Prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

Zoltan Simandi, Attila Horvath, Peter Nagy, Laszlo Nagy

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article numbere53978
JournalJournal of Visualized Experiments
Issue number112
StatePublished - Jun 21 2016
Externally publishedYes


  • ChIP-seq
  • Developmental biology
  • ERNA
  • Embryonic stem cell
  • Enhancer
  • Gene expression
  • Issue 112
  • Luciferase assay
  • Retinoic acid
  • Retinoic acid receptor
  • Transcription

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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