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12 pages, 7 figures.-- PMID: 19355788 [PubMed].-- Supporting information (Suppl. figures S1-S3, movies S1-S7) available at: http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.1000079#toclink6 ; Regulation of cell proliferation has been extensively studied in cultured cell systems that are characterized by coordinated growth and cell-cycle progression and relatively uniform cell size distribution. During the development of multicellular organisms, however, growth and division can be temporally uncoupled, and the signaling pathways that regulate these growth programs are poorly understood. A good model for analyzing proliferation control in such systems is the morphogenesis of the Drosophila adult abdominal epidermis by histoblasts. These cells undergo a series of temporally regulated transitions during which neither cell size nor division rate is constant. The proliferation of histoblasts during metamorphosis is uniquely amenable to clonal analysis in combination with live imaging. Thereby, we show that abdominal histoblasts, which grow while in G2 arrest during larval stages, enter a proliferative stage in the pupal period that is initiated by ecdysone-dependent string/Cdc25 phosphatase transcription. The proliferating histoblasts have preaccumulated stores of Cyclin E, which trigger an immediate S phase onset after mitosis. These rapid cell cycles lack a G1 phase and result in a progressive reduction of cell size. Eventually, the histoblasts proceed to a stage of slower proliferation that, in contrast to the preceding, depends on epidermal growth factor receptor (EGFR) signaling for progression through the G2/M transition and on insulin receptor/PI3K-mediated signaling for growth. These results uncover the developmentally programmed changes coupling the growth and proliferation of the histoblasts that form the abdominal epidermis of Drosophila. Histoblasts proceed through three distinct stages: growth without division, division without growth, and growth-coupled proliferation. Our identification of the signaling pathways and cell-cycle regulators that control these programs illustrates the power of in vivo time-lapse analyses after clone induction. It sets the stage for the comprehensive understanding of the coordination of cell growth and cell-cycle progression in complex multicellular eukaryotes. ; [Author summary] A fundamental issue in biology is the question of how the rate of cell division is coupled to cell growth. Coordination of these processes has been studied extensively in cultured cell systems but to a much lesser extent in intact organisms. To study this phenomenon in a physiological setting, we developed a methodology to observe and manipulate cell division and growth in a population of Drosophila abdominal cells called histoblasts. The various developmental stages of histoblast morphogenesis include exit from cell-cycle arrest, initially rapid growth in the absence of cell division, and subsequent coupling of proliferation and growth. We identified several critical developmental signaling pathways (including signaling via ecdysone, the EGF receptor, and PI 3-kinase) that regulate and coordinate cell growth and division cycles during these different types of cell-cycle phenomena. We propose that the internal logic of the Drosophila histoblast system may serve as a basic framework for understanding not only how coordinated cell growth and division operate in a number of other developmental contexts, but also how misregulation of cell growth and division occurs in contexts such as cancer cell populations. ; NN was supported by a Formación de Profesorado Universitario (FPU) fellowship from the Spanish Ministry of Education and Science (MEC) and CM by the "Juan de la Cierva" MEC program. Work in EM-B's lab is supported by grants of the DGICYT (MEC) and the European Union (EU) (WOUND-STREP Project). ; Peer reviewed

[Abstract] This paper proposes a new model for music prediction based on Variational Autoencoders (VAEs). In this work, VAEs are used in a novel way to address two different issues: music representation into the latent space, and using this representation to make predictions of the future note events of the musical piece. This approach was trained with different songs of Handel. As a result, the system can represent the music in the latent space, and make accurate predictions. Therefore, the system can be used to compose new music either from an existing piece or from a random starting point. An additional feature of this system is that a small dataset was used for training. However, results show that the system is able to return accurate representations and predictions on unseen data ; This work is supported by Instituto de Salud Carlos III, grant number PI17/01826 (Collaborative Project in Genomic Data Integration CICLOGEN) funded by the Instituto de Salud Carlos III from the Spanish National plan for Scientific and Technical Research and Innovation 2013-2016 and the European Regional Development Funds (FEDER). This project was also supported by the General Directorate of Culture, Education and University Management of Xunta de Galicia ED431D 2017/16 and Drug Discovery Galician Network Ref. ED431G/01 and the Galician Network for Colorectal Cancer Research (Ref. ED431D 2017/23), and by the Spanish Ministry of Economy and Competitiveness through the funding of the unique installation BIOCAI (UNLC08-1E-002, UNLC13-13-3503) and the European Regional Development Funds (FEDER) by the European Union. This work was also funded by the grant for the consolidation and structuring of competitive research units (ED431C 2018/49) from the General Directorate of Culture, Education and University Management of Xunta de Galicia, and the CYTED network (PCI2018_093284) funded by the Spanish Ministry of Ministry of Innovation and Science. The experiments described in this section were carried out using the equipment of the ...

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells. We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization. These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch. ; AMP held a Spanish FPU PhD studentship and CT was supported by the Marie Curie Research Training Network IMMUNANOMAP (MRTNCT-2006-035946). Research in the EMB laboratory is funded by grants of the Spanish Ministry of Science (DGI and Consolider) and the European Union. Research in the VS laboratory is supported by "Stichting voor Fundamenteel Onderzoek der Materie", the "Nederlandse Organisatie voor Wetenschappelijk Onderzoek", and the European Union. ; Peer reviewed

Cells display versatile responses to mechanical inputs and recent studies have identified the mitogen-activated protein kinase (MAPK) cascades mediating the biological effects observed upon mechanical stimulation. Although, MAPK pathways can act insulated from each other, several mechanisms facilitate the crosstalk between the components of these cascades. Yet, the combinatorial complexity of potential molecular interactions between these elements have prevented the understanding of their concerted functions. To analyze the plasticity of the MAPK signaling network in response to mechanical stress we performed a non-saturating epistatic screen in resting and stretched conditions employing as readout a JNK responsive dJun-FRET biosensor. By knocking down MAPKs, and JNK pathway regulators, singly or in pairs in Drosophila S2R+ cells, we have uncovered unexpected regulatory links between JNK cascade kinases, Rho GTPases, MAPKs and the JNK phosphatase Puc. These relationships have been integrated in a system network model at equilibrium accounting for all experimentally validated interactions. This model allows predicting the global reaction of the network to its modulation in response to mechanical stress. It also highlights its context-dependent sensitivity. © 2014 Pereira et al. ; A.M.P held a Spanish FPU PhD studentship, and C.T. was supported by the Marie Curie Research Training Network IMMUNANOMAP. Research in the E.M.-B. laboratory is funded by grants of the Ministry of Economy and Competitivity (DGI and Consolider). Research in the V.S. laboratory is supported by "Stitching voor Fundamenteel Onderzoek der Materie" (FOM), the Nederlandse Organisatie voor Wetenschappelik Onderzoek (N.W.O.), and the European Union ; Peer Reviewed

Profiling the chromatin-bound proteome (chromatome) in a simple, direct, and reliable manner might be key to uncovering the role of yet uncharacterized chromatin factors in physiology and disease. Here, we have designed an experimental strategy to survey the chromatome of proliferating cells by using the DNA-mediated chromatin pull-down (Dm-ChP) technology. Our approach provides a global view of cellular chromatome under normal physiological conditions and enables the identification of chromatin-bound proteins de novo. Integrating Dm-ChP with genomic and functional data, we have discovered an unexpected chromatin function for adenosylhomocysteinase, a major one-carbon pathway metabolic enzyme, in gene activation. Our study reveals a new regulatory axis between the metabolic state of pluripotent cells, ribosomal protein production, and cell division during the early phase of embryo development, in which the metabolic flux of methylation reactions is favored in a local milieu. ; We acknowledge support from the Spanish Ministry of Economy, Industry and Competitiveness to the EMBL partnership, Centro de Excelencia Severo Ochoa, the CERCA Programme/Generalitat de Catalunya, the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (to S.A. and A.A.-C.). The CRG/UPF Proteomics Unit is a member of the ProteoRed PRB3 consortium that was supported by grant PT17/0019 of the PE I+D+i 2013–2016 from the Instituto de Salud Carlos III (ISCIII), ERDF, and "Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya" (2017SGR595). The Di Croce Laboratory was supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2016-75008-P), AGAUR, and La Marato TV3

Polycomb group proteins (PcG) are transcriptional repressors that control cell identity and development. In mammals, five different CBX proteins associate with the core Polycomb repressive complex 1 (PRC1). In mouse embryonic stem cells (ESCs), CBX6 and CBX7 are the most highly expressed CBX family members. CBX7 has been recently characterized, but little is known regarding the function of CBX6. Here, we show that CBX6 is essential for ESC identity. Its depletion destabilizes the pluripotency network and triggers differentiation. Mechanistically, we find that CBX6 is physically and functionally associated to both canonical PRC1 (cPRC1) and non-canonical PRC1 (ncPRC1) complexes. Notably, in contrast to CBX7, CBX6 is recruited to chromatin independently of H3K27me3. Taken together, our findings reveal that CBX6 is an essential component of ESC biology that contributes to the structural and functional complexity of the PRC1 complex. ; This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2016-75008-P), Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208), AGAUR, Fundació "La Marató de TV3," CERCA Programme/Generalitat de Catalunya, and EU FP7 Programs 4DCellFate (277899) to L.D.C. This work aided by collaboration with the National Center for Dynamic Interactome Research and the National Resource for the Mass Spectrometric Analysis of Biological Macromolecules, supported in part by NIH grant P41 GM109824 to Michael P. Rout and grant P41 GM103314 to Brian T. Chait. This paper is subject to the NIH Public Access Policy.

The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis. ; The S.A.B. laboratory research is supported by the European Research Council (ERC), the Worldwide Cancer Research Foundation, the Foundation La Marató de TV3, the Spanish Ministry of Economy and Development, the Foundation Vencer el Cancer (''Beat Cancer''), the Government of Cataluña (SGR and Mario Salvia' grants), the Foundation Fundación Botín, and the Institute for Research in Biomedicine (IRB-Barcelona). L.R. is a La Caixa Foundation Ph.D. fellow. G.S. was supported by an AXA postdoctoral fellowship. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). L.D.C. was supported by grants from the Spanish Ministerio de Educación y Ciencia (SAF2013-48926-P) and the European Commission's 7th Framework Program 4DCellFate grant number 277899. We are grateful to the Common Fund's Epigenomic Program from the NIH (USA) for providing the bisulphite whole genome sequencing data of human EpSCs

Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generating all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of Phf19 increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage-specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis. ; The work in the Di Croce laboratory is supported by grants from the Spanish of Economy, Industry, and Competitiveness (MEIC) (BFU2016-75008-P), "Fundación Vencer El Cancer" (VEC), the European Regional Development Fund (FEDER), Fundació "La Marató de TV3," and from AGAUR. The laboratory of A.B. is supported by SAF2016-75613-R from the Ministerio de Ciencia, Innovación y Universidades. H.H. is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII) and received funding from the European Union's Horizon 2020 research and innovation programme (MSCA-ITN-2015-675752) and the Ministerio de Ciencia, Innovación y Universidades (SAF2017-89109-P; AEI/FEDER, UE). P.V. was supported by "Fundación Científica de la Asociación Española Contra el Cáncer."

Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1–induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors. ; S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC).