3C-digital PCR for quantification of chromatin interactions

Abstract

BackgroundChromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls.ResultsTo demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40–1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41–2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76–1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145).ConclusionsCompared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate multiple and complex normalization controls and provide accurate calculation of proximity-based fragment ligation frequency. Therefore, we recommend 3C-dPCR as a preferred method for sensitive detection of low frequency chromatin interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0076-6) contains supplementary material, which is available to authorized users.

Trans-interaction of risk loci 6p24.1 and 10q11.21 is associated with endothelial damage in coronary artery disease

“…Furthermore, the inherent bias posed by 3C technique toward cis interactions due to the power-law decay model makes it only possible for the most frequent long-range interactions to be accurately quantified [56]. Therefore, instead of conventional TaqMan qPCR, we switched to droplet digital PCR, which delivered high precision of absolute ligation product copy numbers, without the numerous normalization controls that 3C-qPCR requires [40], allowing us to accurately quantify ligation frequency using 3C. To make up the large number of cells (10 million) required for 3C, we pooled five independently differentiated batches of iPSC-derived endothelial cells, at the same time controlling for batch variations.…”

“…Probe-based ddPCR was performed following manufacturer’s protocol with reference to previously described 3C-digital PCR protocol [40]. Reactions were performed in total 24ul volume using 12ul of 2 x ddPCR Supermix for Probes (No dUTP) (Bio-Rad Laboratories, catalog no.…”

“…Furthermore, the inherent 34 bias posed by 3C technique toward cis interactions due to the power-law decay model makes it only possible for the most frequent long-range interactions to be accurately quantified [56]. Therefore, instead of conventional TaqMan qPCR, we switched to droplet digital PCR, which delivered high precision of absolute ligation product copy numbers, without the numerous normalization controls that 3C-qPCR requires [40],…”

Targeting the chromatin structural changes of antitumor immunity

“…Primers that might interact with the target segments are used to conduct general PCR and quantitative PCR to determine whether there is interaction [ 202 ]. Moreover, 3C technology assumes that the connection frequency of physically interacting DNA segments is the highest, uses site-specific PCR to detect the physical contact between DNA segments in the genome, and finally determines whether there is interaction based on the abundance of PCR products [ 202 , 203 ]. The use of 3C technology is suitable for studying the interaction between 5 kb and hundreds of kb chromatin [ 204 ].…”

An improved method for quantitative ChIP studies of nuclear receptor function

“…Exploiting these advantages of digital PCR, the technique has been combined with ChIP-related techniques such as chromosome conformation capture (3C) (Link et al 2013, Du & Wang 2016, and in quantifying binding of Cas9 endonuclease to its targets (Chen et al 2017). In recent years, ChIP-ddPCR itself has been employed to study histone marks (Kim et al 2016) and the binding of transactivator protein CIITA (Wong et al 2014).…”