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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 while NuRD is not required to silence all genes, its activity is important to restrict expression of genes primed for activation upon exit from the pluripotent state, and that NuRD activity facilitates their subsequent transcriptional activation. 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.

Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local regions of the genome to which it binds.

Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The NuRD complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. We investigated the structure and function of NuRD in human induced pluripotent stem cells (hiPSCs). Using immunoprecipitation followed by mass-spectrometry in hiPSCs and in naive or primed mouse pluripotent stem cells, we find that NuRD structure and biochemical interactors are generally conserved. Using RNA sequencing, we find that, whereas in mouse primed stem cells and in mouse naïve ES cells, NuRD is required for an appropriate level of transcriptional response to differentiation signals, hiPSCs require NuRD to initiate these responses. This difference indicates that mouse and human cells interpret and respond to induction of differentiation differently.Graphical AbstractNuRD acts like a conductor in an orchestra.A. In the presence of NuRD (pink blob figure, centre) differentiation occurs in an ordered fashion in both mouse (left) and human (right) ES cells. Gene expression changes in both cell types are tightly controlled with down-regulation of pluripotency genes and up-regulation of lineage appropriate genes. This is akin to a group of musicians producing musical notes in the right order and at the right amplitude to create a coherent piece of music. B. Loss of “the conductor” NuRD results in increased transcriptional noise in both systems, indicated here as a low-level blanket of sound in both systems. Consequences of MBD3/NuRD loss differs between human and mouse ES cells. In mouse ES cells, differentiation cues lead to some down-regulation of pluripotency genes and incomplete progression along a lineage appropriate pathway. This is like musicians who know that they should be making music but who lose their way without a conductor’s influence. In human iPS cells the background level of noise without NuRD results in a lack of order to gene expression changes in response to differentiation. The noise from these “musicians” would be truly awful.

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop.

Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial–facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels., PWWP2A is a chromatin-binding transcriptional regulator that mediates mitosis-progression. Here, the authors provide evidence that PWWP2A directly interacts with H2A.Z nucleosomes, DNA and H3K36me3, binds to an MTA1-specific subcomplex of the NuRD complex (M1HR) and promotes changes to histone acetylation.

Graphical abstract, Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The Nucleosome Remodelling and Deacetylation (NuRD) complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. Here we find that while NuRD activity is required for lineage commitment from primed pluripotency in both human and mouse cells, the nature of this requirement is surprisingly different. While mouse embryonic stem cells (mESC) and epiblast stem cells (mEpiSC) require NuRD to maintain an appropriate differentiation trajectory as judged by gene expression profiling, human induced pluripotent stem cells (hiPSC) lacking NuRD fail to even initiate these trajectories. Further, while NuRD activity is dispensable for self-renewal of mESCs and mEpiSCs, hiPSCs require NuRD to maintain a stable self-renewing state. These studies reveal that failure to properly fine-tune gene expression and/or to reduce transcriptional noise through the action of a highly conserved chromatin remodeller can have different consequences in human and mouse pluripotent stem cells.

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.

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often present 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 ES cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. Nevertheless, ES cells lacking all three MTA proteins represent a complete NuRD null and are viable, allowing us to identify a previously undetected function for NuRD in maintaining differentiation trajectory during early stages of lineage commitment.

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.

The very first cell divisions in mammalian embryogenesis produce a ball of cells, each with the potential to form any cell in the developing embryo or placenta. At some point, the embryo produces enough cells that some are located on the outside of the embryo, while others are completely surrounded by other cells. It is at this point that cells undergo the very first lineage commitment event: outer cells form the trophectoderm and lose the potential to form embryonic lineages, while inner cells form the Inner Cell Mass, which retain embryonic potential. Cell identity is defined by gene expression patterns, and gene expression is largely controlled by how the DNA is packaged into chromatin. A number of protein complexes exist which are able to use the energy of ATP to remodel chromatin: that is, to alter the nucleosome topology of chromatin. Here, we summarise the evidence that chromatin remodellers play essential roles in the successful completion of preimplantation development in mammals and describe recent efforts to understand the molecular mechanisms through which chromatin remodellers facilitate the successful completion of the first cell fate decisions in mammalian embryogenesis.

The very first cell divisions in mammalian embryogenesis produce a ball of cells, each with the potential to form any cell in the developing embryo or placenta. At some point, the embryo produces enough cells that some are located on the outside of the embryo, while others are completely surrounded by other cells. It is at this point that cells undergo the very first lineage commitment event: outer cells form the trophectoderm and lose the potential to form embryonic lineages, while inner cells form the Inner Cell Mass, which retain embryonic potential. Cell identity is defined by gene expression patterns, and gene expression is largely controlled by how the DNA is packaged into chromatin. A number of protein complexes exist which are able to use the energy of ATP to remodel chromatin: that is, to alter the nucleosome topology of chromatin. Here, we summarise the evidence that chromatin remodellers play essential roles in the successful completion of preimplantation development in mammals and describe recent efforts to understand the molecular mechanisms through which chromatin remodellers facilitate the successful completion of the first cell fate decisions in mammalian embryogenesis.

The enhancer-binding zinc finger transcription factor Sall4 is essential for early mammalian postimplantation development and plays important roles in lineage commitment of embryonic stem cells. Enhancer binding by Sall4 results in transcriptional activation of some genes, but repression of others. Exactly how cells in preimplantation stage embryos use this transcriptional modulatory activity of Sall4 during early developmental transitions has not been determined. Using single cell gene expression analyses we show that Sall4 is required to maintain the gene regulatory network in inner cell mass (ICM) cells prior to lineage commitment. Although Sall4 is not required for ICM cells to adopt a correct epiblast or primitive endoderm gene expression profile, in the absence of Sall4 early ICM cells commit to either lineage at reduced frequency. We propose a model whereby Sall4 activity sets the stage for efficient progression from the uncommitted ICM progenitor state by modulating the gene regulatory network in early ICM cells.

B cell specification requires the establishment of accessible transcriptional enhancers and promoters by lineage-specific transcription factors. Loughran et al. show that Mbd3/NuRD chromatin remodeling restricts the accessibility of these regions and therefore controls B versus T cell lineage fate by preventing B cell programming transcription factors from prematurely enacting lineage commitment., 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.

Turning genes on and off is essential for development and homeostasis, yet little is known about the sequence and causal role of chromatin state changes during the repression of active genes. This is surprising, as defective gene silencing underlies developmental abnormalities and disease. Here we delineate the sequence and functional contribution of transcriptional repression mechanisms at high temporal resolution. Inducible entry of the NuRD-interacting transcriptional regulator Ikaros into mouse pre-B cell nuclei triggered immediate binding to target gene promoters. Rapid RNAP2 eviction, transcriptional shutdown, nucleosome invasion, and reduced transcriptional activator binding required chromatin remodeling by NuRD-associated Mi2beta/CHD4, but were independent of HDAC activity. Histone deacetylation occurred after transcriptional repression. Nevertheless, HDAC activity contributed to stable gene silencing. Hence, high resolution mapping of transcriptional repression reveals complex and interdependent mechanisms that underpin rapid transitions between transcriptional states, and elucidates the temporal order, functional role and mechanistic separation of NuRD-associated enzymatic activities. DOI: http://dx.doi.org/10.7554/eLife.22767.001

Summary The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for embryonic development and pluripotent stem cell differentiation. In this study, we investigated whether NuRD is also involved in the reverse biological process of induction of pluripotency in neural stem cells. By knocking out MBD3, an essential scaffold subunit of the NuRD complex, at different time points in reprogramming, we found that efficient formation of reprogramming intermediates and induced pluripotent stem cells from neural stem cells requires NuRD activity. We also show that reprogramming of epiblast-derived stem cells to naive pluripotency requires NuRD complex function and that increased MBD3/NuRD levels can enhance reprogramming efficiency when coexpressed with the reprogramming factor NANOG. Our results therefore show that the MBD3/NuRD complex plays a key role in reprogramming in certain contexts and that a chromatin complex required for cell differentiation can also promote reversion back to a naive pluripotent cell state., Graphical Abstract, Highlights • Mbd3 facilitates the initiation of neural stem cell reprogramming • Mbd3 is also required for efficient iPSC generation from EpiSCs and preiPSCs • Overexpression of Mbd3/NuRD facilitates reprogramming in a context-dependent manner, dos Santos et al. show that Mbd3/NuRD plays a positive role in reprogramming in certain contexts and that overexpression of Mbd3 facilitates Nanog-mediated reprogramming.

A number of different chromatin remodelling complexes in mammalian cells are implicated in the control of gene expression. The genetic requirements for many such complex components have been described, and the biochemical activities of complex components characterised in vitro, yet the molecular mechanisms by which these biochemical activities impact transcriptional regulation in vivo remain ill-defined. Using an inducible system with fine temporal resolution, we show that the Nucleosome Remodelling and Deacetylation (NuRD) complex directly regulates chromatin architecture at enhancer regions in ES cells, in turn influencing the activity of RNA polymerase II via Mediator. Through this mechanism NuRD restricts Mediator access to enhancer chromatin during lineage commitment, thereby enabling appropriate transcriptional regulation. In contrast, acetylation levels of histone H3 lysine 27 are not immediately impacted by NuRD activity, correlating with transcriptional response only after expression levels have changed. These findings provide a detailed, molecular picture of genome-wide modulation of lineage-specific transcription by an abundant chromatin remodelling complex.

Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic development.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

Embryonic Stem (ES) cells are able to give rise to the three germ layers of the embryo but are prevented from contributing to the trophoblast. The molecular nature of this barrier between embryonic and trophectodermal cell fates is not clear, but is known to involve DNA methylation. Here we demonstrate that the Nucleosome Remodeling and Deacetylation (NuRD) co-repressor complex maintains the developmental barrier between embryonic and trophectodermal cell fates by maintaining transcriptional silencing of trophectoderm determinant genes in ES cells. We further show that NuRD activity facilitates DNA methylation of several of its target promoters, where it acts non-redundantly with DNA methylation to enforce transcriptional silencing. NuRD-deficient ES cells fail to completely silence expression of the trophectoderm determinant genes Elf5 and Eomes, but this alone is not sufficient to induce transdifferentiation towards the trophectoderm fate. Rather this leaves ES cells capable of activating expression of trophectoderm-specific genes in response to appropriate extracellular signals, enabling them to commit to a trophectodermal cell fate. Our findings clarify the molecular nature of the developmental barrier between the embryonic and trophoblast cell fates, and establish a role for NuRD activity in specifying sites for de novo DNA methylation. (C) 2012. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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.

Cells of early mammalian embryos have the potential to develop into any adult cell type, and are thus said to be pluripotent. Pluripotency is lost during embryogenesis as cells commit to specific developmental pathways. Although restriction of developmental potential is often associated with repression of inappropriate genetic programmes1, the role of epigenetic silencing during early lineage commitment remains undefined. Here, we used mouse embryonic stem cells to study the function of epigenetic silencing in pluripotent cells. Embryonic stem cells lacking Mbd3 — a component of the nucleosome remodelling and histone deacetylation (NuRD) complex2,3 — were viable but failed to completely silence genes that are expressed before implantation of the embryo. Mbd3-deficient embryonic stem cells could be maintained in the absence of leukaemia inhibitory factor (LIF) and could initiate differentiation in embryoid bodies or chimeric embryos, but failed to commit to developmental lineages. Our findings define a role for epigenetic silencing in the cell-fate commitment of pluripotent cells.

DNA methylation occurs in bacteria, fungi, plants and animals, however its role varies widely among different organisms. Even within animal genomes, methylation patterns vary substantially from undetectable in nematodes, to global methylation in vertebrate genomes. The number and variety of proteins containing methyl-CpG binding domains (MBDs) that are encoded in animal genomes also varies, with a general correlation between the extent of genomic methylation and the number of MBD proteins. We describe here the evolution of the MBD proteins and argue that the vertebrate MBD complement evolved to exploit the benefits and protect against the dangers of a globally methylated genome.

Rais et al. (Nature 502: 65-70) reported that suppressing formation of the Nucleosome Remodelling and Deacetylase (NuRD) complex by depleting its structural component Mbd3 promotes near 100% induction of somatic cells reprogrammed to pluripotency. These results have not been independently reproduced and conflicting data were obtained in a recent study by our groups (Cell Stem Cell 15: 102-10). Here we investigate the source of these disparities through analysis of the genomic data accompanying Rais et al. In contrast to the conditions described in the study, Mbd3 remained expressed at high levels in both heterozygous and incompletely ablated knockout cell lines used to assess pluripotency induction in an Mbd3-depleted state, calling into question the involvement of Mbd3 in the reported improvements to reprogramming efficiency. Mismatched reporter constructs were found to be applied to Oct4-GFP experiment and control cells, where the primary enhancer element regulating Oct4 expression is present only in the test group where apparent enhancements in reprogramming kinetics were observed. This setup led to inappropriate comparison of test cells harboring a promiscuous Oct4-GFP reporter and control cells transformed with a much more stringent version. Such conditions are expected to incur substantial differences in GFP output and misleading interpretation of fluorescence data. We relay our findings to inform the community of these issues and to express our concerns regarding the conclusions presented in Rais et al.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13064-015-0040-z) contains supplementary material, which is available to authorized users.

Proteins that modify the structure of chromatin are known to be important for various aspects of metazoan biology including development, disease and possibly ageing. Yet functional details of why these proteins are important, i.e. how their action influences a given biological process, are lacking. While it is now possible to describe the biochemistry of how these proteins remodel chromatin, their chromatin binding profiles in cell lines, or gene expression changes upon loss of a given protein, in very few cases has this easily translated into an understanding of how the function of that protein actually influences a developmental process. Given that many chromatin modifying proteins will largely exert their influence through control of gene expression, it is useful to consider developmental processes as changes in the gene regulatory network (GRN), with each cell type exhibiting a unique gene expression profile. In this essay we consider the impact of two abundant and highly conserved chromatin modifying complexes, namely the nucleosome remodelling and deacetylation (NuRD) complex and the polycomb repressive complex 2 (PRC2), on the change in GRNs associated with lineage commitment during early mammalian development. We propose that while the NuRD complex limits the stability of cell states and defines the developmental trajectory between two stable states, PRC2 activity is important for stabilizing a new GRN once established. Although these two complexes display different biochemical activities, chromatin binding profiles and mutant phenotypes, we propose a model to explain how they cooperate to facilitate the transition through cell states that is development.

MBD2 and MBD3 are closely related proteins with consensus methyl-CpG binding domains. MBD2 is a transcriptional repressor that specifically binds to methylated DNA and is a component of the MeCP1 protein complex. In contrast, MBD3 fails to bind methylated DNA in murine cells, and is a component of the Mi-2/NuRD corepressor complex. We show by gene targeting that the two proteins are not functionally redundant in mice, as Mbd3(−/−) mice die during early embryogenesis, whereas Mbd2(−/−) mice are viable and fertile. Maternal behavior of Mbd2(−/−) mice is however defective and, at the molecular level, Mbd2(−/−) mice lack a component of MeCP1.Mbd2-mutant cells fail to fully silence transcription from exogenous methylated templates, but inappropriate activation of endogenous imprinted genes or retroviral sequences was not detected. Despite their differences, Mbd3 and Mbd2 interact genetically suggesting a functional relationship. Genetic and biochemical data together favor the view that MBD3 is a key component of the Mi-2/NuRD corepressor complex, whereas MBD2 may be one of several factors that can recruit this complex to DNA.

Rett syndrome (RTT) is an inherited neurodevelopmental disorder of females that occurs once in 10,000–15,000 births1,2. Affected females develop normally for 6–18 months, but then lose voluntary movements, including speech and hand skills. Most RTT patients are heterozygous for mutations in the Xlinked gene MECP2 (refs. 3–12), encoding a protein that binds to methylated sites in genomic DNA and facilitates gene silencing13– 17. Previous work with Mecp2-null embryonic stem cells indicated that MeCP2 is essential for mouse embryogenesis18. Here we generate mice lacking Mecp2 using Cre-loxP technology. Both Mecp2-null mice and mice in which Mecp2 was deleted in brain showed severe neurological symptoms at approximately six weeks of age. Compensation for absence of MeCP2 in other tissues by MeCP1 (refs. 19,20) was not apparent in genetic or biochemical tests. After several months, heterozygous female mice also showed behavioral symptoms. The overlapping delay before symptom onset in humans and mice, despite their profoundly different rates of development, raises the possibility that stability of brain function, not brain development per se, is compromised by the absence of MeCP2.

Two human genetic diseases have recently been shown to be due to mutations in genes encoding proteins involved in DNA methylation. The phenotypes of these two diseases are surprisingly distinct from each other and provide insights into the functions of DNA methylation in mammals.

Defects of mismatch repair are thought to be responsible for carcinogenesis in hereditary non-polyposis colorectal cancer and about 15% of sporadic colon cancers. The phenotype is seen as microsatellite instability and is known to be caused either by mutations in mismatch repair genes or by aberrant methylation of these genes stabilizing their downregulation. Lack of repair of microsatellite sequence errors, created during replication, leads to a mutation-prone phenotype. Where mutations occur within mononucleotide tracts within exons they cause translation frameshifts, premature cessation of translation and abnormal protein expression. Such mutations have been observed in the TGFbetaRII, BAX, IGFIIR, MSH3 and MSH6 genes in colon and other cancers. We describe here frameshift mutations affecting the gene for the methyl-CpG binding thymine glycosylase, MBD4, in over 40% of microsatellite unstable sporadic colon cancers. The mutations all appear heterozygous but their location would ensure truncation of the protein between the methyl-CpG binding and glycosylase domains, thus potentially generating a dominant negative effect. It is thus possible that such mutations enhance mutation frequency at other sites in these tumours. A suggestion has been made that MBD4 (MED1) mutations may lead to an increased rate of microsatellite instability but this mechanism appears unlikely due to the nature of mutations we have found.

The CHD4 (chromodomain-helicase-DNA-binding 4) (or Mi-2β) protein is a founding component of the NuRD (nucleosome remodelling and deacetylation) complex. NuRD has long been known to function in transcriptional regulation, and is conserved throughout the animal and plant kingdoms. In recent years, evidence has steadily accumulated indicating that CHD4 can both function outside of the NuRD complex and also play important roles in cellular processes other than transcriptional regulation. A number of loss-of-function studies have identified important roles for CHD4 in the DNA-damage response and in cell cycle progression through S-phase and into G2. Furthermore, as part of NuRD, it participates in regulating acetylation levels of p53, thereby indirectly regulating the G1/S cell cycle checkpoint. Although CHD4 has a somewhat complicated relationship with the cell cycle, recent evidence indicates that CHD4 may exert some tumour-suppressor functions in human carcinogenesis. CHD4 is a defining member of the NuRD complex, but evidence is accumulating that CHD4 also plays important NuRD-independent roles in the DNA-damage response and cell cycle progression, as well as in transcriptional regulation.

Through decades of research it has been established that some chromatin-modifying proteins can repress transcription, and thus are generally termed ‘repressors’. Although classic repressors undoubtedly silence transcription, genome-wide studies have shown that many repressors are associated with actively transcribed loci and that this is a widespread phenomenon. Here, we review the evidence for the presence of repressors at actively transcribed regions and assess what roles they might be playing. We propose that the modulation of expression levels by chromatin-modifying, co-repressor complexes provides transcriptional fine-tuning that drives development.

Epigenetic gene regulation refers to different states of phenotypic expression caused by differential effects of chromosome or chromatin packaging rather than by differences in DNA sequence. Examples of epigenetic regulation can be found in organisms as diverse as the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and mammals. Three major types of epigenetic regulation are considered in this review : dosage compensation, imprinting and position effect variegation. While the specific details and mechanisms of each is quite different, they all involve either local or extensive alterations in chromatin structure. A number of genes implicated in epigenetic regulation have been isolated and their products identified as proteins or RNA molecules involved at various levels in DNA, chromatin or chromosome binding. While in general our understanding of mammalian epigenetic phenomena is not as advanced as that in model systems, the detailed molecular and genetic understanding of processes responsible for conditional gene silencing in invertebrate systems provides strong models for consideration of such effects in human and mouse genetics.

The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2(-/y)) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.

Summary Transcriptional heterogeneity within embryonic stem cell (ESC) populations has been suggested as a mechanism by which a seemingly homogeneous cell population can initiate differentiation into an array of different cell types. Chromatin remodeling proteins have been shown to control transcriptional variability in yeast and to be important for mammalian ESC lineage commitment. Here we show that the Nucleosome Remodeling and Deacetylation (NuRD) complex, which is required for ESC lineage commitment, modulates both transcriptional heterogeneity and the dynamic range of a set of pluripotency genes in ESCs. In self-renewing conditions, the influence of NuRD at these genes is balanced by the opposing action of self-renewal factors. Upon loss of self-renewal factors, the action of NuRD is sufficient to silence transcription of these pluripotency genes, allowing cells to exit self-renewal. We propose that modulation of transcription levels by NuRD is key to maintaining the differentiation responsiveness of pluripotent cells., Graphical Abstract Highlights ► NuRD directly regulates the transcription of pluripotency genes ► The repressive activity of NuRD is required for ESC differentiation ► NuRD mediates transcriptional heterogeneity in ESC populations ► Transcription levels are determined by balanced activation and silencing

DNA methylation patterns are dynamic in cleavage-stage embryos of a number of mammalian species. A failure to properly recapitulate preimplantation DNA methylation patterns in embryos derived by nuclear transfer may contribute to the low efficiency of nuclear transfer in producing live offspring.

The Sin3a/HDAC co-repressor complex is a critical regulator of transcription networks that govern cell cycle control and apoptosis throughout development. Previous studies have identified Sin3a as essential for embryonic development around the time of implantation, during which the epiblast cell cycle is uniquely structured to achieve very rapid divisions with little tolerance of DNA damage. This study investigates the specific requirement for Sin3a in the early mouse embryo and shows that embryos lacking Sin3a suffer unresolved DNA damage and acute p53-independent apoptosis specifically in the E3.5–4.5 epiblast. Surprisingly, Myc and E2F targets in Sin3a-null ICMs are downregulated, suggesting a central but non-canonical role for Sin3a in regulating the pluripotent embryonic cell cycle. ES cells deleted for Sin3a mount a DNA damage response indicative of unresolved double-strand breaks, profoundly arrest at G2, and undergo apoptosis. These results indicate that Sin3a protects the genomic integrity of pluripotent embryonic cells and governs their unusual cell cycle., Highlights ► Sin3a is required for proliferation and survival of pluripotent embryonic cells. ► Sin3a-/- mouse ICM cells are eliminated by p53-independent apoptosis. ► Sin3a is a critical regulator of the unusual pluripotent embryonic cell cycle. ► Sin3a prevents or repairs DSBs that arise in ES cells, perhaps during replication. ► The Sin3a interactome provides insights into function Sin3a in ES cells.

NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression The NURD and Polycomb complexes PRC1 and PRC2 have been implicated in stem cell differentiation although their molecular roles are unclear. This study identifies a common set of genes that are sequentially regulated by these complexes, the NURD complex deacetylates H3K27 as a prerequisite for subsequent methylation by PRC2., 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.

The human XIST gene, a candidate for a role in X chromosome inactivation, has recently been cloned and sequenced, yielding a 17 kb cDNA with no apparent significant, conserved open reading frame. In addition, the XIST transcript has been localized within the nucleus to the Barr body by RNA in situ hybridization. This subnuclear localization and lack of any significant protein-coding potential suggest that XIST may act as a functional RNA within the nucleus. In the absence of a conserved open reading frame, we have turned to evolutionary studies as a first step toward elucidating a function for XIST in the process of X inactivation. While probes for XIST detect homologues in numerous eutherians, sequence comparisons require significant gapping and reveal identity levels intermediate between those seen for coding and non-coding regions in other genes. Further, sequence comparison of the most likely candidate open reading frame among several primate species reveals sequence changes not normally associated with protein-coding regions. Other features of XIST are conserved in different species, however, including the position of a major transcription start site and active X chromosome-specific DNA methylation patterns at the gene's 5' end. Finally, a possible molecular basis for differing propensity toward X inactivation between Xce alleles in mouse is investigated by comparing the sequence of the Xist conserved 5' repeats in mouse strains carrying different Xce alleles.

AP-1 (activator protein 1) activity is strongly induced in response to numerous signals, including growth factors, cytokines and extracellular stresses. The proto-oncoprotein c-Jun belongs to the AP-1 group of transcription factors and it is a crucial regulator of intestinal progenitor proliferation and tumorigenesis. An important mechanism of AP-1 stimulation is phosphorylation of c-Jun by the Jun amino-terminal kinases (JNKs). N-terminal phosphorylation of the c-Jun transactivation domain increases target gene transcription, but a molecular explanation was elusive. Here we show that unphosphorylated, but not N-terminally phosphorylated c-Jun, interacts with Mbd3 and thereby recruits the nucleosome remodelling and histone deacetylation (NuRD) repressor complex. Mbd3 depletion in colon cancer cells increased histone acetylation at AP-1-dependent promoters, which resulted in increased target gene expression. The intestinal stem cell marker lgr5 was identified as a novel target gene controlled by c-Jun/Mbd3. Gut-specific conditional deletion of mbd3 (mbd3(ΔG/ΔG) mice) stimulated c-Jun activity and increased progenitor cell proliferation. In response to inflammation, mdb3 deficiency resulted in colonic hyperproliferation and mbd3(ΔG/ΔG) mice showed markedly increased susceptibility to colitis-induced tumorigenesis. Notably, concomitant inactivation of a single allele of c-jun reverted physiological and pathological hyperproliferation, as well as the increased tumorigenesis in mbd3(ΔG/ΔG) mice. Thus the transactivation domain of c-Jun recruits Mbd3/NuRD to AP-1 target genes to mediate gene repression, and this repression is relieved by JNK-mediated c-Jun N-terminal phosphorylation.

Background: The precise molecular changes that occur when a neural stem (NS) cell switches from a programme of self-renewal to commit towards a specific lineage are not currently well understood. However it is clear that control of gene expression plays an important role in this process. DNA methylation, a mark of transcriptionally silent chromatin, has similarly been shown to play important roles in neural cell fate commitment in vivo. While DNA methylation is known to play important roles in neural specification during embryonic development, no such role has been shown for any of the methyl-CpG binding proteins (Mecps) in mice.Methodology/Principal Findings: To explore the role of DNA methylation in neural cell fate decisions, we have investigated the function of Mecps in mouse development and in neural stem cell derivation, maintenance, and differentiation. In order to test whether the absence of phenotype in singly-mutant animals could be due to functional redundancy between Mecps, we created mice and neural stem cells simultaneously lacking Mecp2, Mbd2 and Zbtb33. No evidence for functional redundancy between these genes in embryonic development or in the derivation or maintenance of neural stem cells in culture was detectable. However evidence for a defect in neuronal commitment of triple knockout NS cells was found.Conclusions/Significance: Although DNA methylation is indispensable for mammalian embryonic development, we show that simultaneous deficiency of three methyl-CpG binding proteins genes is compatible with apparently normal mouse embryogenesis. Nevertheless, we provide genetic evidence for redundancy of function between methyl-CpG binding proteins in postnatal mice.

Transcription of the Xist gene triggers X chromosome inactivation in cis and is therefore silenced on the X chromosome that remains active. DNA methylation contributes to this silencing, but the mechanism is unknown. As methylated DNA binding proteins (MBPs) are potential mediators of gene silencing by DNA methylation, we asked whether MBP-deficient cell lines could maintain Xist repression. The absence of Mbd2 caused significant low-level reactivation of Xist, but silencing was restored by exogenous Mbd2. In contrast, deficiencies of Mbd1, MeCP2, and Kaiso had no detectable effect, indicating that MBPs are not functionally redundant at this locus. Xist repression in Mbd2-null cells was hypersensitive to the histone deacetylase inhibitor trichostatin A and to depletion of the DNA methyltransferase Dnmtl. These synergies implicate Mbd2 as a mediator of the DNA methylation signal at this locus. The presence of redundant mechanisms to enforce repression at Xist and other loci is compatible with the hypothesis that "stacking" of imperfect repressive tendencies may be an evolutionary strategy to ensure leakproof gene silencing.

Mbd3 is a core component of the NuRD (Nucleosome Remodeling and Histone Deacetylation) co-repressor complex, and NuRD-mediated silencing has been implicated in cell fate decisions in a number of contexts. Mbd3-deficient embryonic stem (ES) cells made by gene targeting are viable but fail to form a stable NuRD complex, are severely compromised in the ability to differentiate,and show LIF-independent self-renewal. Mbd3 is known to be essential for postimplantation embryogenesis in mice, but the function of Mbd3 in vivo has not previously been addressed. Here we show that the inner cell mass (ICM) of Mbd3-deficient blastocysts fails to develop into mature epiblast after implantation. Unlike Mbd3-null ES cells, Mbd3-deficient ICMs grown ex vivo fail to expand their Oct4-positive, pluripotent cell population despite producing robust endoderm outgrowths. Additionally, we identify a set of genes showing stage-specific expression in ICM cells during preimplantation development, and show that Mbd3 is required for proper gene expression patterns in pre- and peri-implantation embryos and in ES cells. These results demonstrate the importance of Mbd3/NuRD for the development of pluripotent cells in vivo and for their ex vivo progression into embryonic stem cells, and highlight the differences between ES cells and the ICM cells from which they are derived.