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) ...

The MBD2-NuRD (Nucleosome Remodeling and Deacetylase) complex is an epigenetic reader of DNA methylation that regulates genes involved in normal development and neoplastic diseases. To delineate the architecture and functional interactions of the MBD2-NuRD complex, we previously solved the structures of MBD2 bound to methylated DNA and a coiled-coil interaction between MBD2 and p66α that recruits the CHD4 nucleosome remodeling protein to the complex. The work presented here identifies novel structural and functional features of a previously uncharacterized domain of MBD2 (MBD2IDR). Biophysical analyses show that the MBD2IDR is an intrinsically disordered region (IDR). However, despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg286 and Leu287. Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.

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.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.Neurodevelopmental disorders (NDDs) and intellectual disability (ID) affect approximately 1%-3% of the general population. 1-3 NDDs/ID are largely genetically determined, but identification of the underlying molecular causes has been hampered by clinical and genetic heterogeneity. During the last decades, the use of high-resolution array-based copy number variant (CNV) analysis and second-generation sequencing techniques has improved our knowledge of the genetic basis of both syndromic and non-syndromic NDDs/ID. [2][3][4] Notwithstanding these achievements, the genetic basis of NDDs/ID is still unsolved in a large proportion of affected individuals.Deletion 1p36 (del1p36) syndrome, first described by Shapira and colleagues in 1997, 5 is the most common autosomal terminal deletion syndrome in humans, occurring in about 1 in 5,000 births. [6][7][8] This disorder is characterized by developmental delay (DD)/ID, behavioral abnormalities, hypotonia, seizures, brain anomalies, vision problems, hearing loss, orofacial clefting, congenital heart defects (CHDs), cardiomyopathy, renal anomalies, short