Polycomb regulation is coupled to cell cycle transition in pluripotent stem cells

When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifier genes and cell differentiation is confined to the G1 phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are markedly accumulated at target promoters during S and G2 phases, while the transcriptionally activating subunits EPOP and EloB are enriched during G1 phase. Fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 −/− mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G1 phase.This study was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-40891-R and BFU2016-75233-P) and the Andalusian Regional Government (PC-0246-2017). D.L. is a Ramón y Cajal researcher of the Spanish Ministry of Economy and Competitiveness (RYC-2012-10019)

The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells

Mammals optimize their physiology to the light–dark cycle by synchronization of the master circadian clock in the brain with peripheral clocks in the rest of the tissues of the body. Circadian oscillations rely on a negative feedback loop exerted by the molecular clock that is composed by transcriptional activators Bmal1 and Clock, and their negative regulators Period and Cryptochrome. Components of the molecular clock are expressed during early development, but onset of robust circadian oscillations is only detected later during embryogenesis. Here, we have used na¨ıve pluripotent mouse embryonic stem cells (mESCs) to study the role of Bmal1 during early development. We found that, compared to wild-type cells, Bmal12/2 mESCs express higher levels of Nanog protein and altered expression of pluripotencyassociated signalling pathways. Importantly, Bmal12/2 mESCs display deficient multi-lineage cell differentiation capacity during the formation of teratomas and gastrula-like organoids. Overall, we reveal that Bmal1 regulates pluripotent cell differentiation and propose that the molecular clock is an hitherto unrecognized regulator of mammalian development.Ramon y Cajal grant of the Spanish ministry of economy and competitiveness RYC2012-10019Spanish ministry of economy and competitiveness BFU2016-75233-PAndalusian regional government PC-0246-2017Fundacion Progreso y Salud (FPS)Instituto de Salud Carlos III European Union (EU) CPII17/00032 PI17/01574University of Granad

Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells

We thank the core facilities at GENYO for excellent technical support. We also thank the genomics unit at the CRG for assistance with RNA-seq and ChIP-seq experiments. The Landeira lab is supported by the Spanish ministry of science and innovation (PID2019-108108-100, EUR2021- 122005), the Andalusian regional government (PIER-0211-2019, PY20_00681) and the University of Granada (A-BIO-6-UGR20) grants. Research in the Klose lab is supported by the Wellcome Trust (209400/ Z/17/Z) and the European Research Council (681440). A.F. was sup- ported by a Sir Henry Wellcome Post-doctoral fellowship (110286/Z/15/ Z). Work in the Rada-Iglesias lab is funded by the Ministerio de Ciencia e Innovación, the Agencia Española de Investigación and the European Regional Development Fund (PGC2018-095301-B-I00 and RED2018- 102553-T); by the European Research Council (862022); and by the European Commission (H2020-MSCA-ITN-2019-860002).The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-35859-9The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimu- lation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to develop- mental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.Ministry of Science and Innovation, Spain (MICINN) Spanish Government PID2019-108108-100, EUR2021-122005Andalusian regional government PIER-0211-2019, PY20_00681University of Granada A-BIO-6-UGR20Wellcome Trust 209400/Z/17/ZEuropean Research Council (ERC) European Commission 862022Wellcome Trust PGC2018-095301-B-I00Ministry of Science and Innovation, Spain (MICINN) Instituto de Salud Carlos III Spanish GovernmentEuropean Commission RED2018-102553-T, H2020-MSCA-ITN-2019-860002European Commission European Commission Joint Research Centre 681440Agencia Española de Investigación110286/Z/15/

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patientsThis work was supported by the Ramon y Cajal (RYC-2015-18382) to PJR founded by the Ministry of Economy and Competitiveness; the Instituto de Salud Carlos III-FEDER (CP12/03175 and CPII17/00032) to V.R-M., (PI17/01311) to M.L.L and J.R., (PI17/01966; Fundación Mutua Madrileña AP172142019; Premio Lopez Borrasca SETH 2019; GRS2061/A/19) to J.M.B. and (CPII15/00018 and PI16/01340) to PJR; by the Chair "Doctors Galera-Requena in cancer stem cell research" (CMC-CTS963) to J.A.M. and C.G-L

Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary

8 pages, 5 figures.-- PMID: 18171682 [PubMed].-- Printed version published Feb 2008.-- Supporting information (Suppl. table S1, figure S1) available at: http://dev.biologists.org/cgi/content/full/135/3/533/DC1The existence of specialised regulatory microenvironments or niches that sustain stable stem cell populations is well documented in many tissues. However, the specific mechanisms by which niche support (or stromal) cells govern stem cell maintenance remain largely unknown. Here we demonstrate that removal of the Jak/Stat pathway in support cells of the Drosophila ovarian niche leads to germline stem cell loss by differentiation. Conversely, ectopic Jak/Stat activation in support cells induces stem cell tumours, implying the presence of a signal relay between the stromal compartment and the stem cell population. We further show that ectopic Jak/Stat signalling in support cells augments dpp mRNA levels and increases the range of Dpp signalling, a Bmp2 orthologue known to act as a niche extrinsic factor required for female germline stem cell survival and division. Our results provide strong evidence for a model in which Jak/Stat signalling in somatic support cells regulates dpp transcription to define niche size and to maintain the adjacent germline stem cells in an undifferentiated state.[To be completed upon receiving the author full-text version of the paper]Peer reviewe

Genetic dissection of a stem cell niche: the case of the Drosophila ovary

11 páginas, 6 figuras, 2 tablas.-- El material suplementario referido en este artículo puede verse en www.interscience.wiley.com/jpages/1058-8388/suppmatIn this work, we demonstrate a powerful new tool for the manipulation of the stromal component of a well-established Drosophila stem cell niche. We have generated a bric-a-brac 1 (bab1)-Gal4 line that drives UAS expression in many somatic ovary cell types from early larval stages. Using this Gal4 line, we could effectively induce FLP/FRT-mediated recombination in the stromal cells of the ovarian germline stem cell niche. Mutant clones were observed in the developing ovary of larvae and pupae, including in somatic cell types that do not divide in the adult, such as the cap cells and the terminal filament cells. Exploiting the ability of bab1-Gal4 to generate large clones, we demonstrate that bab1-Gal4 is an effective tool for analyzing stem cell niche morphogenesis and cyst formation in the germarium. We have identified a novel requirement for engrailed in the correct organization of the terminal filaments. We also demonstrate an involvement for integrins in cyst formation and follicle cell encapsulation. Finally using bab1-Gal4 in conjunction with the Gal80 system, we show that while ectopic dpp expression from stromal cells is sufficient to induce hyperplastic stem cell growth, neither activation nor inactivation of the BMP pathway within stromal cells affects germline stem cell maintenance.Grant sponsor: Spanish Ministerio de Ciencia y Tecnología; Grant number: BMC2003-01512; Grant sponsor: Junta de Andalucía; Grant number: CVI-280.Peer reviewe

Recent advances in Drosophila stem cell biology

11 páginas, 3 figuras.Stem cells possess the unique properties of self-renewal and the ability to give rise to multiple types of differentiated tissue. The fruit fly Drosophila melanogaster retains several populations of stem cells during adulthood as well as transient populations of stem cells during development. Studies of these different populations of stem cells using the genetic tools available to Drosophila researchers have played an important role in understanding many conserved stem cell characteristics. This review aims highlight some of the recent contributions from this important model system to our understanding of the myriad of processes that interact to control stem cell biology.Peer reviewe

Polycomb regulation is coupled to cell cycle transition in pluripotent stem cells

When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifier genes and cell differentiation is confined to the G1 phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are markedly accumulated at target promoters during S and G2 phases, while the transcriptionally activating subunits EPOP and EloB are enriched during G1 phase. Fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 -/- mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G1 phase.This study was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-40891-R and BFU2016-75233-P) and the Andalusian Regional Government (PC-0246-2017). D.L. is a Ramón y Cajal researcher of the Spanish Ministry of Economy and Competitiveness (RYC-2012-10019)Ye

Generation of human pluripotent stem cell lines with suppressed expression of the Notch ligand DLL4 using short hairpin RNAs.

Studies with different animal models have shown that the Notch ligand DLL4 has a key role in the development of the embryonic vasculature. Here we describe the generation and characterization of a human embryonic stem cell line and an induced pluripotent stem cell line that constitutively express short hairpin RNAs targeting DLL4 mRNA. These cells present reduced DLL4 expression at both protein and mRNA level, as well as a reduced induction of DLL4 target genes. They represent an ideal tool to study the role of DLL4 in human embryonic vascular and hematopoietic development

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266