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.

The NuRD complex and macrocephaly associated neurodevelopmental disorders.

The nucleosome remodeling and deacetylase (NuRD) complex is a major regulator of gene expression involved in pluripotency, lineage commitment, and corticogenesis. This important complex is composed of seven different proteins, with mutations in CHD3, CHD4, and GATAD2B being associated with neurodevelopmental disorders presenting with macrocephaly and intellectual disability similar to other overgrowth and intellectual disability (OGID) syndromes. Pathogenic variants in CHD3 and CHD4 primarily involve disruption of enzymatic function. GATAD2B variants include loss-of-function mutations that alter protein dosage and missense variants that involve either of two conserved domains (CR1 and CR2) known to interact with other NuRD proteins. In addition to macrocephaly and intellectual disability, CHD3 variants are associated with inguinal hernias and apraxia of speech; whereas CHD4 variants are associated with skeletal anomalies, deafness, and cardiac defects. GATAD2B-associated neurodevelopmental disorder (GAND) has phenotypic overlap with both of these disorders. Of note, structural models of NuRD indicate that CHD3 and CHD4 require direct contact with the GATAD2B-CR2 domain to interact with the rest of the complex. Therefore, the phenotypic overlaps of CHD3- and CHD4-related disorders with GAND are consistent with a loss in the ability of GATAD2B to recruit CHD3 or CHD4 to the complex. The shared features of these neurodevelopmental disorders may represent a new class of OGID syndrome: the NuRDopathies.

The Nucleosome Remodelling and Deacetylation complex suppresses transcriptional noise during lineage commitment.

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often found as multiple paralogous proteins in vertebrate complexes. In some cases, these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here, we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within embryonic stem (ES) cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. ES cells lacking all three MTA proteins exhibit complete NuRD loss of function and are viable, allowing us to identify a previously unreported function for NuRD in reducing transcriptional noise, which is essential for maintaining a proper differentiation trajectory during early stages of lineage commitment.

CHD4 and the NuRD complex directly control cardiac sarcomere formation.

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.

Chromatin remodeling by the NuRD complex regulates development of follicular helper and regulatory T cells.

Lineage commitment and differentiation into CD4+ T cell subsets reflect an interplay between chromatin regulators and transcription factors (TF). Follicular T cell development is regulated by the Bcl6 TF, which helps determine the phenotype and follicular localization of both CD4+ follicular helper T cells (TFH) and follicular regulatory T cells (TFR). Here we show that Bcl6-dependent control of follicular T cells is mediated by a complex formed between Bcl6 and the Mi-2ß-nucleosome-remodeling deacetylase complex (Mi-2ß-NuRD). Formation of this complex reflects the contribution of the intracellular isoform of osteopontin (OPN-i), which acts as a scaffold to stabilize binding between Bcl6 and the NuRD complex that together regulate the genetic program of both TFH and TFR cells. Defective assembly of the Bcl6-NuRD complex distorts follicular T cell differentiation, resulting in impaired TFR development and skewing of the TFH lineage toward a TH1-like program that includes expression of Blimp1, Tbet, granzyme B, and IFNγ. These findings define a core Bcl6-directed transcriptional complex that enables CD4+ follicular T cells to regulate the germinal center response.

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program.

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro-B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell-programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation.

Epigenomic regulation of oncogenesis by chromatin remodeling.

Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

Perspective on unraveling the versatility of 'co-repressor' complexes.

A multitude of post-translational modifications take place on histones, one of the best studied being acetylation on lysine residues, which is generally associated with gene activation. During the last decades, several so-called co-repressor protein complexes that carry out the reverse process, histone deacetylation, have been identified and characterized, such as the Sin3, N-CoR/SMRT and NuRD complexes. Although a repressive role for these complexes in regulating gene expression is well established, accumulating evidence also points to a role in gene activation. Here, we argue that integration of various state-of-the-art technologies, addressing different aspects of transcriptional regulation, is essential to unravel this apparent biological versatility of 'co-repressor' complexes.

The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.

BACKGROUND: Chromatin-modifying complexes have key roles in regulating various aspects of neural stem cell biology, including self-renewal and neurogenesis. The methyl binding domain 3/nucleosome remodelling and deacetylation (MBD3/NuRD) co-repressor complex facilitates lineage commitment of pluripotent cells in early mouse embryos and is important for stem cell homeostasis in blood and skin, but its function in neurogenesis had not been described. Here, we show for the first time that MBD3/NuRD function is essential for normal neurogenesis in mice. RESULTS: Deletion of MBD3, a structural component of the NuRD complex, in the developing mouse central nervous system resulted in reduced cortical thickness, defects in the proper specification of cortical projection neuron subtypes and neonatal lethality. These phenotypes are due to alterations in PAX6+ apical progenitor cell outputs, as well as aberrant terminal neuronal differentiation programmes of cortical plate neurons. Normal numbers of PAX6+ apical neural progenitor cells were generated in the MBD3/NuRD-mutant cortex; however, the PAX6+ apical progenitor cells generate EOMES+ basal progenitor cells in reduced numbers. Cortical progenitor cells lacking MBD3/NuRD activity generate neurons that express both deep- and upper-layer markers. Using laser capture microdissection, gene expression profiling and chromatin immunoprecipitation, we provide evidence that MBD3/NuRD functions to control gene expression patterns during neural development. CONCLUSIONS: Our data suggest that although MBD3/NuRD is not required for neural stem cell lineage commitment, it is required to repress inappropriate transcription in both progenitor cells and neurons to facilitate appropriate cell lineage choice and differentiation programmes.

Zic2 is an enhancer-binding factor required for embryonic stem cell specification.

The Zinc-finger protein of the cerebellum 2 (Zic2) is one of the vertebrate homologs of the Drosophila pair-rule gene odd-paired (opa). Our molecular and biochemical studies demonstrate that Zic2 preferentially binds to transcriptional enhancers and is required for the regulation of gene expression in embryonic stem cells. Detailed genome-wide and molecular studies reveal that Zic2 can function with Mbd3/NuRD in regulating the chromatin state and transcriptional output of genes linked to differentiation. Zic2 is required for proper differentiation of embryonic stem cells (ESCs), similar to what has been previously reported for Mbd3/NuRD. Our study identifies Zic2 as a key factor in the execution of transcriptional fine-tuning with Mbd3/NuRD in ESCs through interactions with enhancers. Our study also points to the role of the Zic family of proteins as enhancer-specific binding factors functioning in development.

Specificity in circadian clock feedback from targeted reconstitution of the NuRD corepressor.

Mammalian circadian rhythms are generated by a negative feedback loop in which PERIOD (PER) proteins accumulate, form a large nuclear complex (PER complex), and bind the transcription factor CLOCK-BMAL1, repressing their own expression. We found that mouse PER complexes include the Mi-2/nucleosome remodelling and deacetylase (NuRD) transcriptional corepressor. Unexpectedly, two NuRD subunits, CHD4 and MTA2, constitutively associate with CLOCK-BMAL1, with CHD4 functioning to promote CLOCK-BMAL1 transcriptional activity. At the onset of negative feedback, the PER complex delivers the remaining complementary NuRD subunits to DNA-bound CLOCK-BMAL1, thereby reconstituting a NuRD corepressor that is important for circadian transcriptional feedback and clock function. The PER complex thus acquires full repressor activity only upon successful targeting of CLOCK-BMAL1. Our results show how specificity is generated in the clock despite its dependence on generic transcriptional regulators and reveal the existence of active communication between the positive and negative limbs of the circadian feedback loop.

Physiological functions of MTA family of proteins.

Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

Role of MTA1 in cancer progression and metastasis.

The MTA1 protein contributes to the process of cancer progression and metastasis through multiple genes and protein targets and interacting proteins with roles in transformation, anchorage-independent growth, invasion, survival, DNA repair, angiogenesis, hormone independence, metastasis, and therapeutic resistance. Because the roles and clinical significance of MTA proteins in human cancer are discussed by other contributors in this issue, this review will focus on our current understanding of the underlying principles of action behind the biological effects of MTA1. MTA proteins control a spectrum of cancer-promoting processes by modulating the expression of target genes and/or the activity of MTA-interacting proteins. In the case of MTA1, these functions are manifested through posttranslational modifications of MTA1 in response to upstream signals, MTA1 interaction with binding proteins, and the expression of target gene products. Studies delineating the molecular basis of dual functionality of MTA1 reveal that the functions of MTA1-chromatin-modifying complexes in the context of target gene regulation are dynamic in nature. The nature and targets of MTA1-chromatin-modifying complexes are also governed by the dynamic plasticity of the nucleosome landscape as well as kinetics of activation and inactivation of enzymes responsible for posttranslational modifications on the MTA1 protein. These broadly applicable functions also explain why MTA1 may be a "hub" gene in cancer. Because the deregulation of enzymes and their substrates with roles in MTA1 biology is not necessarily limited to cancer, we speculate that the lessons from MTA1 as a prototype dual master coregulator will be relevant for other human diseases. In this context, the concept of the dynamic nature of corepressor versus coactivator complexes and the MTA1 proteome as a function of time to signal is likely to be generally applicable to other multiprotein regulatory complexes in living systems.

CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation.

BACKGROUND: Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. RESULTS: We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. CONCLUSIONS: Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.

NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of a defined set of transcription factors requires epigenetic changes in pluripotency genes. Nuclear reprogramming is an inefficient process and the molecular mechanisms that reset the epigenetic state during iPSC generation are largely unknown. Here, we show that downregulation of the nucleosome remodeling and deacetylation (NuRD) complex is required for efficient reprogramming. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs by establishing heterochromatic features and silencing embryonic stem cell-specific marker genes, including Oct4 and Nanog. Depletion of Mbd3, on the other hand, improves reprogramming efficiency and facilitates the formation of pluripotent stem cells that are capable of generating viable chimeric mice, even in the absence of c-Myc or Sox2. The results establish Mbd3/NuRD as an important epigenetic regulator that restricts the expression of key pluripotency genes, suggesting that drug-induced downregulation of Mbd3/NuRD may be a powerful means to improve the efficiency and fidelity of reprogramming.

Establishment of epigenetic patterns in development.

The distinct cell types of the body are established from the fertilized egg in development and assembled into functional tissues. Functional characteristics and gene expression patterns are then faithfully maintained in somatic cell lineages over a lifetime. On the molecular level, transcription factors initiate lineage-specific gene expression programmmes and epigenetic regulation contributes to stabilization of expression patterns. Epigenetic mechanisms are essential for maintaining stable cell identities and their disruption can lead to disease or cellular transformation. Here, we discuss the role of epigenetic regulation in the early mouse embryo, which presents a relatively well-understood system. A number of studies have contributed to the understanding of the function of Polycomb group complexes and the DNA methylation system. The role of many other chromatin regulators in development remains largely unexplored. Albeit the current picture remains incomplete, the view emerges that multiple epigenetic mechanisms cooperate for repressing critical developmental regulators. Some chromatin modifications appear to act in parallel and others might repress the same gene at a different stage of cell differentiation. Studies in pluripotent mouse embryonic stem cells show that epigenetic mechanisms function to repress lineage specific gene expression and prevent extraembryonic differentiation. Insights into this epigenetic "memory" of the first lineage decisions help to provide a better understanding of the function of epigenetic regulation in adult stem cell differentiation.

The nucleosome remodeling and deacetylase chromatin remodeling (NuRD) complex is required for peripheral nerve myelination.

Several key transcription factors and coregulators important to peripheral nerve myelination have been identified, but the contributions of specific chromatin remodeling complexes to peripheral nerve myelination have not been analyzed. Chromodomain helicase DNA-binding protein 4 (Chd4) is the core catalytic subunit of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex. Previous studies have shown Chd4 interacts with Nab (NGFI-A/Egr-binding) corepressors, which are required for early growth response 2 (Egr2/Krox20), to direct peripheral nerve myelination by Schwann cells. In this study, we examined the developmental importance of the NuRD complex in peripheral nerve myelination through the generation of conditional Chd4 knock-out mice in Schwann cells (Chd4(loxP/loxP); P0-cre). Chd4 conditional null mice were found to have delayed myelination, radial sorting defects, hypomyelination, and the persistence of promyelinating Schwann cells. Loss of Chd4 leads to elevated expression of immature Schwann cell genes (Id2, c-Jun, and p75), and sustained expression of the promyelinating Schwann cell gene, Oct6/Scip, without affecting the levels of Egr2/Krox20. Furthermore, Schwann cell proliferation is upregulated in Chd4-null sciatic nerve. In vivo chromatin immunoprecipitation studies reveal recruitment of Chd4 and another NuRD component, Mta2, to genes that are positively and negatively regulated by Egr2 during myelination. Together, these results underscore the necessity of Chd4 function to guide proper terminal differentiation of Schwann cells and implicate the NuRD chromatin remodeling complex as a requisite factor in timely and stable peripheral nerve myelination.

Metastasis-associated protein 3 (MTA3) regulates G2/M progression in proliferating mouse granulosa cells.

Metastasis-associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (NuRD) protein complex that regulates gene expression by altering chromatin structure and can facilitate cohesin loading onto DNA. The biological function of MTA3 within the NuRD complex is unknown. Herein, we show that MTA3 was expressed highly in granulosa cell nuclei of all ovarian follicle stages and at lower levels in corpora lutea. We tested the hypothesis that MTA3-NuRD complex function is required for granulosa cell proliferation. In the ovary, MTA3 interacted with NuRD proteins CHD4 and HDAC1 and the core cohesin complex protein RAD21. In cultured mouse primary granulosa cells, depletion of endogenous MTA3 using RNA interference slowed cell proliferation; this effect was rescued by coexpression of exogenous MTA3. Slowing of cell proliferation correlated with a significant decrease in cyclin B1 and cyclin B2 expression. Granulosa cell populations lacking MTA3 contained a significantly higher percentage of cells in G2/M phase and a lower percentage in S phase compared with control cells. Furthermore, MTA3 depletion slowed entry into M phase as indicated by reduced phosphorylation of histone H3 at serine 10. These findings provide the first evidence to date that MTA3 interacts with NuRD and cohesin complex proteins in the ovary in vivo and regulates G2/M progression in proliferating granulosa cells.

NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression.

Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.