Nucleosome Remodeling And Deacetylase Research Articles

CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration

SummaryRegeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.

PAX9 Determines Epigenetic State Transition and Cell Fate in Cancer.

Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and cofunctioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacologic HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that reactivation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9. SIGNIFICANCE: A genome-wide screen in small cell lung cancer reveals PAX9/NuRD-mediated epigenetic enhancer silencing and tumor progression, supporting the development of novel personalized therapeutic approaches targeting the PAX9-regulated network.

Unique protein interaction networks define the chromatin remodelling module of the NuRD complex.

The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1 and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodelling module of the nucleosome remodelling and deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue‐specific function and specificity have not been addressed. Understanding the protein–protein interaction (PPI) network of the MGCC subunits is essential for defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD‐MS), we report a proteome‐wide interaction network of the MGCC module in a paralogue‐specific manner. Our data also demonstrate that the disordered C‐terminal region of CHD3/4/5 is a gateway to incorporate remodelling activity into both ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation‐prone region (APR) within the C‐terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the nuclear receptor co‐repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development.

Simple SummaryMost epithelial ovarian cancer (EOC) patients, although initially responsive to standard treatment with platinum-based chemotherapy, develop platinum resistance over the clinical course and succumb due to drug-resistant metastases. It has long been hypothesized that resistance to platinum develops as a result of epigenetic changes within tumor cells evolving over time. In this study, we investigated epigenomic changes in EOC patient samples, as well as in cell lines, and showed that profound changes at enhancers result in a platinum-resistant phenotype. Through correlation of the epigenomic alterations with changes in the transcriptome, we could identify potential novel prognostic biomarkers for early patient stratification. Furthermore, we applied a combinatorial RNAi screening approach to identify suitable targets that prevent the enhancer remodeling process. Our results advance the molecular understanding of epigenetic mechanisms in EOC and therapy resistance, which will be essential for the further exploration of epigenetic drug targets and combinatorial treatment regimes.Epithelial ovarian cancer (EOC) is the most lethal disease of the female reproductive tract, and although most patients respond to the initial treatment with platinum (cPt)-based compounds, relapse is very common. We investigated the role of epigenetic changes in cPt-sensitive and -resistant EOC cell lines and found distinct differences in their enhancer landscape. Clinical data revealed that two genes (JAK1 and FGF10), which gained large enhancer clusters in resistant EOC cell lines, could provide novel biomarkers for early patient stratification with statistical independence for JAK1. To modulate the enhancer remodeling process and prevent the acquisition of cPt resistance in EOC cells, we performed a chromatin-focused RNAi screen in the presence of cPt. We identified subunits of the Nucleosome Remodeling and Deacetylase (NuRD) complex as critical factors sensitizing the EOC cell line A2780 to platinum treatment. Suppression of the Methyl-CpG Binding Domain Protein 3 (MBD3) sensitized cells and prevented the establishment of resistance under prolonged cPt exposure through alterations of H3K27ac at enhancer regions, which are differentially regulated in cPt-resistant cells, leading to a less aggressive phenotype. Our work establishes JAK1 as an independent prognostic marker and the NuRD complex as a potential target for combinational therapy.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

High-Resolution CRISPR Functional Genomics Reveals Critical Vulnerabilities of the NuRD Complex for Fetal Hemoglobin Control

Hemoglobinopathies resulting from mutations of the adult beta-globin gene affect millions worldwide. Persistently elevated levels of fetal hemoglobin (HbF) are without consequence in healthy individuals but convey a major clinical benefit in patients with sickle cell disease and beta-thalassemia. Fetal globin silencing relies on interactions between DNA binding factors and chromatin readers, writers, and erasers acting within multiprotein nuclear complexes. The nucleosome remodeling and deacetylase (NuRD) complex is an epigenetic chromatin modifier, including ATP-dependent nucleosome remodeling and histone deacetylase activities, that has been implicated in HbF control. BCL11A and ZBTB7A, two key transcription factors required for HbF repression in adult erythroid cells, physically interact with NuRD complex members. In order to identify potential target sites within the NuRD complex with maximal HbF de-repression and minimal cellular toxicity potential we set out to dissect the function of the NuRD complex in adult erythroid precursors by dense mutagenesis. We performed CRISPR-Cas9 mediated saturating mutagenesis of all coding regions of human NuRD complex members in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) adult erythroid cells with HbF expression as the primary readout and cell fitness as a counter-screen. This approach distinguished four classes of NuRD complex members: those required for HbF repression but dispensable for cellular fitness (such as MTA2, MBD2, HDAC2, and GATAD2A), those required for both HbF repression and cell fitness (such as CHD4), those only required for cell fitness (such as RBBP4), and many not essential for either HbF repression or cell fitness. The NuRD paralogs essential for HbF repression correlated with the abundance of peptides retrieved by MTA2 immunoprecipitation followed by mass spectrometry, suggesting a NuRD sub-complex that silences fetal globin. We demonstrated that this tiling CRISPR screen approach identifies residues at which short in-frame deletions result in loss of function, including at catalytic residues of CHD4 and HDAC2, additional NuRD domains and conserved regions of unknown significance. By mapping functional data onto existing structures we detected sequences essential for HbF repression within the ELM2 and BAH domains of MTA2 that mediate key protein-protein interactions with HDAC1/2 and with chromatin. At CHD4, we found numerous critical positions where in-frame alleles resulted in robust HbF elevation to a greater magnitude than disruption of any other NuRD member. However nearly all of these HbF-inducing perturbations at CHD4 were inversely correlated with cell fitness. In contrast, we observed that in-frame deletions within the CHDCT2 domain of CHD4 resulted in profound elevation of HbF level but spared the negative impact on cell growth. Overall, these results demonstrate a sub-complex of NuRD essential for HbF silencing and identify key interfaces including at the potent HbF repressor CHD4 which could serve as selective small molecule development targets for therapeutic HbF re-induction. DisclosuresOrkin:Epizyme Inc.: Consultancy; Bioverativ: Consultancy.

Identification of retinoblastoma binding protein 7 (Rbbp7) as a mediator against tau acetylation and subsequent neuronal loss in Alzheimer\u2019s disease and related tauopathies

Evidence indicates that tau hyper-phosphorylation and subsequent neurofibrillary tangle formation contribute to the extensive neuronal death in Alzheimer’s disease (AD) and related tauopathies. Recent work has identified that increased tau acetylation can promote tau phosphorylation. Tau acetylation occurs at lysine 280 resulting from increased expression of the lysine acetyltransferase p300. The exact upstream mechanisms mediating p300 expression remain elusive. Additional work highlights the role of the epigenome in tau pathogenesis, suggesting that dysregulation of epigenetic proteins may contribute to acetylation and hyper-phosphorylation of tau. Here, we identify and focus on the histone-binding subunit of the Nucleosome Remodeling and Deacetylase (NuRD) complex: Retinoblastoma-Binding Protein 7 (Rbbp7). Rbbp7 chaperones chromatin remodeling proteins to their nuclear histone substrates, including histone acetylases and deacetylases. Notably, Rbbp7 binds to p300, suggesting that it may play a role in modulating tau acetylation. We interrogated Rbbp7 in post-mortem brain tissue, cell lines and mouse models of AD. We found reduced Rbbp7 mRNA expression in AD cases, a significant negative correlation with CERAD (neuritic plaque density) and Braak Staging (pathogenic tau inclusions) and a significant positive correlation with post-mortem brain weight. We also found a neuron-specific downregulation of Rbbp7 mRNA in AD patients. Rbbp7 protein levels were significantly decreased in 3xTg-AD and PS19 mice compared to NonTg, but no decreases were found in APP/PS1 mice that lack tau pathology. In vitro, Rbbp7 overexpression rescued TauP301L-induced cytotoxicity in immortalized hippocampal cells and primary cortical neurons. In vivo, hippocampal Rbbp7 overexpression rescued neuronal death in the CA1 of PS19 mice. Mechanistically, we found that increased Rbbp7 reduced p300 levels, tau acetylation at lysine 280 and tau phosphorylation at AT8 and AT100 sites. Collectively, these data identify a novel role of Rbbp7, protecting against tau-related pathologies, and highlight its potential as a therapeutic target in AD and related tauopathies.

Thpok promotes CD4+-lineage differentiation by recruiting the NuRD chromatin remodeling complex

Abstract T cell precursors develop into helper CD4 or cytotoxic CD8 T cells in thymus. Thpok, a BTB/POZ family transcription factor, enforces MHC II restricted thymocytes commitment to the CD4 lineage, while Runx3 protein drives MHC I restricted thymocytes to the CD8 lineage. However, the molecular mechanisms underlying Thpok functions during CD4 T cell development are not understood. Replacing the BTB domain from Thpok with that from Bcl6, another BTB/POZ zinc finger transcription factor that recruits NCOR-family co-repressors through its BTB domain, abrogated CD4 T cell development in thymus; this suggests a unique feature associated with Thpok BTB domain. Here, using mass-spectrometry, we identified nucleosome remodeling and deacetylase (NuRD) complex as a novel Thpok cofactor. We demonstrated that the Thpok BTB domain is essential for NuRD recruitment. Moreover, we identified BTB domain amino acid residues critical for NuRD complex interaction, and showed that mutation of these Thpok residues disrupts CD4 T cell development in vivo. Reciprocally, a fusion protein reconstituting NuRD binding to a BTB-less version of Thpok (which cannot bind NuRD) restored CD4 T cells in vivo. In addition, we found that CD4-lineage thymocytes expressing that fusion protein have a transcriptome similar to that of thymocytes expressing wild type Thpok. Finally, we found that NuRD binding is required for Thpok to antagonize Runx3 expression in thymocytes. Thus, our results demonstrate that NuRD recruitment is both necessary and sufficient for the functions of the Thpok BTB domain in CD4 T cell development and it is also required to antagonize Runx3-dependent differentiation towards CD8 lineage.

Chromatin Remodeling in the Brain-a NuRDevelopmental Odyssey.

During brain development, the genome must be repeatedly reconfigured in order to facilitate neuronal and glial differentiation. A host of chromatin remodeling complexes facilitates this process. At the genetic level, the non-redundancy of these complexes suggests that neurodevelopment may require a lexicon of remodelers with different specificities and activities. Here, we focus on the nucleosome remodeling and deacetylase (NuRD) complex. We review NuRD biochemistry, genetics, and functions in neural progenitors and neurons.

Role of Chromodomain-Helicase-DNA-Binding Protein 4 (CHD4) in Breast Cancer.

Chromodomain-helicase-DNA-binding protein 4 (CHD4) is an epigenetic regulator identified as an oncogenic element that may provide a novel therapeutic target for the treatment of breast cancer (BC). CHD4—the core component of the nucleosome remodeling and deacetylase (NuRD) complex—may be mutated in patients with this disease. However, information on CHD4 mutants that might allow their use as biomarkers of therapeutic success and prognosis is lacking. The present work examines mutations in CHD4 reported in patients with breast cancer and included in public databases and attempts to identify their roles in its development. The databases revealed 81 point mutations across different types of breast cancer (19 of which also appeared in endometrial, intestinal, nervous system, kidney, and lymphoid organ cancers). 71.6% of the detected mutations were missense mutations, 13.6% were silent, and 6.2% nonsense. Over 50% affected conserved residues of the ATPase motor (ATPase and helicase domains), and domains of unknown function in the C-terminal region. Thirty one mutations were classified in the databases as either ‘deleterious’, ‘probably/possibly damaging’ or as ‘high/medium pathogenic’; another five nonsense and one splice-site variant were predicted to produce potentially harmful truncated proteins. Eight of the 81 mutations were categorized as putative driver mutations and have been found in other cancer types. Some mutations seem to influence ATPase and DNA translocation activities (R1162W), while others may alter protein stability (R877Q/H, R975H) or disrupt DNA binding and protein activity (R572*, X34_splice) suggesting CHD4 function may be affected. In vivo tumorigenecity studies in endometrial cancer have revealed R975H and R1162W as mutations that lead to CHD4 loss-of-function. Our study provides insight into the molecular mechanism whereby CHD4, and some of its mutants could play a role in breast cancer and suggest important implications for the biological comprehension and prognosis of breast cancer, identifying CHD4 as a novel therapeutic target for BC patients.

Targeting ZNF410 as a potential β-hemoglobinopathy therapy.

The nucleosome remodeling and deacetylase (NuRD) complex is a chromatin modifier with a key role in the switch from fetal to adult hemoglobin. In a new study, Vinjamur et al. identify a fetal hemoglobin repressor, ZNF410, which does not directly bind the γ-globin promoter but acts through highly specific regulation of CHD4, a protein subunit of the NuRD complex, thus presenting a potential approach for therapeutic reactivation of fetal hemoglobin.

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung

BackgroundTbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes.MethodsWe used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses.ResultsWe identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4.ConclusionOur data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

A Casz1\u2013NuRD complex regulates temporal identity transitions in neural progenitors

Neural progenitor cells undergo identity transitions during development to ensure the generation different types of neurons and glia in the correct sequence and proportions. A number of temporal identity factors that control these transitions in progenitor competence have been identified, but the molecular mechanisms underlying their function remain unclear. Here, we asked how Casz1, the mammalian orthologue of Drosophila castor, regulates competence during retinal development. We show that Casz1 is required to control the transition between neurogenesis and gliogenesis. Using BioID proteomics, we reveal that Casz1 interacts with the nucleosome remodeling and deacetylase (NuRD) complex in retinal cells. Finally, we show that both the NuRD and the polycomb repressor complexes are required for Casz1 to promote the rod fate and suppress gliogenesis. As additional temporal identity factors have been found to interact with the NuRD complex in other contexts, we propose that these factors might act through this common biochemical process to regulate neurogenesis.

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.

ZBTB2 protein is a new partner of the Nucleosome Remodeling and Deacetylase (NuRD) complex

ZBTB2 is a protein belonging to the BTB/POZ zinc-finger family whose members typically contain a BTB/POZ domain at the N-terminus and several zinc-finger domains at the C-terminus. Studies have been carried out to disclose the role of ZBTB2 in cell proliferation, in human cancers and in regulating DNA methylation. Moreover, ZBTB2 has been also described as an ARF, p53 and p21 gene repressor as well as an activator of genes modulating pluripotency. In this scenario, ZBTB2 seems to play many functions likely associated with other proteins. Here we report a picture of the ZBTB2 protein partners in U87MG cell line, identified by high-resolution mass spectrometry (MS) that highlights the interplay between ZBTB2 and chromatin remodeling multiprotein complexes.In particular, our analysis reveals the presence, as ZBTB2 candidate interactors, of SMARCA5 and BAZ1B components of the chromatin remodeling complex WICH and PBRM1, a subunit of the SWI/SNF complex. Intriguingly, we identified all the subunits of the NuRD complex among the ZBTB2 interactors. By co-immunoprecipitation experiments and ChIP-seq analysis we definitely identify ZBTB2 as a new partner of the NuRD complex.

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 Has an Asymmetric, Dynamic, and Modular Architecture

SUMMARYThe nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.

Regulating the Regulators: The Role of Histone Deacetylase 1 (HDAC1) in Erythropoiesis.

Histone deacetylases (HDACs) play important roles in transcriptional regulation in eukaryotic cells. Class I deacetylase HDAC1/2 often associates with repressor complexes, such as Sin3 (Switch Independent 3), NuRD (Nucleosome remodeling and deacetylase) and CoREST (Corepressor of RE1 silencing transcription factor) complexes. It has been shown that HDAC1 interacts with and modulates all essential transcription factors for erythropoiesis. During erythropoiesis, histone deacetylase activity is dramatically reduced. Consistently, inhibition of HDAC activity promotes erythroid differentiation. The reduction of HDAC activity not only results in the activation of transcription activators such as GATA-1 (GATA-binding factor 1), TAL1 (TAL BHLH Transcription Factor 1) and KLF1 (Krüpple-like factor 1), but also represses transcription repressors such as PU.1 (Putative oncogene Spi-1). The reduction of histone deacetylase activity is mainly through HDAC1 acetylation that attenuates HDAC1 activity and trans-repress HDAC2 activity through dimerization with HDAC1. Therefore, the acetylation of HDAC1 can convert the corepressor complex to an activator complex for gene activation. HDAC1 also can deacetylate non-histone proteins that play a role on erythropoiesis, therefore adds another layer of gene regulation through HDAC1. Clinically, it has been shown HDACi can reactivate fetal globin in adult erythroid cells. This review will cover the up to date research on the role of HDAC1 in modulating key transcription factors for erythropoiesis and its clinical relevance.

STEM-13. FUNCTIONAL CHARACTERIZATION OF THE ZFHX4-CHD4 INTERACTION IN GLIOBLASTOMA CANCER STEM CELLS

Abstract Glioblastoma stem cells (GSCs) they may be one reason for inevitable recurrence of GBM. We previously discovered that Zinc Finger Homeobox 4 (ZFHX4), a 450kD transcription factor, is required to maintain the GSC state. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex, which activates or represses gene expression via two distinct functions - histone deacetylation and ATP-dependent chromatin remodeling. CHD4 suppression phenocopies ZFHX4 suppression. The precise nature and function of the ZFHX4 interaction with CHD4 is not understood. Here we report that the ZFHX4-CHD4 interaction requires a single zinc-finger domain. An incremental truncation screen revealed that ZFHX4 amino acids 1838 to 2387, which contains zinc fingers 14 and 15, are required to bind CHD4. Disrupting the zinc coordination of zinc finger 14 impaired the ZFHX4-CHD4 interaction. Moreover, by overexpressing ZFHX4 amino acids 1838 to 2487, we decreased CHD4 recruitment to transcription regulatory regions of the stem cell genes SOX2 and SOX9, decreased transcription, and reduced clonogenic self-renewal. These results may provide the structural basis for new treatments to target GSCs and prevent recurrence in this devastating disease.

P38 and Casein Kinase 2 Mediate Ribonuclease 1 Repression in Inflamed Human Endothelial Cells via Promoter Remodeling Through Nucleosome Remodeling and Deacetylase Complex.

Vascular pathologies, such as thrombosis or atherosclerosis, are leading causes of death worldwide and are strongly associated with the dysfunction of vascular endothelial cells. In this context, the extracellular endonuclease Ribonuclease 1 (RNase1) acts as an essential protective factor in regulation and maintenance of vascular homeostasis. However, long-term inflammation causes strong repression of RNase1 expression, thereby promoting endothelial cell dysfunction. This inflammation-mediated downregulation of RNase1 in human endothelial cells is facilitated via histone deacetylase (HDAC) 2, although the underlying molecular mechanisms are still unknown. Here, we report that inhibition of c-Jun N-terminal kinase by small chemical compounds in primary human endothelial cells decreased physiological RNase1 mRNA abundance, while p38 kinase inhibition restored repressed RNase1 expression upon proinflammatory stimulation with tumor necrosis factor alpha (TNF-α) and poly I:C. Moreover, blocking of the p38 kinase- and HDAC2-associated kinase casein kinase 2 (CK2) by inhibitor as well as small interfering RNA (siRNA)-knockdown restored RNase1 expression upon inflammation of human endothelial cells. Further downstream, siRNA-knockdown of chromodomain helicase DNA binding protein (CHD) 3 and 4 of the nucleosome remodeling and deacetylase (NuRD) complex restored RNase1 repression in TNF-α treated endothelial cells implicating its role in the HDAC2-containing repressor complex involved in RNase1 repression. Finally, chromatin immunoprecipitation in primary human endothelial cells confirmed recruitment of the CHD4-containing NuRD complex and subsequent promoter remodeling via histone deacetylation at the RNASE1 promoter in a p38-dependent manner upon human endothelial cell inflammation. Altogether, our results suggest that endothelial RNase1 repression in chronic vascular inflammation is regulated by a p38 kinase-, CK2-, and NuRD complex-dependent pathway resulting in complex recruitment to the RNASE1 promoter and subsequent promoter remodeling.