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Developmental Biology

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Published: June 21st, 2016

DOI:

10.3791/53978

1Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona, 2Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, 3MTA-DE “Lendulet” Immunogenomics Research Group, University of Debrecen
* These authors contributed equally

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

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.

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3' of the gene or even within the introns and exons2.

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1. Enhancer Selection Based on Chip-seq Analysis

  1. Download the RXR ChIP-seq raw data fastq file (mm_ES_RXR_24h_ATRA.fastq.gz) from http://ngsdebftp.med.unideb.hu/bioinformatics/
  2. Download and extract the required BWA index file for the alignment (in our case: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz
    NOTE: Visit to https://github.com/ahorvath/Bioinformatics_scripts for more information regarding the steps of bioin.......

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We used a pan-specific RXR antibody in order to identify genome-wide which RA-regulated genes have receptor enrichment in their close proximity. Bioinformatics analysis of RXR ChIP-seq data obtained from ES cells treated with retinoic acid revealed the enrichment of the nuclear receptor half site (AGGTCA) under the RXR occupied sites (Figure 1). Using a bioinformatics algorithm we mapped back the motif search result for the half site to the RXR ChIP-seq data (Figu.......

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In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.

The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP.......

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The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-I/9.

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Name Company Catalog Number Comments
KOD DNA polymerase Merck Millipore 71085-3 for PCR amplification of enhancer from gDNA
DNeasy Blood & Tissue kit  Qiagen 69504 for genomic DNA isolation
QIAquick PCR Purification kit Qiagen 28106 for PCR product purification
Gel extraction kit  Qiagen 28706 for gel extraction if there are more PCR product
HindIII NEB R3104L restriction enzyme
BamHI NEB R3136L restriction enzyme
FastAP Thermo Scientific EF0651 release of 5'- and 3'-phosphate groups from DNA
T4 DNA ligase NEB M0202 for ligation
QIAprep Spin Miniprep kit Qiagen 27106 for plasmid isolation
DMEM Gibco 31966-021 ES media
FBS Hyclone SH30070.03 ES media
MEM Non-Essential Amino Acid Sigma M7145 ES media
Penicillin-Streptomycin Sigma P4333 ES media
Beta Mercaptoethanol Sigma M6250 ES media
FuGENE HD  Promega E2311 transfection reagent
Opti-MEM® I Reduced Serum Medium Life Technologies 31985-062 for transfection
All-trans retinoic acid Sigma R2625 ligand, for activation of RAR/RXR
96-well clear plate Greiner 655101 for Beta galactosidase assay
96-well white plate Greiner 655075 for Luciferase assay
D-luciferin, potassium salt Goldbio.com 115144-35-9 for Luciferase assay
ATP salt Sigma A7699-1G for Luciferase assay
MgSO4x 7H2O Sigma 230391-25G for Luciferase assay
HEPES Sigma H3375-25G for Luciferase assay
Na2HPO4 x 7H2O Sigma 431478-50G for Beta galactosidase assay
NaH2PO4 x H2O Sigma S9638-25G for Beta galactosidase assay
MgSO4 x 7H2O Sigma 230391-25G for Beta galactosidase assay
KCl Sigma P9541-500G for Beta galactosidase assay
ONPG (o-nitrophenyl-β-D-galactosidase) Sigma N1127-1G for Beta galactosidase assay
TRIzol® Life Technologies 15596-026 RNA isolation
High-Capacity cDNA Reverse Transcription Kit Life Technologies 4368814 reverse transcription of eRNA
Rnase-free Dnase Promega M6101 Dnase treatment
SsoFast Eva Green BioRad 750000105 RT-qPCR mastermix
CFX384 Touch™ Real-Time PCR Detection System BioRad qPCR machine
BioTek Synergy 4 microplate reader BioTek luminescent counter

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