David C. Williams

Methyl-CpG binding domain 2 (MBD2) is a critical epigenetic regulator that selectively binds methylated CpG dinucleotides, key marks controlling gene regulation and chromatin organization. Understanding the interactions and conformational dynamics underlying this high selectivity is essential to elucidate MBD2’s regulatory role. Here, using extensive classical MD simulations, we explored the formation of the MBD2–mCpG recognition complex. By initially positioning MBD2 one base pair downstream of its target, we observed its transition to the target site within microseconds. Notably, upon binding, MBD2 adopts two distinct stable conformations: a primary state closely resembling the X-ray crystal structure, and a secondary state of reduced affinity that nevertheless retains comparable selectivity for mCpG. Our results establish S189 as a key macro-switch; loss of its interaction with the methylcytosine backbone shifts the equilibrium toward the secondary state. This is corroborated by MD simulations of the S189A mutant, which preferentially adopts the secondary state-like conformation. Complementary NMR experiments confirm that S189A mutation does not alter mCpG selectivity, while fluorescence polarization measurements reveal a reduced binding affinity, consistent with our MD simulations results. Together, these findings indicate that MBD2 binding to methylated CpG involves a bistable equilibrium, providing new insights into how high affinity and adaptability are balanced in epigenetic recognition. In a broader context, our findings suggest that such alternative bound-state equilibria may represent an inherent feature of specific protein–DNA complexes.

The Nucleosome Remodeling and Deacetylase complex (NuRD) plays a key role in regulating hemoglobin expression in adult erythroid cells. Selectively disrupting this complex potently induces the expression of fetal hemoglobin, a proven therapeutic strategy for treating beta-hemoglobinopathies such as sickle cell anemia. In these studies, we have used mRNA display to identify small macrocyclic peptides that inhibit the interaction between two core components of NuRD, the SANT-SLIDE domain of CHD4 and the CR2 domain of GATAD2A. In addition, the screen suggested a second binding site on the CHD4 domain. Based on this observation, we hypothesized and confirmed that CDK2AP1 bound to this region of CHD4, leading us to purify and determine the structure of the ternary complex between CHD4, GATAD2A, and CDK2AP1. The results of our studies show that the SANT-SLIDE domain of CHD4 functions as a critical interaction hub in the formation of NuRD and suggest a strategy to block NuRD function for therapy.

Fetal hemoglobin (HbF) expression is silenced postnatally in adult erythroid cells. Sufficiently increased expression of HbF has been shown to overcome the pathophysiologic sequelae of both sickle cell disease and beta-thalassemia. As the MBD2a-NuRD chromatin remodeling complex is required for silencing of HbF, the present studies were aimed at exploring a potential therapeutic approach for disrupting this complex. AlphaFold 3 and a recent crystal structure were employed to predict the critical interaction domains linking GATAD2A in the histone deacetylase core subcomplex (HDCC) of NuRD and the CHD4 ATPase which has been shown to be required for silencing of the fetal gamma-globin ( HBG ) genes. The two predicted critical domains, the CR2 helical domain of GATAD2A and the C-terminal domains 1 and 2 (C1b and C2ab) of CHD4, were validated by in vitro biophysical studies. Mutation of two amino acids in the CR2 helical domain of the endogenous GATAD2A gene in HUDEP-2 cells resulted in dissociation of CHD4, loss of repressive chromatin over the HBG promoter and ~40% HbF levels compared to < 1% in control cells. Strikingly, enforced expression of a peptide containing the helical portion of the CR2 domain of GATAD2A in both HUDEP-2 cells and primary adult erythroid cells resulted in high levels of HbF, with up to ~75% HbF compared to mutant peptide control level of ~9% in the latter without perturbing erythroid differentiation. These results suggest that targeting the critical interaction domains of GATAD2A and CHD4 with a macrocyclic peptide or small molecule may lead to much needed small molecule therapeutics for sickle cell disease. Key Points: Association of CHD4 with the HDCC core of the MBD2-NuRD chromatin remodeling complex is required for silencing of HbF expression in adult human erythroid cells Genetic alteration or enforced peptide expression of a critical helical domain of GATAD2A results in dissociation of CHD4 from the MBD2-NuRD complex and high-level expression of HbF.

The Nucleosome Remodeling and Deacetylase complex (NuRD) plays a key role in regulating hemoglobin expression in adult erythroid cells. Selectively disrupting this complex potently induces the expression of fetal hemoglobin, a proven therapeutic strategy for treating beta-hemoglobinopathies such as sickle cell anemia. In these studies, we have used mRNA display to identify small macrocyclic peptides that inhibit the interaction between two core components of NuRD, the SANT-SLIDE domain of CHD4 and the CR2 domain of GATAD2A. In addition, the screen suggested a second binding site on the CHD4 domain. Based on this observation, we hypothesized and confirmed that CDK2AP1 bound to this region of CHD4, leading us to purify and determine the structure of the ternary complex between CHD4, GATAD2A, and CDK2AP1. The results of our studies show that the SANT-SLIDE domain of CHD4 functions as a critical interaction hub in the formation of NuRD and suggest a strategy to block NuRD function for therapy.

Sufficient levels of fetal hemoglobin (HbF) can ameliorate the pathophysiologic basis of sickle cell disease and β-thalassemia postnatally. DNA methylation has long been posited to mediate silencing of HbF expression, but this has been controversial. Recent publications provide definitive evidence for the critical role of HBG gene promoter methylation in silencing and insight into the mechanisms involved. The data support a model in which methylation of CpG sites in the HBG promoters and repressive transcription factors recruit the MBD2-NuRD chromatin remodeling complex, which enforces silencing. These findings have implications for the treatment of β-globin disorders.

The evolution of the methyl-binding domain (MBD) suggests a shared function in gene regulation, from plants to humans, with the conservation of non-DNA-interacting amino acids critical for protein-protein interactions. The MBD-regulated NuRD complex localizes to promoters of housekeeping genes with hypomethylated CpG islands. Expression profiles suggest a shared role for NuRD complex components in neurodevelopment, where the MBD3 and GATAD2B subcomplex of NuRD may be underexplored due to its non-DNA-binding biology.

Over the past 50 years, research has uncovered the co-regulatory proteins and complexes that silence the expression of the γ-globin gene in a developmental stage-specific manner. Recent research expanded the list of these regulatory factors by showing that the eight twenty-one protein 2 (ETO2) helps recruit the nucleosome remodeling and deacetylase (NuRD) complex to the globin locus. Furthermore, ETO2 regulates hematopoietic differentiation and is a potential therapeutic target for acute leukemia. In this work, we identify critical interactions between ETO2 and the GATA Zn finger domain containing the 2A (GATAD2A) component of NuRD. The ETO2 nervy homology region 4 (NHR4) domain interacts with multiple polyproline-leucine motifs within GATAD2A. We demonstrate that oligomerization of the ETO2 nervy homology region 3 (NHR3) enhances its affinity for peptides containing at least two polyproline-leucine motifs. Replacing the native motifs from GATAD2A with a higher-affinity sequence from known-binder N-CoR markedly enhances binding affinity, yielding a peptide that disrupts the interaction between ETO2 and target proteins. Enforced peptide expression elevates γ-globin expression levels and induces differentiation of HUDEP-2 and K562 cells. These findings provide insight into ETO2-mediated recruitment of co-regulatory proteins and yield a novel approach for ETO2 inhibition through multivalent binding of the NHR4 domain.

The nucleosome remodeling and deacetylase (NuRD) complex modifies nucleosome positioning and chromatin compaction to regulate gene expression. The methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) play a critical role in complex formation; however, the molecular details of how they interact with other NuRD components have yet to be fully elucidated. We previously showed that an intrinsically disordered region (IDR) of MBD2 is necessary and sufficient to bind to the histone deacetylase core of NuRD. Building on that work, we have measured the inherent structural propensity of the MBD2-IDR using solvent and site-specific paramagnetic relaxation enhancement measurements. We then used the AlphaFold2 machine learning software to generate a model of the complex between MBD2 and the histone deacetylase core of NuRD. This model is remarkably consistent with our previous studies, including the current paramagnetic relaxation enhancement data. The latter suggests that the free MBD2-IDR samples conformations similar to the bound structure. We tested this model of the complex extensively by mutating key contact residues and measuring binding using an intracellular bioluminescent resonance energy transfer assay. Furthermore, we identified protein contacts that, when mutated, disrupted gene silencing by NuRD in a cell model of fetal hemoglobin regulation. Hence, this work provides insights into the formation of NuRD and highlights critical binding pockets that may be targeted to block gene silencing for therapy. Importantly, we show that AlphaFold2 can generate a credible model of a large complex that involves an IDR that folds upon binding.

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 manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.