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

Summary

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

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.

Introduction

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.

Protocol

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 bioinformatics analysis and to download the scripts used below.
3. Align the example fastq file to mm10 genome (use the script: perform_alignment.sh....

Results

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.......

Discussion

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.......

Materials

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