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

Mostra el registre complet Registre parcial de l'ítem

• dc.contributor.author Tian, Tian V.
• dc.contributor.author Vidal Ocabo, Enrique
• dc.contributor.author Graf, T. (Thomas)
• dc.contributor.author Stik, Grégoire
• dc.date.accessioned 2020-10-20T05:53:19Z
• dc.date.available 2020-10-20T05:53:19Z
• dc.date.issued 2020
• dc.description.abstract 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.
• dc.description.sponsorship 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
• dc.format.mimetype application/pdf
• dc.identifier.citation Tian TV, Vidal E, Graf T, Stik G. Identification of enhancer-promoter contacts in embryoid bodies by quantitative chromosome conformation capture (4C). J Vis Exp. 2020 Apr 29; (158). DOI: 10.3791/60960
• dc.identifier.doi http://dx.doi.org/10.3791/60960
• dc.identifier.issn 1940-087X
• dc.identifier.uri http://hdl.handle.net/10230/45510
• dc.language.iso eng
• dc.publisher MyJove Corporation
• dc.relation.ispartof Journal of Visualized Experiments. 2020 Apr 29;(158)
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/747539
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/609989
• dc.rights © 2020 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/
• dc.subject.other Embrions
• dc.subject.other Genètica
• dc.subject.other Cromosomes
• dc.title Identification of enhancer-promoter contacts in embryoid bodies by quantitative chromosome conformation capture (4C)
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion