High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

Summary

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Abstract

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

Introduction

The identification of regulatory elements for gene expression has been facilitated by the Encyclopedia of DNA Elements (ENCODE) Project that comprehensively annotated functional activity for 80% of the human genome^1,2. The identification of the sites for in vivo transcription factor binding, DNaseI hypersensitivity, and epigenetic histone and DNA methylation modifications in individual cell types paved the way for the functional analyses of candidate regulatory elements for target gene expression. Armed with these findings, we are faced with the challenge of determining the functional interconnectivit....

Protocol

1. Restriction Enzyme Selection

1. Identify a region of interest (e.g., gene promoter, single nucleotide polymorphism (SNP), enhancer) and obtain the DNA sequence from repositories such as National Center for Biotechnology Information (NCBI).
2. Identify the candidate restriction enzymes (REs) for the first restriction enzyme digestion (RE1) that do not cut within the sequence of interest, which produce sticky ends (DNA termini with overhangs) after RE digestion, and whose activities are not inhibited by CpG methylation.
   Note: The resolution of the data is determined by RE1. An enzyme with a 6 base pair (bp) recognition sequence produce....

Results

Primary human keratinocytes were isolated from 2–3 discarded neonatal foreskins, pooled, and cultured in KSFM supplemented with 30 µg/mL bovine pituitary extract, 0.26 ng/mL recombinant human epidermal growth factor, and 0.09 mM calcium chloride (CaCl₂) at 37 °C, 5% carbon dioxide. The cells were split into two flasks, and one flask was differentiated by the addition of CaCl₂ to a final concentration of 1.2 mM for 72 h. 10⁷ cells each from proliferating an.......

Discussion

4C results have the potential to reveal chromatin interactions that can identify previously unknown regulatory elements and/or target genes that are important in a specific biological context^24,25,26. However, technical hurdles may limit the data obtained from these experiments. PCR bias stemming from over-amplification of template in 4C protocols is likely. This protocol addresses this issue by utilizing qPCR to determine the o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HindIII	NEB	R0104S
CviQI	NEB	R0639S
DNA oligonucleotide primers	IDT		To be designed by the reader
50 mL conical centrifuge tubes	Fisher Scientific	06-443-19
1.7 mL microcentrifuge tubes	MidSci	AVSS1700
Phosphate buffered saline	Thermo Fisher	14190-136
Formaldehyde, methanol free	Electron Microscopy Sciences	15710
Nutator	VWR	15172-203
Glycine	JT Baker	4059-00
Benchtop centrifuge
Refrigerated microcentrifuge
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	ED2SS
20% SDS solution	Sigma-Aldrich	05030
Trypan Blue	Thermo Fisher	15250061
Glass slides	Fisher Scientific	12-550-143
Cover slips	VWR	16004-094
Light microscope
Triton X-100	Alfa Aesar	A16046
Shaking heat block
2 M Tris-HCl	Quality Biological	351-048-101
Proteinase K	NEB	P8107S
Phenol:chloroform:isoamyl alcohol (25:24:1)	Sigma-Aldrich	P2069
Sodium acetate	Sigma-Aldrich	M5661
20 mg/mL glycogen	Thermo Fisher	R0561
Ethanol	Fisher Scientific	04-355-223
Nuclease-free water	Fisher Scientific	MT-46-000-CM
qPCR cycler	Thermo Fisher	4453536
qPCR plates	Thermo Fisher	4309849
Thermocycler	Thermo Fisher	4375786
PCR strip tubes	MidSci	AVSST-FL
1 M Magnesium chloride	Quality Biological	351-033-721
Dithiothreotol	Sigma-Aldrich	43815
Adenosine triphosphate	Sigma-Aldrich	A2383
T4 DNA Ligase	NEB	M0202S
Agarose	Sigma-Aldrich	A6013
RNase A	Thermo Fisher	EN0531
Qiaquick PCR purification kit	Qiagen	28104
MinElute PCR Purification kit	Qiagen	28004
Spectrophotometer
Expand Long Template PCR System	Sigma-Aldrich	11681834001
dNTP mix	Thermo Fisher	R0191
SYBR Green I	Sigma-Aldrich	S9430
ROX	BioRad	172-5858
Sodium chloride	Sigma-Aldrich	S5886
End-It DNA End-Repair Kit	Lucigen	ER0720
LigaFast Rapid DNA Ligation System	Promega	M8221
SYBR Safe	Thermo Fisher	S33102
Taq Polymerase	NEB	M0267S
UltraSieve Agarose	IBI Scientific	IB70054
Qiaquick Gel Extraction Kit	Qiagen	28704