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Martello G.,University of Cambridge | Martello G.,Research Council Stem Cell Institute | Bertone P.,University of Cambridge | Bertone P.,European Bioinformatics Institute | And 3 more authors.
EMBO Journal | Year: 2013

Self-renewal of pluripotent mouse embryonic stem (ES) cells is sustained by the cytokine leukaemia inhibitory factor (LIF) acting through the transcription factor Stat3. Several targets of Stat3 have previously been identified, most notably the reprogramming factor Klf4. However, such factors are neither required nor sufficient for the potent effect of LIF. We took advantage of Stat3 null ES cells to confirm that Stat3 mediates the self-renewal response to LIF. Through comparative transcriptome analysis intersected with genome location data, we arrived at a set of candidate transcription factor effectors. Among these, Tfcp2l1 (also known as Crtr-1) was most abundant. Constitutive expression of Tfcp2l1 at levels similar to those induced by LIF effectively substituted for LIF or Stat3 in sustaining clonal self-renewal and pluripotency. Conversely, knockdown of Tfcp2l1 profoundly compromised responsiveness to LIF. We further found that Tfcp2l1 is both necessary and sufficient to direct molecular reprogramming of post-implantation epiblast stem cells to naïve pluripotency. These results establish Tfcp2l1 as the principal bridge between LIF/Stat3 input and the transcription factor core of naïve pluripotency. © 2013 European Molecular Biology Organization.

Caren H.,University College London | Caren H.,Gothenburg University | Stricker S.H.,University College London | Bulstrode H.,Genome Biology and Developmental Biology Units | And 12 more authors.
Stem Cell Reports | Year: 2015

Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stem cell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stem cells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Here we find only a subset of GSC cultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy for GBM. © 2015 The Authors.

Foti R.,Mouse Biology Unit | Gnan S.,Mouse Biology Unit | Cornacchia D.,Mouse Biology Unit | Cornacchia D.,Sloan Kettering Cancer Center | And 17 more authors.
Molecular Cell | Year: 2016

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals. © 2016 The Authors.

Metzakopian E.,NIMR | Lin W.,NIMR | Salmon-Divon M.,European Bioinformatics Institute | Salmon-Divon M.,Cancer Research Center | And 8 more authors.
Development (Cambridge) | Year: 2012

The transcription factors Foxa1 and Foxa2 promote the specification of midbrain dopaminergic (mDA) neurons and the floor plate. Whether their role is direct has remained unclear as they also regulate the expression of Shh, which has similar roles. We characterized the Foxa2 cis-regulatory network by chromatin immunoprecipitation followed by high-throughput sequencing of mDA progenitors. This identified 9160 high-quality Foxa2 binding sites associated with 5409 genes, providing mechanistic insights into Foxa2-mediated positive and negative regulatory events. Foxa2 regulates directly and positively key determinants of mDA neurons, including Lmx1a, Lmx1b, Msx1 and Ferd3l, while negatively inhibiting transcription factors expressed in ventrolateral midbrain such as Helt, Tle4, Otx1, Sox1 and Tal2. Furthermore, Foxa2 negatively regulates extrinsic and intrinsic components of the Shh signaling pathway, possibly by binding to the same enhancer regions of co-regulated genes as Gli1. Foxa2 also regulates the expression of floor plate factors that control axon trajectories around the midline of the embryo, thereby contributing to the axon guidance function of the floor plate. Finally, this study identified multiple Foxa2-regulated enhancers that are active in the floor plate of the midbrain or along the length of the embryo in mouse and chick. This work represents the first comprehensive characterization of Foxa2 targets in mDA progenitors and provides a framework for elaborating gene regulatory networks in a functionally important progenitor population. © 2012. Published by The Company of Biologists Ltd.

Reynolds N.,University of Cambridge | Salmon-Divon M.,European Bioinformatics Institute | Salmon-Divon M.,Cancer Research Center | Dvinge H.,European Bioinformatics Institute | And 8 more authors.
EMBO Journal | Year: 2012

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. © 2012 European Molecular Biology Organization | All Rights Reserved.

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