CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC.

Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

Clinicopathological Significance of Nucleosome Remodeling and Deacetylase Complex Expression in Endometrial Carcinoma.

BACKGROUND/AIM: Only a few studies have examined the expression of nucleosome remodeling and deacetylase complex in endometrial carcinoma (EC). The aim of this study was to analyze the expressions of histone deacetylase (HDAC1), HDAC2, and chromodomain helicase DNA-binding protein 4 (CHD4) in EC. PATIENTS AND METHODS: Sixty cases of EC were categorized into two clusters based on the expression levels of the three proteins. RESULTS: Cluster 1 (C1) exhibited elevated expressions of HDAC2 and CHD4 compared with cluster 2 (C2). Notably, 75% of cases in C2 represented non-aggressive histological types, whereas 37.5% of cases in C1 manifested aggressive types. C2 exclusively comprised pathological tumor stage 1 (pT1) tumors, whereas C1 included pT2 and pT3 tumors. In C1, 25% of cases displayed aberrant p53 expression, contrasting with the absence of such expression in C2. Furthermore, only one patient in C2 experienced disease recurrence, whereas 20.8% of patients in C1 developed recurrent tumors. CONCLUSION: High HDAC2 and CHD4 expression may be associated with adverse clinicopathological characteristics in EC. Further studies are needed to validate these results.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

Fbxw7 suppresses carcinogenesis and stemness in triple-negative breast cancer through CHD4 degradation and Wnt/ß-catenin pathway inhibition.

BACKGROUND: Cancer stem cells (CSCs) are a small population of cells in tumor tissues that can drive tumor initiation and promote tumor progression. A small number of previous studies indirectly mentioned the role of F-box and WD repeat domain-containing 7 (FBXW7) as a tumor suppressor in Triple-negative breast cancer (TNBC). However, few studies have focused on the function of FBXW7 in cancer stemness in TNBC and the related mechanism. METHODS: We detected FBXW7 by immunohistochemistry (IHC) in 80 TNBC patients. FBXW7 knockdown and overexpression in MD-MBA-231 and HCC1937 cell models were constructed. The effect of FBXW7 on malignant phenotype and stemness was assessed by colony assays, flow cytometry, transwell assays, western blot, and sphere formation assays. Immunoprecipitation-Mass Spectrometry (IP-MS) and ubiquitination experiments were used to find and verify potential downstream substrate proteins of FBXW7. Animal experiments were constructed to examine the effect of FBXW7 on tumorigenic potential and cancer stemness of TNBC cells in vivo. RESULTS: The results showed that FBXW7 was expressed at low levels in TNBC tissues and positively correlated with prognosis of TNBC patients. In vitro, FBXW7 significantly inhibited colony formation, cell cycle progression, cell migration, EMT process, cancer stemness and promotes apoptosis. Further experiments confirmed that chromodomain-helicase-DNA-binding protein 4 (CHD4) is a novel downstream target of FBXW7 and is downregulated by FBXW7 via proteasomal degradation. Moreover, CHD4 could promote the nuclear translocation of ß-catenin and reverse the inhibitory effect of FBXW7 on ß-catenin, and ultimately activate the Wnt/ß-catenin pathway. Rescue experiments confirmed that the FBXW7-CHD4-Wnt/ß-catenin axis was involved in regulating the maintenance of CSC in TNBC cells. In animal experiments, FBXW7 reduced CSC marker expression and suppressed TNBC cell tumorigenesis in vivo. CONCLUSIONS: Taken together, these results highlight that FBXW7 degrades CHD4 protein through ubiquitination, thereby blocking the activation of the Wnt/ß-catenin pathway to inhibit the stemness of TNBC cells. Thus, targeting FBXW7 may be a promising strategy for therapeutic intervention against TNBC.

The Chromatin Remodeler CHD4 Sustains Ewing Sarcoma Cell Survival by Controlling Global Chromatin Architecture.

Ewing sarcoma is an aggressive cancer with a defective response to DNA damage leading to an enhanced sensitivity to genotoxic agents. Mechanistically, Ewing sarcoma is driven by the fusion transcription factor EWS-FLI1, which reprograms the tumor cell epigenome. The nucleosome remodeling and deacetylase (NuRD) complex is an important regulator of chromatin function, controlling both gene expression and DNA damage repair, and has been associated with EWS-FLI1 activity. Here, a NuRD-focused CRISPR/Cas9 inactivation screen identified the helicase CHD4 as essential for Ewing sarcoma cell proliferation. CHD4 silencing induced tumor cell death by apoptosis and abolished colony formation. Although CHD4 and NuRD colocalized with EWS-FLI1 at enhancers and super-enhancers, CHD4 promoted Ewing sarcoma cell survival not by modulating EWS-FLI1 activity and its oncogenic gene expression program but by regulating chromatin structure. CHD4 depletion led to a global increase in DNA accessibility and induction of spontaneous DNA damage, resulting in an increased susceptibility to DNA-damaging agents. CHD4 loss delayed tumor growth in vivo, increased overall survival, and combination with PARP inhibition by olaparib treatment further suppressed tumor growth. Collectively, these findings highlight the NuRD subunit CHD4 as a therapeutic target in Ewing sarcoma that can potentiate the antitumor activity of genotoxic agents. SIGNIFICANCE: CRISPR/Cas9 screening in Ewing sarcoma identifies a dependency on CHD4, which is crucial for the maintenance of chromatin architecture to suppress DNA damage and a promising therapeutic target for DNA damage repair-deficient malignancies.

Inhibition of the chromatin remodeling factor NURF rescued sterility by a clinic variant of NuRD.

The nucleosome remodeling and deacetylase (NuRD) complex is essential for gene expression and cell fate determination, and missense mutations of NuRD caused neurodevelopmental diseases. However, the molecular pathogenesis of clinic NuRD variants is unknown. Here, we introduced a clinic CHD3 (L915F) variant into Caenorhabditis elegans homologue LET-418, impairing germline and vulva development and ultimately causing animal sterility. Our ATAC-seq and RNA-seq analyses revealed that this variant generated an abnormal open chromatin structure and disrupted the expression of developmental genes. Through genetic suppressor screens, we uncovered that intragenic mutations, likely renovating NuRD activity, restored animal viability. We also found that intergenic mutations in nucleosome remodeling factor NURF that counteracts NuRD rescued abnormal chromatin structure, gene expression, and animal sterility. We propose that two antagonistic chromatin-remodeling factors coordinate to establish the proper chromatin status and transcriptome and that inhibiting NURF may provide insights for treatment of NuRD mutation-related diseases.

RUNX1-IT1 acts as a scaffold of STAT1 and NuRD complex to promote ROS-mediated NF-κB activation and ovarian cancer progression.

Dysregulated expression of long-stranded non-coding RNAs is strongly associated with carcinogenesis. However, the precise mechanisms underlying their involvement in ovarian cancer pathogenesis remain poorly defined. Here, we found that lncRNA RUNX1-IT1 plays a crucial role in the progression of ovarian cancer. Patients with high RUNX1-IT1 expression had shorter survival and poorer outcomes. Notably, knockdown of RUNX1-IT1 suppressed the proliferation, migration and invasion of ovarian cancer cells in vitro, and reduced the formation of peritoneum metastasis in vivo. Mechanistically, RUNX1-IT1 bound to HDAC1, the core component of the NuRD complex, and STAT1, acting as a molecular scaffold of the STAT1 and NuRD complex to regulate intracellular reactive oxygen homeostasis by altering the histone modification status of downstream targets including GPX1. Consequently, RUNX1-IT1 activated NF-κB signaling and altered the biology of ovarian cancer cells. In conclusion, our findings demonstrate that RUNX1-IT1 promotes ovarian malignancy and suggest that targeting RUNX1-IT1 represents a promising therapeutic strategy for ovarian cancer treatment.

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

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.

A severe neurocognitive phenotype caused by biallelic CHD3 variants in two siblings.

CHD3 heterozygous variants are associated with Snijders Blok-Campeau syndrome (SBCS) which consists of intellectual disability (ID), macrocephaly, and dysmorphic facies. Most reported variants are missense or loss of function clustered within the ATPase/helicase domain of the protein. We report a severe neurocognitive phenotype caused by biallelic CHD3 variants in two siblings, each inherited from a mildly affected parent. Male and female siblings were referred to the Genetics Clinic due to severe ID and profound dysmorphism. The parents are first cousins of Iranian descent with borderline intellectual abilities. Exome sequencing was performed for the affected female and her parents. A single homozygous candidate variant in the CHD3 gene was detected in the proband: c.5384_5389dup. p.Arg1796_Phe1797insTrpArg, resulting in an in-frame insertion of 2 amino acids located outside the ATPase/helicase domain at the C-terminal region of CHD3-encoding residues. This variant is classified as likely pathogenic according to ACMG guidelines. The variant was detected in a heterozygous state in each parent. Both affected siblings were homozygous, while their unaffected brother did not carry the variant. Biallelic CHD3 variants cause a severe neurodevelopmental syndrome that is distinguishable from SBCS. We assume that the variant type (in-frame insertion) and location may enable CHD3 biallelic variants.

Evidence of a synthetic lethality interaction between SETDB1 histone methyltransferase and CHD4 chromatin remodeling protein in a triple negative breast cancer cell line.

During the tumorigenic process, cancer cells may become overly dependent on the activity of backup cellular pathways for their survival, representing vulnerabilities that could be exploited as therapeutic targets. Certain molecular vulnerabilities manifest as a synthetic lethality relationship, and the identification and characterization of new synthetic lethal interactions may pave the way for the development of new therapeutic approaches for human cancer. Our goal was to investigate a possible synthetic lethal interaction between a member of the Chromodomain Helicase DNA binding proteins family (CHD4) and a member of the histone methyltransferases family (SETDB1) in the molecular context of a cell line (Hs578T) representing the triple negative breast cancer (TNBC), a subtype of breast cancer lacking validated molecular targets for treatment. Therefore, we employed the CRISPR-Cas9 gene editing tool to individually or simultaneously introduce indels in the genomic loci corresponding to the catalytic domains of SETDB1 and CHD4 in the Hs578T cell line. Our main findings included: a) introduction of indels in exon 22 of SETDB1 sensitized Hs578T to the action of the genotoxic chemotherapy doxorubicin; b) by sequentially introducing indels in exon 22 of SETDB1 and exon 23 of CHD4 and tracking the percentage of the remaining wild-type sequences in the mixed cell populations generated, we obtained evidence of the existence of a synthetic lethality interaction between these genes. Considering the lack of molecular targets in TNBC, our findings provided valuable insights for development of new therapeutic approaches not only for TNBC but also for other cancer types.

ß1 integrin signaling governs necroptosis via the chromatin-remodeling factor CHD4.

Fibrosis, characterized by sustained activation of myofibroblasts and excessive extracellular matrix (ECM) deposition, is known to be associated with chronic inflammation. Receptor-interacting protein kinase 3 (RIPK3), the central kinase of necroptosis signaling, is upregulated in fibrosis and contributes to tumor necrosis factor (TNF)-mediated inflammation. In bile-duct-ligation-induced liver fibrosis, we found that myofibroblasts are the major cell type expressing RIPK3. Genetic ablation of ß1 integrin, the major profibrotic ECM receptor in fibroblasts, not only abolished ECM fibrillogenesis but also blunted RIPK3 expression via a mechanism mediated by the chromatin-remodeling factor chromodomain helicase DNA-binding protein 4 (CHD4). While the function of CHD4 has been conventionally linked to the nucleosome-remodeling deacetylase (NuRD) and CHD4-ADNP-HP1(ChAHP) complexes, we found that CHD4 potently repressed a set of genes, including Ripk3, with high locus specificity but independent of either the NuRD or the ChAHP complex. Thus, our data uncover that ß1 integrin intrinsically links fibrotic signaling to RIPK3-driven inflammation via a novel mode of action of CHD4.

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

BCL11B and the NuRD complex cooperatively guard T-cell fate and inhibit OPA1-mediated mitochondrial fusion in T cells.

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.

Snijders Blok-Campeau Syndrome: Description of 20 Additional Individuals with Variants in CHD3 and Literature Review.

Snijders Blok-Campeau syndrome (SNIBCPS, OMIM# 618205) is an extremely infrequent disease with only approximately 60 cases reported so far. SNIBCPS belongs to the group of neurodevelopmental disorders (NDDs). Clinical features of patients with SNIBCPS include global developmental delay, intellectual disability, speech and language difficulties and behavioral disorders like autism spectrum disorder. In addition, patients with SNIBCPS exhibit typical dysmorphic features including macrocephaly, hypertelorism, sparse eyebrows, broad forehead, prominent nose and pointed chin. The severity of the neurological effects as well as the presence of other features is variable among subjects. SNIBCPS is caused likely by pathogenic and pathogenic variants in CHD3 (Chromodomain Helicase DNA Binding Protein 3), which seems to be involved in chromatin remodeling by deacetylating histones. Here, we report 20 additional patients with clinical features compatible with SNIBCPS from 17 unrelated families with confirmed likely pathogenic/pathogenic variants in CHD3. Patients were analyzed by whole exome sequencing and segregation studies were performed by Sanger sequencing. Patients in this study showed different pathogenic variants affecting several functional domains of the protein. Additionally, none of the variants described here were reported in control population databases, and most computational predictors suggest that they are deleterious. The most common clinical features of the whole cohort of patients are global developmental delay (98%) and speech disorder/delay (92%). Other frequent features (51-74%) include intellectual disability, hypotonia, hypertelorism, abnormality of vision, macrocephaly and prominent forehead, among others. This study expands the number of individuals with confirmed SNIBCPS due to pathogenic or likely pathogenic variants in CHD3. Furthermore, we add evidence of the importance of the application of massive parallel sequencing for NDD patients for whom the clinical diagnosis might be challenging and where deep phenotyping is extremely useful to accurately manage and follow up the patients.

Analysis of the complex between MBD2 and the histone deacetylase core of NuRD reveals key interactions critical for gene silencing.

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.

Collateral lethality between HDAC1 and HDAC2 exploits cancer-specific NuRD complex vulnerabilities.

Transcriptional co-regulators have been widely pursued as targets for disrupting oncogenic gene regulatory programs. However, many proteins in this target class are universally essential for cell survival, which limits their therapeutic window. Here we unveil a genetic interaction between histone deacetylase 1 (HDAC1) and HDAC2, wherein each paralog is synthetically lethal with hemizygous deletion of the other. This collateral synthetic lethality is caused by recurrent chromosomal deletions that occur in diverse solid and hematological malignancies, including neuroblastoma and multiple myeloma. Using genetic disruption or dTAG-mediated degradation, we show that targeting HDAC2 suppresses the growth of HDAC1-deficient neuroblastoma in vitro and in vivo. Mechanistically, we find that targeted degradation of HDAC2 in these cells prompts the degradation of several members of the nucleosome remodeling and deacetylase (NuRD) complex, leading to diminished chromatin accessibility at HDAC2-NuRD-bound sites of the genome and impaired control of enhancer-associated transcription. Furthermore, we reveal that several of the degraded NuRD complex subunits are dependencies in neuroblastoma and multiple myeloma, providing motivation to develop paralog-selective HDAC1 or HDAC2 degraders that could leverage HDAC1/2 synthetic lethality to target NuRD vulnerabilities. Altogether, we identify HDAC1/2 collateral synthetic lethality as a potential therapeutic target and reveal an unexplored mechanism for targeting NuRD-associated cancer dependencies.

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

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.