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Castaño J, Menendez P, Bruzos-Cidon C, Straccia M, Sousa A, Zabaleta L, Vazquez N, Zubiarrain A, Sonntag KC, Ugedo L, Carvajal-Vergara X, Canals JM, Torrecilla M, Sanchez-Pernaute R, Giorgetti A

Fast and Efficient Neural Conversion of Human Hematopoietic Cells.

Stem Cell Reports 9 Dec 2014, 3 (6) 1118-1131. Epub 13 Nov 2014
Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.
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Ost A, Lempradl A, Casas E, Weigert M, Tiko T, Deniz M, Pantano L, Boenisch U, Itskov PM, Stoeckius M, Ruf M, Rajewsky N, Reuter G, Iovino N, Ribeiro C, Alenius M, Heyne S, Vavouri T, Pospisilik JA

Paternal diet defines offspring chromatin state and intergenerational obesity.

Cell 4 Dec 2014, 159 (6) 1352-64.
The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.
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Creppe C, Palau A, Malinverni R, Valero V, Buschbeck M

A Cbx8-Containing Polycomb Complex Facilitates the Transition to Gene Activation during ES Cell Differentiation.

PLoS Genet. Dec 2014, 10 (12) e1004851. Epub 11 Dec 2014
Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq). Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.
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Freije A, Molinuevo R, Ceballos L, Cagigas M, Alonso-Lecue P, Rodriguez R, Menendez P, Aberdam D, De Diego E, Gandarillas A

Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

Cell Rep 20 Nov 2014, 9 (4) 1349-60. Epub 6 Nov 2014
Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear.
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Guiu J, Bergen DJ, De Pater E, Islam AB, Ayllón V, Gama-Norton L, Ruiz-Herguido C, González J, López-Bigas N, Menendez P, Dzierzak E, Espinosa L, Bigas A

Identification of Cdca7 as a novel Notch transcriptional target involved in hematopoietic stem cell emergence.

J. Exp. Med. 17 Nov 2014, 211 (12) 2411-23. Epub 10 Nov 2014
Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence.
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