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

Zoltan Simandi, Attila Horvath, Peter Nagy, L. Nagy

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Article numbere53978
JournalJournal of Visualized Experiments
Volume2016
Issue number112
DOIs
Publication statusPublished - Jun 21 2016

Fingerprint

Chromatin Immunoprecipitation
Regulator Genes
Embryonic Stem Cells
Tretinoin
Stem cells
Chromatin
Cell Differentiation
Workflow
Genes
Nucleic Acid Regulatory Sequences
RNA
Gene expression
Acids
Genome
Cloning
Gene Expression Regulation
Microarrays
Embryonic Development
Organism Cloning
Assays

Keywords

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

ASJC Scopus subject areas

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

Cite this

Prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation. / Simandi, Zoltan; Horvath, Attila; Nagy, Peter; Nagy, L.

In: Journal of Visualized Experiments, Vol. 2016, No. 112, e53978, 21.06.2016.

Research output: Contribution to journalArticle

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