine residues in DNA has an important role in the regulation of gene expression in higher eukaryotes. The first descriptions of an inverse correlation between DNA methylation and expression of a protein-coding eukaryotic gene were those made for the vertebrate globin genes of the chicken, rabbit, and human. [1][2][3][4] The tremendous interest in DNA methylation seen within the past decade stems from its important role in the silencing of tumor suppressor genes in cancer. 5,6 Despite the prediction of a widespread role for DNA methylation in gene regulation, 7 the expression of only a small number of vertebrate genes has been shown to be tissue-restricted or developmentally restricted by DNA methylation. 8 Studies showing tissue-restricted or developmentally restricted expression in nontransformed, primary cells are even more scarce and are limited to 4 main examples: the Gallus gallus -globin gene, 9,10 the Mus musculus interleukin-4 and interferon-␥ genes, 11 and the Xenopus laevis mesodermal genes. 12 Intense study has been directed toward elucidating the mechanisms through which DNA methylation represses transcription. The discovery of a family of proteins that specifically recognize methylated DNA, the methyl-CpG-binding proteins (MCBPs), 13,14 has led to the general observation of these proteins and their associated corepressor complexes as the mediators of DNA methylation-induced gene silencing in a variety of systems. [15][16][17][18][19] Biochemical studies have shown that the MCBPs are members of distinct and nonoverlapping transcriptional repression complexes. MBD1 was identified as a critical component of an S phase-specific complex that propagates the DNA methylation signal into a dimethylation of lysine 9 of histone H3 (H3-K9-Me 2 ) signal during DNA replication. 17 MBD2 is the methyl-CpG-binding component of the MeCP1 transcriptional repression complex. 20 MBD3 is a core component of the NuRD transcriptional repression complex that can be recruited by ...

The selectivity of coupling of m1, m3, and m5 muscarinic receptors to activation of the neuronal type of nitric oxide synthase was investigated. Stimulation with the agonist carbachol of all three receptor subtypes expressed in Chinese hamster ovary cells resulted in a rapid and transient activation of the enzyme, as measured by stimulation of guanylate cyclase in reporter neuroblastoma cells. Carbachol was more potent and efficacious at m5 receptors than at the other two receptor subtypes. Stimulation of all three muscarinic receptors resulted in an increased concentration of intracellular calcium, with a time course that preceded activation of nitric oxide synthase. At each receptor subtype, there was a close relationship between the magnitude of the maximal calcium response and that of enzyme activation.