title: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation.
abstract: Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.
authors.affiliation: Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Simandi
authors.affiliation: Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen.
authors.forename: Attila
authors.initials: A
authors.lastname: Horvath
authors.affiliation: Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona.
authors.forename: Peter
authors.initials: P
authors.lastname: Nagy
authors.affiliation: Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona; Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen; MTA-DE "Lendulet" Immunogenomics Research Group, University of Debrecen; lnagy@sbpdiscovery.org.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Nagy
mesh_terms: D002454:Cell Differentiation; D047369:Chromatin Immunoprecipitation; D053595:Embryonic Stem Cells; D004742:Enhancer Elements, Genetic; D014212:Tretinoin
chemical_list: D014212:Tretinoin