Brian Hendrich

The NuRD component Mbd3 is required for pluripotency of embryonic stem cells

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 programmes, 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) complex — 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.

Gene Silencing Quantitatively Controls the Function of a Developmental trans-Activator

How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.

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.

NuRD Suppresses Pluripotency Gene Expression to Promote Transcriptional Heterogeneity and Lineage Commitment

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.

Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells

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.

Kaiso-deficient mice show resistance to intestinal cancer.

ABSTRACT Kaiso is a BTB domain protein that associates with the signaling molecule p120-catenin and binds to the methylated sequence mCGmCG or the nonmethylated sequence CTGCNA to modulate transcription. In Xenopus laevis, xKaiso deficiency leads to embryonic death accompanied by premature gene activation in blastulae and upregulation of the xWnt11 gene. Kaiso has also been proposed to play an essential role in mammalian synapse-specific transcription. We disrupted the Kaiso gene in mice to assess its role in mammalian development. Kaiso-null mice were viable and fertile, with no detectable abnormalities of development or gene expression. However, when crossed with tumor-susceptible Apc Min/+ mice, Kaiso-null mice showed a delayed onset of intestinal tumorigenesis. Kaiso was found to be upregulated in murine intestinal tumors and is expressed in human colon cancers. Our data suggest that Kaiso plays a role in intestinal cancer and may therefore represent a potential target for therapeutic intervention.

3D structures of individual mammalian genomes studied by single-cell Hi-C

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.

Somatic frameshift mutations in the MBD4 gene of sporadic colon cancers with mismatch repair deficiency

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 TGFβRII, 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.

Keeping things quiet: Roles of NuRD and Sin3 co-repressor complexes during mammalian development

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.

MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner.

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.