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The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8–ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.

Gene regulation; Somatic cell reprogramming; Trophectoderm Regulación de genes; Reprogramación de células somáticas; Trofoectodermo Regulació de gens; Reprogramació de cèl·lules somàtiques; Trofectoderma IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6. We thank C. Berenguer for help with B cell reprogramming and bone marrow collection; S. Nakagawa and B. Pernaute for advice on pre-implantation embryo culture and manipulation, and Kyle M. Loh for his valuable discussions; the flow cytometry and microscopy units of UPF-CRG for technical assistance; the CRG genomics core facility for sequencing and Graf laboratory members for critical discussions. Work in the laboratory of T.G. was supported by the Spanish Ministry of Economy, Industry and Competitiveness (Plan Estatal PID2019-109354GB-I00), the CRG, AGAUR (SGR 726), and a European Research Council Synergy grant (4D-Genome). M.P.-C. was supported by an FPI fellowship (BES-2016-076900). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), la Caixa Foundation, and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282).

Enhancer activity drives cell differentiation and cell fate determination, but it remains unclear how enhancers cooperate during these processes. Here we investigate enhancer cooperation during transdifferentiation of human leukemia B-cells to macrophages. Putative enhancers are established by binding of the pioneer factor C/EBPα followed by chromatin opening and enhancer RNA (eRNA) synthesis from H3K4-monomethylated regions. Using eRNA synthesis as a proxy for enhancer activity, we find that most putative enhancers cooperate in an additive way to regulate transcription of assigned target genes. However, transcription from 136 target genes depends exponentially on the summed activity of its putative paired enhancers, indicating that these enhancers cooperate synergistically. The target genes are cell type-specific, suggesting that enhancer synergy can contribute to cell fate determination. Enhancer synergy appears to depend on cell type-specific transcription factors, and such interacting enhancers are not predicted from occupancy or accessibility data that are used to detect superenhancers. Fuinding: Max-Planck-Gesellschaft (Open-access funding), Deutsche Forschungsgemeinschaft (SFB860), Deutsche Forschungsgemeinschaft (SPP1935), Deutsche Forschungsgemeinschaft (SPP2191), European Research Council (693023)

Extracellular vesicles (EVs) are emerging as crucial mediators in cell-to-cell communication. Stik et al. provide evidence that EVs released by supportive stromal cells target hematopoietic stem and progenitor cells in vivo and in vitro and influence their gene expression and potential., Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver–derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

Three-dimensional organization of the genome is important for transcriptional regulation1-7. In mammals, CTCF and the cohesin complex create submegabase structures with elevated internal chromatin contact frequencies, called topologically associating domains (TADs)8-12. Although TADs can contribute to transcriptional regulation, ablation of TAD organization by disrupting CTCF or the cohesin complex causes modest gene expression changes13-16. In contrast, CTCF is required for cell cycle regulation17, embryonic development and formation of various adult cell types18. To uncouple the role of CTCF in cell-state transitions and cell proliferation, we studied the effect of CTCF depletion during the conversion of human leukemic B cells into macrophages with minimal cell division. CTCF depletion disrupts TAD organization but not cell transdifferentiation. In contrast, CTCF depletion in induced macrophages impairs the full-blown upregulation of inflammatory genes after exposure to endotoxin. Our results demonstrate that CTCF-dependent genome topology is not strictly required for a functional cell-fate conversion but facilitates a rapid and efficient response to an external stimulus. We thank M. T. Kanemaki for the degron plasmids; R. Guigó’s laboratory, and S. Pérez-Lluch in particular, for the H3K27ac and H3K4me1 ChIP–seq, produced in the framework of the RNA-MAPS project (ERC-2011-AdG-294653-RNA-MAPS); Y. Cuartero for help with sequencing and CTCF ChIP–seq; C. Segura for help with immunofluorescence microscopy; the CRG Genomics and flow cytometry core facilities and the CRG-CNAG Sequencing Unit for sequencing; and members of T.G.’s laboratory for discussions. This work was supported by the European Research Council under the 7th Framework Programme FP7/2007-2013 (ERC Synergy Grant 4D-Genome, grant agreement 609989, to T.G. and M.A.M.-R.), the Ministerio de Educación y Ciencia (SAF.2012-37167, to T.G., and BFU2017-85926-P, to M.A.M.-R.), the AGAUR (to T.G.) and the Marató TV3 (201611) (to M.A.M.-R.). P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935, EXC 2067/1-390729940), the European Research Council (advanced investigator grant TRANSREGULON, grant agreement no. 693023) and the Volkswagen Foundation.

Cèl·lules mare embrionàries; Seqüenciació d'alt rendiment Células madre embrionarias; Secuenciación de alto rendimiento Embryonic stem cells; High-throughput sequencing During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial patterns of gene expression. Embryoid bodies (EBs) derived from pluripotent stem cells have been a popular model to study the differentiation of the main three germ layers and to define regulatory circuits during cell fate specification. Although it is well-known that tissue-specific enhancers play an important role in these networks by interacting with promoters, assigning them to their relevant target genes still remains challenging. To make this possible, quantitative approaches are needed to study enhancer-promoter contacts and their dynamics during development. Here, we adapted a 4C method to define enhancers and their contacts with cognate promoters in the EB differentiation model. The method uses frequently cutting restriction enzymes, sonication, and a nested-ligation-mediated PCR protocol compatible with commercial DNA library preparation kits. Subsequently, the 4C libraries are subjected to high-throughput sequencing and analyzed bioinformatically, allowing detection and quantification of all sequences that have contacts with a chosen promoter. The resulting sequencing data can also be used to gain information about the dynamics of enhancer-promoter contacts during differentiation. The technique described for the EB differentiation model is easy to implement. We would like to thank F. Le Dily, R. Stadhouders and members of the Graf laboratory for their advice and discussions. G.S. was supported by a Marie Sklodowska-Curie fellowship (H2020-MSCA-IF-2016, miRStem), T.V.T by a Juan de la Cierva postdoctoral fellowship (MINECO, FJCI-2014-22946). This work was supported by the European Research Council under the 7th Framework Programme FP7 (ERC Synergy Grant 4D-Genome, grant agreement 609989 to T.G.), the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, Centro de Excelencia Severo Ochoa 2013-2017 and CERCA Program Generalitat de Catalunya.

How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages. J.L.S. were supported by Juan de la Cierva postdoctoral fellowships (MINECO; FJCI-2014-22946 and IJCI-2014-21872), B.D.S. by an EMBO long-term fellowship (number ALTF 1143-2015), G.S. by a Marie Sklodowska-Curie fellowship (H2020-MSCA-IF-2016, miRStem), A.D. by a Severo Ochoa fellowship and J.G. by a Boehringer Ingelheim Graduate Student Fellowship. R.J. was supported by NIH grants R01 NS088538-01 and 2R01MH104610-15. This work was supported by the EU-FP7 project BLUEPRINT, the Spanish Ministry of Economy, Industry and Competitiveness to the EMBL partnership, Centro de Excelencia Severo Ochoa 2013–2017 and the CERCA Program Generalitat de Catalunya. T.V.T., J.L.S. and B.D.S. were supported by a CRG award for junior collaborative projects

International audience

The transcription factor Hoxb5 is expressed specifically in hematopoietic stem cells, yet its ectopic expression in mouse B cell progenitors induces their conversion into functional T cells in vivo.

Hematopoietic stem cells (HSCs) are at the basis of the adult hematopoietic system. During development, HSCs emerge autonomously in the aorta-gonad-mesonephros region at mid gestation. They massively amplify in the fetal liver and placenta and, by the end of gestation, colonize the bone marrow where they will be maintained throughout life. In these successive hematopoietic microenvironments, HSCs develop in close association with surrounding cells that provide a supportive microenvironment for their survival, proliferation, and differentiation. In this chapter, we will discuss our current knowledge on the cellular and molecular identity of the HSC niches during ontogeny and will explore the molecular requirements for the HSC supportive capacity of the niche cells and their crosstalk with HSCs.

Gallid herpesvirus 2 (GaHV-2) is an oncogenic herpesvirus that causes T lymphoma in chicken. GaHV-2 encodes a basic leucine zipper (bZIP) protein of the AP-1 family, Meq. Upon formation of homo- or heterodimers with c-Jun, Meq may modulate the expression of viral and cellular genes involved in lymphomagenesis. GaHV-2 also encodes viral microRNAs (miRNAs) involved in latency and apoptosis escape. However, little is known about cellular miRNA deregulation during the development of GaHV-2-associated lymphoma. We determined the cellular miRNA expression profiles of chickens infected with a very virulent strain (RB-1B) or a vaccine strain (CVI988) or noninfected. Among the most deregulated cellular miRNAs, we focused our efforts on gga-miR-21, which is upregulated during GaHV-2 infection. We mapped the gga-miR-21 promoter to the 10th intron of the TMEM49 gene and found it to be driven by AP-1- and Ets-responsive elements. We show here that the viral oncoprotein Meq binds to this promoter, thereby transactivating gga-miR-21 expression. We confirmed that this miRNA targets chicken programmed death cell 4 (PDCD4) and promotes tumor cell growth and apoptosis escape. Finally, gga-miR-21 was overexpressed only during infection with a very virulent strain (RB-1B) and not during infection with a nononcogenic strain (CVI988), providing further evidence for its role in GaHV-2 lymphomagenesis. Our data therefore suggest an additional role for Meq in GaHV-2-mediated lymphomagenesis through the induction of miR-21 expression.

Marek’s disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 5′ end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 3′ end and allowed identification of the 5′ end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 3′ UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70 % when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.

The tumor suppressor protein p53 plays a role in cellular responses to cancer-initiating events by regulating progress through the cell cycle. Several recent studies have shown that p53 transactivates expression of the members of the proapoptotic microRNA-34 family, which are underexpressed in several cancers. We demonstrate here that the latency-associated cluster of microRNAs (miRNA) encoded by an oncogenic herpesvirus, gallid herpesvirus 2 (GaHV-2), is a direct target of p53. Robust transcriptional activity was induced in three avian cell lines by a sequence mapping 600 base pairs (bp) upstream of the cluster of miRNAs. We found transcription start sites for the pri-miRNA transcript at the 3′ end of this transcription-inducing sequence. The promoter has no consensus core promoter element, but is organized into a variable number of tandem repeats of 60-bp harboring p53-responsive elements (RE). The minimal functional construct consists of two tandem repeats. Mutagenesis to change the sequence of the p53 RE abolished transcriptional activity, whereas p53 induction enhanced mature miRNA expression. The identification of a viral miRNA promoter regulated by p53 is biologically significant, because all avirulent GaHV-2 strains described to date lack the corresponding regulatory sequence, whereas all virulent, very virulent, and hypervirulent strains possess at least two tandem repeats harboring the p53 RE.

Hematopoietic stem cells (HSCs) are identified by their ability to self-renew and to differentiate into all blood cell lineages. In vivo, hematopoietic stem/progenitor cells (HSPCs) are in close association with stromal cells that constitute a supportive microenvironment also called niche. Recently, exosomes that are small microvesicles enclosed by a lipid bilayer and enriched in cytoplasmic proteins, mRNAs, microRNAs, have emerged as major communication mediators between cells. However, their implication in the cross-talk between HSCs and stromal cells is still largely unknown. This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support. To address this issue, we used two murine stromal cell lines derived from the fetal liver and with differing capacity to maintain HSPCs ex vivo as revealed by repopulation assay and long-term cultures. AFT024 (AFT) harbors a potent HSPC supporting capacity in vitro whereas BFC012 (BFC) is non supportive. For each cell line, the exosome fractions were isolated from culture supernatant by ultra-centrifugation. Electron microscopy, western blot, and flow cytometry analyses revealed that both AFT and BFC stromal cells secrete exosomes. Interestingly, using PKH67 stained exosomes, we demonstrated that bone marrow Lin-Sca-1+c-kit+ (LSK) cells preferentially uptake AFT-derived exosomes. This observation might be related to the different tetraspanin compositions of AFT and BFC derived exosomes as observed by flow cytometry. We then showed an increase in cell viability and clonogenic potential when LSK cells were exposed to AFT-derived exosomes for 96 hours in cytokine-free medium as compared to controls. Moreover, cultures with AFT-derived exosomes exhibited a 3.5 fold increase in the number of LSK cells as compared to untreated conditions. We then used high-throughput sequencing to explore the molecular signatures of AFT and BFC derived exosomes, as well as their cells of origin. We identified a list of 394 mRNAs and 6 microRNAs specifically expressed in exosomes and correlated to the HSPC support. Gene ontology analysis revealed that the apoptotic regulation, cell survival and proliferation pathways were significantly enriched in the AFT-derived exosomal signature. In addition, we showed the transfer of mRNAs involved in these pathways from the AFT-exosomes to the LSK recipient cells. Together with our observation of a decrease in the LSK apoptotic cells after co-culture with AFT-derived exosomes, these data suggest that exosomes released by AFT cells may protect HSPCs from apoptosis. Collectively, our results revealed an important role for exosomes in the HSPC supporting capacity of stromal cells. This work provides new insights in our understanding of the molecular and cellular mechanisms involved in the cross-talk between HSPCs and their niches. It may also have interesting applications in regenerative medicine, regarding the ex vivo manipulation of HSCs in stromal-free conditions for cell therapy. Disclosures No relevant conflicts of interest to declare.

The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.

International audience; Despite progress in identifying the cellular composition of hematopoietic stem/progenitor cell (HSPC) niches, little is known about the molecular requirements of HSPC support. To address this issue, we used a panel of six recognized HSPC-supportive stromal lines and less-supportive counterparts originating from embryonic and adult hematopoietic sites. Through comprehensive transcriptomic meta-analyses, we identified 481 mRNAs and 17 microRNAs organized in a modular network implicated in paracrine signaling. Further inclusion of 18 additional cell strains demonstrated that this mRNA subset was predictive of HSPC support. Our gene set contains most known HSPC regulators as well as a number of unexpected ones, such as Pax9 and Ccdc80, as validated by functional studies in zebrafish embryos. In sum, our approach has identified the core molecular network required for HSPC support. These cues, along with a searchable web resource, will inform ongoing efforts to instruct HSPC ex vivo amplification and formation from pluripotent precursors.