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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.

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

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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.