The trophectoderm acts as a niche for the inner cell mass through C/EBPα-regulated IL-6 signaling

Gene regulation; Somatic cell reprogramming; TrophectodermRegulación de genes; Reprogramación de células somáticas; TrofoectodermoRegulació de gens; Reprogramació de cèl·lules somàtiques; TrofectodermaIL-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)

3D chromatin reprogramming primes human memory T_H2 cells for rapid recall and pathogenic dysfunction

Memory T cells provide long-lasting defense responses through their ability to rapidly reactivate, but how they efficiently "recall" an inflammatory transcriptional program remains unclear. Here, we show that human CD4+ memory T helper 2 (TH2) cells carry a chromatin landscape synergistically reprogrammed at both one-dimensional (1D) and 3D levels to accommodate recall responses, which is absent in naive T cells. In memory TH2 cells, recall genes were epigenetically primed through the maintenance of transcription-permissive chromatin at distal (super)enhancers organized in long-range 3D chromatin hubs. Precise transcriptional control of key recall genes occurred inside dedicated topologically associating domains ("memory TADs"), in which activation-associated promoter-enhancer interactions were preformed and exploited by AP-1 transcription factors to promote rapid transcriptional induction. Resting memory TH2 cells from patients with asthma showed premature activation of primed recall circuits, linking aberrant transcriptional control of recall responses to chronic inflammation. Together, our results implicate stable multiscale reprogramming of chromatin organization as a key mechanism underlying immunological memory and dysfunction in T cells.</p

Transcriptional regulation of viral and cellular microRNA during infection by oncogenic Marek's disease virus

Le Gallid Herpesvirus 2 (GaHV-2) est un herpesvirus responsable de lymphomes T chez le poulet qui encode 25 miARN matures regroupés en deux clusters localisés au sein des régions TRL/IRL et TR/IR. Mes travaux ont consisté en l’étude de la régulation transcriptionnelle du cluster mdvl-miR-M8-M10 situé au sein de la région IR5/TR. Le promoteur est constitué d’au moins 2 séquences répétées de 60 pb contenant des éléments de réponse à la protéine p53. Nous avons montré que cette protéine classiquement suppresseur de tumeur était détournée de sa fonction par le virus au profit de l’expression du cluster de miR viraux. D’autre part, le promoteur de l’oncomiR-21 cellulaire, surexprimé au cours de la lymphomagenèse induite par GaHV-2 et localisé au niveau d’une région hyper conservée chez les vertébrés est piloté en partie par les protéines de la famille AP-1. Nous avons montré que l’oncoprotéine virale Meq surexprimée au cours de l’infection transactivait le promoteur de miR-21. Enfin, l’étude des cibles potentielles virale ou cellulaire du miR-21 semble indiquer que ce dernier pourrait être impliqué dans les mécanismes de latence virale et de lymphomagenèseGallid herpes virus 2 (GaHV-2) is an alphaherpesvirus involved in lymphomas on chickens that encodes 25 matures miRNA grouped in 2 clusters localized on IRL and TRL regions. In the first hand, I studied the transcriptional regulation of the mdvl-miR-M8-M10 cluster localized on IRS/TRS region. A promoter constituted of at last 2 repeats of 60 bp harbouring p53 responsive element was identified. We showed that tumour suppressive protein p53 was diverted from its function to permit viral miRNA expression. On the second hand, the promoter of the cellular oncomiR-21, over expressed during GaHV-2 lymphomagenesis localized in a region highly conserved on vertebrate and was piloted by AP-1 proteins. We have shown that viral oncoprotein Meq overexpressed during infection transactivated gga-miR-21 promoter. At last, study ofpotential viral and cellular targets for gga-mir-21 seemed to indicate that gga-miR-21could be involved in viral latency establishment and lymphomageneSis

Vésicules extracellulaires stromales et régulation des cellules souches et progéniteurs hématopoïétiques

International audienc

Identification of enhancer-promoter contacts in embryoid bodies by quantitative chromosome conformation capture (4C)

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.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 Cataluny

CTCF chromatin residence time controls three-dimensional genome organization, gene expression and DNA methylation in pluripotent cells

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

A p53-dependent promoter associated with polymorphic tandem repeats controls the expression of a viral transcript encoding clustered microRNAs

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

Severe COVID-19-associated variants linked to chemokine receptor gene control in monocytes and macrophages

International audienceGenome-wide association studies have identified 3p21.31 as the main risk locus for severe COVID-19, although underlying mechanisms remain elusive. We perform an epigenomic dissection of 3p21.31, identifying a CTCF-dependent tissue-specific 3D regulatory chromatin hub that controls the activity of several chemokine receptor genes. Risk SNPs colocalize with regulatory elements and are linked to increased expression of CCR1 , CCR2 and CCR5 in monocytes and macrophages. As excessive organ infiltration of inflammatory monocytes and macrophages is a hallmark of severe COVID-19, our findings provide a rationale for the genetic association of 3p21.31 variants with elevated risk of hospitalization upon SARS-CoV-2 infection

Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures

International audiencetThe establishment of the microRNA (miRNA) expression signatures is the basic element to investigatethe 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. Duringlatency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include threemiRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequenciesobtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effectof small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNAsamples 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 ofthe mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed thehighest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencingapproach was used. Its frequency was 100 times less abundant when determined through the deepsequencing approach. Northern blot analysis showed a better correlation with the miRNA frequenciesfound by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also founda gap between the two sequencing approaches. Collectively, our study indicates that next-generationsequencing 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 differenttechniques 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 functionalstudies

CEBPA phase separation links transcriptional activity and 3D chromatin hubs

We thank the Graf lab members for helpful discussions, and the CRG Genomics, Flow Cytometry and Advanced Light Microscopy core facilities; M.C.-K. was supported by an EMBO postdoctoral fellowship (ALTF 1057-2019) and PCIN-MSCA-fellowship (Ministerio de Ciencia e Innovación PCI2021-122032-2B). S.C. is supported by a "La Caixa" Junior Leader fellowship, by the Jérôme Lejeune Foundation (JLF#1902) and the Spanish Ministry of Science and Innovation (PID2020-117950RA-I00). G.S. was supported by the "Fundación Científica de la Asociación Española Contra el Cáncer. " M.V.N. was supported by People Program (Marie Curie Actions) FP7/2007-2013 under REA grant 6089 and Juan de la Cierva-Incorporación 2017. C.G.-C. acknowledges a graduate fellowship from MINECO (PRE2018-084684). X.S. acknowledges funding from AGAUR (2017 SGR 324), MINECO (BIO2015-70092-R and PID2019-110198RB-I00), and the European Research Council (CONCERT, contract number 648201). This work was funded by the Max Planck Society and partially supported by grants from the Deutsche Forschungsgemeinschaft (DFG) SPP2202 Priority Program Grant HN 4/1-1 (to D.H.) and by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) Plan Estatal 2015, SAF.2015-68740-P (to T.G.). We acknowledge support by the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the Josep Carreras Foundation, and the CERCA Programme / Generalitat de Catalunya.Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell-to-macrophage transdifferentiation. Mechanistically, we find that the intrinsically disordered region (IDR) of CEBPA undergoes in vitro phase separation (PS) dependent on aromatic residues. Both overexpressing B cells and native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors, liver cells, and trophectoderm cells reveal nuclear CEBPA foci and long-range 3D chromatin hubs at CEBPA-bound regions. In short, we show that CEBPA can undergo PS through its IDR, which may underlie in vivo foci formation and suggest a potential role of PS in regulating CEBPA function