Nucleosome Remodeling And Deacetylase Subunit Research Articles

Abstract B010: The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture

Abstract Ewing sarcoma tumorigenesis is tightly linked to epigenetic deregulation. Indeed, the aberrant fusion transcription factor and tumor driver EWS-FLI1 produces extensive rewiring of the cancer cell epigenome. At enhancers containing canonical ETS motifs, the fusion protein represses gene expression but, when binding to GGAA repeats, EWS-FLI1 induces transcription by recruiting activating epigenetic regulators such as the acetyltransferase p300 and the BAF chromatin remodeling complex. The nucleosome remodeling and deacetylase (NuRD) complex is an ATP-dependent multi-subunit complex that modulates chromatin architecture and regulates DNA damage repair, genome stability and gene expression. In Ewing sarcoma, NuRD subunits, such as LSD1, have been suggested to interfere with EWS-FLI1 activity and prevent tumor progression. Hence, we here aimed to comprehensively study the role of NuRD in Ewing sarcoma pathogenesis. First, we performed a NuRD-centered, negative selection, CRISPR/Cas9 screen and identified the chromatin remodeling helicase CHD4 as essential for Ewing sarcoma tumor cell survival. Validation assays using two doxycycline-inducible shRNAs targeting CHD4 demonstrated that silencing of this remodeler in fact drastically reduces tumor cell proliferation and completely prevents colony formation. This cell proliferation impairment was caused by an induction of cell death by apoptosis and not by cell cycle arrest. Surprisingly, this cell death phenotype was not linked to impaired fusion protein activity. CHD4 and NuRD, despite primarily locating to enhancers and super-enhancers in Ewing sarcoma cells, did not preferentially localize to GGAA repeats, unlike EWS-FLI1. Moreover, RNA sequencing assays performed upon CHD4 silencing showed that this ATPase does not regulate the EWS-FLI1 signature. As CHD4 is a chromatin remodeler able to move nucleosomes along the DNA, we performed ATAC sequencing experiments to investigate changes in chromatin status that could explain the dependency of Ewing sarcoma cells on CHD4 for survival. We observed that CHD4 depletion from Ewing sarcoma cells causes a drastic and global chromatin relaxation which renders tumor cells prone to DNA damage and increasingly sensitive to DNA damaging agents. Interestingly, augmented sensitivity to DNA damage was not observed by silencing CHD4 in non-tumorigenic human fibroblasts. Finally, CHD4 depletion also reduced Ewing sarcoma tumor growth in vivo and prolonged mice survival. In conclusion, we demonstrate for the first time that CHD4 is crucial for Ewing sarcoma cell survival and highlight this helicase as a promising therapeutic target for Ewing sarcoma with potential for combination therapy with drugs inducing DNA damage. Importantly, this work has initiated ongoing efforts to develop first-in-class small molecules specifically targeting CHD4 which will be crucial for the future validation of this ATPase as a viable target with clinical application. Citation Format: Joana Graca Marques, Blaz Pavlovic, Quy Ai Ngo, Gloria Pedot, Michaela Roemmele, Larissa Volken, Marco Wachtel, Beat W. Schäfer. The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B010.

Cross-linking mass spectrometry reveals the structural topology of peripheral NuRD subunits relative to the core complex.

The multi-subunit nucleosome remodeling and deacetylase (NuRD) complex consists of seven subunits, each of which comprises two or three paralogs in vertebrates. These paralogs define mutually exclusive and functionally distinct complexes. In addition, several proteins in the complex are multimeric, which complicates structural studies. Attempts to purify sufficient amounts of endogenous complex or recombinantly reconstitute the complex for structural studies have proven quite challenging. Until now, only substructures of individual domains or proteins and low-resolution densities of (partial) complexes have been reported. In this study, we comprehensively investigated the relative orientation of different subunits within the NuRD complex using multiple cross-link IP mass spectrometry (xIP-MS) experiments. Our results confirm that the core of the complex is formed by MTA, RBBP, and HDAC proteins. Assembly of a copy of MBD and GATAD2 onto this core enables binding of the peripheral CHD and CDK2AP proteins. Furthermore, our experiments reveal that not only CDK2AP1 but also CDK2AP2 interacts with the NuRD complex. This interaction requires the C terminus of CHD proteins. Our data provide a more detailed understanding of the topology of the peripheral NuRD subunits relative to the core complex. DATABASE: Proteomics data are available in the PRIDE database under the accession numbers PXD017244 and PXD017378.

Abstract PO-009: Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma

Abstract Cancer-specific chromosomal aberrations producing chimeric fusion genes are recurrently found in pediatric sarcomas. Fusion positive rhabdomyosarcoma (FP-RMS) and Ewing sarcoma (ES) are two rare but lethal pediatric malignancies driven by such chromosomal translocations. PAX3-FOXO1 and EWS-FLI1 are the most common products of the fusion genes found in FP-RMS and ES, respectively, and they are commonly perceived as the founding genetic abnormality driving the development of these malignancies by changing gene expression. Since direct targeting of transcription factors is still very challenging, acting on the activity of these oncogenic transcription factors at the chromatin level presents a robust alternative for targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex subunit CHD4 has been previously identified as an interactor of both PAX3-FOXO1 and EWS-FLI1. Hence, we decided here to further characterize the role of this chromatin remodeler in the regulation of fusion protein-mediated gene expression in both FP-RMS and ES. Our NuRD-centered CRISPR/Cas9 screen demonstrated that both these malignancies are especially dependent on CHD4 amongst all other complex members. In fact, CHD4 silencing in both tumors through shRNA knockdown or CRISPR knockout drastically reduces tumor cell proliferation and induces cell death. In vivo, CHD4 knockdown also impaired tumour growth in both FP-RMS and ES. Mechanistically, our RNA-seq assays demonstrated that silencing of the nucleosome remodeller CHD4 alters gene expression in both tumours and our ChIP-seq experiments show that CHD4 binding sites are highly enriched for the binding motif of PAX3-FOXO1 in FP-RMS and EWS-FLI1 in ES. In FP-RMS, we observed that CHD4 particularly regulates super-enhancer accessibility creating a chromatin architecture permissive to the binding of PAX3-FOXO1 and allowing the expression of the fusion gene signature. Similar studies in ES to further investigate CHD4 as a regulator of gene expression are currently ongoing. Finally, our analysis of genome-wide cancer dependency databases identified CHD4 as general novel cancer vulnerability amongst NuRD subunits and other SNF2-like ATPases. In summary, we have unravelled the prominent role of CHD4 in regulation of super-enhancer driven gene expression in FP-RMS and exposed this chromatin remodeler as novel potential drug target for pediatric sarcoma therapy. Our work has motivated us to establish several collaborations with computational and biophysics experts and we are now currently working to identify the first CHD4 specific small molecule inhibitor. Citation Format: Joana G. Marques, Berkley Gryder, Blaz Pavlovic, Yeonjoo Chung, Quy Ngo, Marco Wachtel, Javed Khan, Beat Schäfer. Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-009.

The nucleosome remodeling and deacetylase complex protein CHD4 regulates neural differentiation of mouse embryonic stem cells by down-regulating p53

Lineage specification of the three germ layers occurs during early embryogenesis and is critical for normal development. The nucleosome remodeling and deacetylase (NuRD) complex is a repressive chromatin modifier that plays a role in lineage commitment. However, the role of chromodomain helicase DNA-binding protein 4 (CHD4), one of the core subunits of the NuRD complex, in neural lineage commitment is poorly understood. Here, we report that the CHD4/NuRD complex plays a critical role in neural differentiation of mouse embryonic stem cells (ESCs). We found that RNAi-mediated Chd4 knockdown suppresses neural differentiation, as did knockdown of methyl-CpG-binding domain protein Mbd3, another NuRD subunit. Chd4 and Mbd3 knockdowns similarly affected changes in global gene expression during neural differentiation and up-regulated several mesendodermal genes. However, inhibition of mesendodermal genes by knocking out the master regulators of mesendodermal lineages, Brachyury and Eomes, through a CRISPR/Cas9 approach could not restore the impaired neural differentiation caused by the Chd4 knockdown, suggesting that CHD4 controls neural differentiation by not repressing other lineage differentiation processes. Notably, Chd4 knockdown increased the acetylation levels of p53, resulting in increased protein levels of p53. Double knockdown of Chd4 and p53 restored the neural differentiation rate. Furthermore, overexpression of BCL2, a downstream factor of p53, partially rescued the impaired neural differentiation caused by the Chd4 knockdown. Our findings reveal that the CHD4/NuRD complex regulates neural differentiation of ESCs by down-regulating p53.

Rational Targeting of a NuRD Sub-Complex for Fetal Hemoglobin Induction Following Comprehensive in Situ Mutagenesis

Sickle cell disease and β-thalassemia are major hemoglobin disorders for which induction of fetal hemoglobin (HbF) can mitigate disease severity. However, the molecular mechanisms underlying the developmental repression of HbF remain incompletely understood. The nucleosome remodeling and deacetylase (NuRD) complex is a major negative regulator of HbF level. In this study, we sought to identify possible rational therapeutic strategies targeting critical NuRD determinants.We employed comprehensive dense mutagenesis using pooled CRISPR screening in HUDEP-2 human erythroid precursors to disrupt protein coding sequences of all 13 genes of the NuRD complex, including CHD, MTA, GATAD2, HDAC, MBD, and RBBP family members. The custom sgRNA library included 5,038 sgRNAs. We found that only 5 genes, CHD4, MTA2, GATAD2A, HDAC2, and MBD2, were required for HbF repression, suggesting that a non-redundant NuRD sub-complex contributes to HbF silencing. We validated the existence of this NuRD sub-complex by mass spectrometry analysis after immunoprecipitation of CHD4 and MTA2 as well as MTA2-BioID2 mediated proximity labeling. Remarkably, 5 of the 6 NuRD subunit proteins commonly detected by these three methods were identified as functional by CRISPR screening (MTA2, RBBP4, CHD4, GATAD2A, HDAC2).Disruption of CHD4 resulted in the highest HbF induction of any of the NuRD subunits. However, unlike the other NuRD genes, CHD4 disruption also led to cellular toxicity. We observed a small group of sgRNAs within the CHDCT2 domain of CHD4 associated with high HbF induction yet relatively modest negative fitness. We validated by electroporation of Cas9:sgRNA to CD34+ HSPC primary erythroid precursors that in-frame mutations of CHD4 CHDCT2 escape cellular toxicity while inducing HbF. Similarly, we targeted homologous amino acid residues within mouse Chd4 CHDCT2 domain by Cas9 mutagenesis in mouse oocytes. While loss of Chd4 is lethal at the blastocyst stage, homozygous in-frame deletions within the Chd4 CHDCT2 domain are tolerated in mouse embryos and result in increased γ-globin expression in mid-gestation embryos bearing transgenic human β-globin gene clusters.To investigate the mechanism whereby in-frame deletions at CHD4 CHDCT2 impact NuRD, we performed glycerol gradient density sedimentation, which revealed that these in-frame mutations impair the recruitment of CHD4 to the NuRD complex. A recent study demonstrated that the previously poorly characterized CHD4 CHDCT2 domain directly binds to GATAD2 factors (Torrado et al, FEBS J, 2017). We observed a cluster of sgRNAs associated with heightened HbF enrichment scores at the C-terminal region of GATAD2A encompassing a C2C2-type GATA zinc finger. We hypothesized that ectopic expression of this GATAD2A zinc finger might competitively bind to CHD4 and displace CHD4 from NuRD. Overexpression of the GATAD2A zinc finger in both HUDEP-2 and CD34+ HSPC derived primary erythroid precursors led to robust induction of HbF without negatively impacting cellular fitness. Immunoprecipitation of the GATAD2A zinc finger enriched CHD4 but not other endogenous NuRD components, such as GATAD2A or MBD2. Moreover, glycerol gradient density sedimentation showed that the GATAD2A zinc finger co-sedimented with sub-NuRD fractions of CHD4. Together these data suggest that expression of the GATAD2A zinc finger sequesters CHD4 from NuRD, yet spares cytotoxicity.In summary, we show that biochemical disruption of the CHD4-GATAD2A interaction could serve as a rational therapeutic strategy to potently induce HbF for the β-hemoglobin disorders while preventing cellular toxicity associated with complete CHD4 inhibition. DisclosuresNo relevant conflicts of interest to declare.

Covalent Modifications of Histone H3K9 Promote Binding of CHD3

Chromatin remodeling is required for genome function and is facilitated by ATP-dependent complexes, such as nucleosome remodeling and deacetylase (NuRD). Among its core components is the chromodomain helicase DNA binding protein 3 (CHD3) whose functional significance is not well established. Here, we show that CHD3 co-localizes with the other NuRD subunits, including HDAC1, near the H3K9ac-enriched promoters of the NuRD target genes. The tandem PHD fingers of CHD3 bind histone H3 tails and posttranslational modifications that increase hydrophobicity of H3K9-methylation or acetylation (H3K9me3 or H3K9ac)-enhance this interaction. Binding of CHD3 PHDs promotes H3K9Cme3-nucleosome unwrapping invitro and perturbs the pericentric heterochromatin structure invivo. Methylation oracetylation of H3K9 uniquely alleviates the intra-nucleosomal interaction of histone H3 tails, increasing H3K9 accessibility. Collectively, our data suggest that the targeting of covalently modified H3K9 by CHD3 might be essential in diverse functions of NuRD.

DOC1-Dependent Recruitment of NURD Reveals Antagonism with SWI/SNF during Epithelial-Mesenchymal Transition in Oral Cancer Cells

The Nucleosome Remodeling and Deacetylase (NURD) complex is a key regulator of cell differentiation that has also been implicated in tumorigenesis. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCC cells leads to a reversal of epithelial-mesenchymal transition (EMT). This is caused by the DOC1-dependent targeting ofNURD to repress key transcriptional regulators ofEMT. NURD recruitment drives extensive epigenetic reprogramming, including eviction of the SWI/SNFremodeler, formation of inaccessible chromatin,H3K27 deacetylation, and binding of PRC2 and KDM1A, followed by H3K27 methylation and H3K4 demethylation. Strikingly, depletion of SWI/SNF mimics the effects of DOC1 re-expression. Our results suggest that SWI/SNF and NURD function antagonistically to control chromatin state and transcription. We propose that disturbance of this dynamic equilibrium may lead to defects in gene expression that promote oncogenesis.

An Embryonic Stem Cell-Specific NuRD Complex Functions through Interaction with WDR5.

SummaryThe Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin regulatory complex that functions as a transcriptional co-repressor in metazoans. The NuRD subunit MBD3 is essential for targeting and assembly of a functional NuRD complex as well as embryonic stem cell (ESC) pluripotency. Three MBD3 isoforms (MBD3A, MBD3B, and MBD3C) are expressed in mouse. Here, we find that the MBD3C isoform contains a unique 50-amino-acid N-terminal region that is necessary for MBD3C to specifically interact with the histone H3 binding protein WDR5. Domain analyses of WDR5 reveal that the H3 binding pocket is required for interaction with MBD3C. We find that while Mbd3c knockout ESCs differentiate normally, MBD3C is redundant with the MBD3A and MBD3B isoforms in regulation of gene expression, with the unique MBD3C N terminus required for this redundancy. Together, our data characterize a unique NuRD complex variant that functions specifically in ESCs.

Structural Basis of Plant Homeodomain Finger 6 (PHF6) Recognition by the Retinoblastoma Binding Protein 4 (RBBP4) Component of the Nucleosome Remodeling and Deacetylase (NuRD) Complex

The NuRD complex is a conserved transcriptional coregulator that contains both chromatin-remodeling and histone deacetylase activities. Mutations of PHF6 are found in patients with Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, or acute myeloid leukemia. Recently, PHF6 was identified to interact with the NuRD complex, and this interaction is mediated by the RBBP4 component. However, little is known about the molecular basis for the interaction. Here, we present the crystal structure of the complex of the NuRD subunit RBBP4 bound to the PHF6 peptide (residues 162-170). The PHF6 peptide binds to the top surface of the RBBP4 β-propeller. A pair of positively charged residues of the PHF6 peptide insert into the negatively charged pocket of RBBP4, which is critical for the interaction between PHF6 and RBBP4. Corresponding PHF6 mutants impair this interaction in vitro and in vivo. Structural comparison shows that the PHF6-binding pocket overlaps with FOG1 and histone H3 on RBBP4/Nurf55, but it is distinct from the pocket recognizing histone H4, Su(z)12, and MTA1. We further show that the middle disordered region (residues 145-207, containing the RBBP4-binding motif) is sufficient for the transcriptional repression mediated by PHF6 on the GAL4 reporter, and knockdown of RBBP4 diminished the PHF6-mediated repression. Our RBBP4-PHF6 complex structure provides insights into the molecular basis of PHF6-NuRD complex interaction and implicates a role for PHF6 in chromatin structure modulation and gene regulation.

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

The NuRD architecture.

The nucleosome remodeling and deacetylase (NuRD) complex regulates chromatin organization, gene transcription, genomic stability and developmental signaling. NuRD has a unique dual enzymatic activity, containing an ATPase and a histone deacetylase among its six core subunits. Recent studies indicate that NuRD composition and the interplay between subunits may dictate the diverse functions of the complex. In this review, we examine the structures and biological roles of the NuRD subunits and discuss new avenues of research to advance our understanding of the NuRD-mediated signaling network.

MBD2 and Multiple Domains of CHD4 Are Required for Transcriptional Repression by Mi-2/NuRD Complexes

Mi-2/nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complexes are important regulators of chromatin structure and DNA accessibility. We examined requirements for individual domains of chromodomain helicase DNA-binding protein 4 (CHD4), a core catalytic component of NuRD complexes, as well as the NuRD subunit methyl-binding domain protein 2 (MBD2) and methylated DNA, for NuRD function in the context of tissue-specific transcription. By itself, loss of NuRD activity is not sufficient for transcriptional activation. However, NuRD complexes greatly reduce activation of the B cell-specific mb-1 (Cd79a) gene by the transcription factors EBF1 and Pax5. Using our B cell model system, we determined that the two chromodomains and ATPase/helicase and C-terminal domains (CTD) of CHD4 are all necessary for repression of mb-1 promoters by NuRD. All of these domains except the CTD are required for efficient association of CHD4 with mb-1 promoter chromatin. Loss of MBD2 expression or of DNA methylation impaired association of CHD4 with mb-1 promoter chromatin and enhanced its transcription. We conclude that repressive functions of MBD2-containing NuRD complexes are dependent on cooperative interactions between the major domains of CHD4 with histones and DNA and on binding of methylated DNA by MBD2.

Different Mi-2 complexes for various developmental functions in Caenorhabditis elegans.

Biochemical purifications from mammalian cells and Xenopus oocytes revealed that vertebrate Mi-2 proteins reside in multisubunit NuRD (Nucleosome Remodeling and Deacetylase) complexes. Since all NuRD subunits are highly conserved in the genomes of C. elegans and Drosophila, it was suggested that NuRD complexes also exist in invertebrates. Recently, a novel dMec complex, composed of dMi-2 and dMEP-1 was identified in Drosophila. The genome of C. elegans encodes two highly homologous Mi-2 orthologues, LET-418 and CHD-3. Here we demonstrate that these proteins define at least three different protein complexes, two distinct NuRD complexes and one MEC complex. The two canonical NuRD complexes share the same core subunits HDA-1/HDAC, LIN-53/RbAp and LIN-40/MTA, but differ in their Mi-2 orthologues LET-418 or CHD-3. LET-418 but not CHD-3, interacts with the Krüppel-like protein MEP-1 in a distinct complex, the MEC complex. Based on microarrays analyses, we propose that MEC constitutes an important LET-418 containing regulatory complex during C. elegans embryonic and early larval development. It is required for the repression of germline potential in somatic cells and acts when blastomeres are still dividing and differentiating. The two NuRD complexes may not be important for the early development, but may act later during postembryonic development. Altogether, our data suggest a considerable complexity in the composition, the developmental function and the tissue-specificity of the different C. elegans Mi-2 complexes.

NAB2 Represses Transcription by Interacting with the CHD4 Subunit of the Nucleosome Remodeling and Deacetylase (NuRD) Complex

Early growth response (EGR) transactivators act as critical regulators of several physiological processes, including peripheral nerve myelination and progression of prostate cancer. The NAB1 and NAB2 (NGFI-A/EGR1-binding protein) transcriptional corepressors directly interact with three EGR family members (Egr1/NGFI-A/zif268, Egr2/Krox20, and Egr3) and repress activation of their target promoters. To understand the molecular mechanisms underlying NAB repression, we found that EGR activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with the CHD4 (chromodomain helicase DNA-binding protein 4) subunit of the NuRD (nucleosome remodeling and deacetylase) chromatin remodeling complex. Both NAB proteins can bind either CHD3 or CHD4, indicating that the interaction is conserved among these two protein families. Furthermore, we show that repression of the endogenous Rad gene by NAB2 involves interaction with CHD4 and demonstrate colocalization of NAB2 and CHD4 on the Rad promoter in myelinating Schwann cells. Finally, we show that interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA.