ChIP-based methods for the identification of long-range chromatin interactions

Abstract

Chromatin immunoprecipitation (ChIP) is an important technique for studying protein-DNA interactions. Whole genome ChIP methods have enjoyed much success, but are limited in that they cannot uncover important long-range chromatin interactions. Chromosome conformation capture (3C) and related methods are capable of detecting remote chromatin interactions, but are tedious, have low signal-to-noise ratios, and are not genome-wide. Although the addition of ChIP to 3C (ChIP-3C) would conceivably reduce noise and increase specificity for chromatin interaction detection, there are concerns that simple mixing of the ChIP and 3C protocols would lead to high levels of false positives. In this essay, we dissect current ChIP- and 3C-based methodologies, discuss the models of specific as opposed to non-specific chromatin interactions, and suggest approaches to separate specific chromatin complexes from non-specific chromatin fragments. We conclude that the combination of sonication-based chromatin fragmentation, ChIP-based enrichment, chromatin proximity ligation and Paired-End Tag ultra-high-throughput sequencing will be a winning implementation for genome-wide, unbiased and de novo discovery of long-range chromatin interactions, which will help to establish an emerging field for studying human chromatin interactomes and genome regulation networks in three-dimensional spaces.

Figure 1. Schematic comparison of 3C, 4C, 5C, ChIP-3C, and ChIA-PET

This schematic shows the differences between the (top to bottom) 3C, 4C, 5C, ChIP-3C and ChIA-PET methodologies. The ChIA-PET method can be implemented with two possible sources of material: ChIP-enriched and non ChIP-enriched sonicated chromatin.

Figure 2. Sonication is a method for breaking up non-specific chromatin interaction complexes

Non-specific interactions from random collisions of chromatin fibers generally involve chromatin interactions brushing past each other at the periphery of chromatin complexes, whereas specific chromatin interactions are tethered to each other by specific factors. Gentle restriction enzyme digestion would not disturb the weak non-specific interactions, but vigorous sonication could “shake off” non-specific, weak, peripheral chromatin and only retain strongly-bound core chromatin interaction complexes that involve specifically-bound chromatin interactions.

Figure 3. Sequencing from different experimental approaches

Here we show expected and actual experimental work from sequencing DNA from different experimental approaches: that of enzymatic fragmentation (left), sonicated fragmentation (center) and sonicated fragmentation with the addition of ChIP enrichment (right). Sequencing reverse cross-linked, fragmented, non ChIP-enriched chromatin would be similar to sequencing the entire genome. Sequencing reverse cross-linked, fragmented, ChIP-enriched chromatin would be similar to a ChIP-Seq experiment.

Figure 4. 3C and 4C from different experimental approaches

Here we show expected and actual experimental work from performing 3C or 4C on proximity-ligated material prepared by different experimental approaches: that of enzymatic fragmentation (left), sonicated fragmentation (center) and sonicated fragmentation with the addition of ChIP enrichment (right). 3C and 4C on enzyme digested chromatin would be quite noisy; 3C and 4C on sonicated chromatin would be less noisy, whilesChIP-3C and ChIP-4C on sonicated chromatin would be the least noisy and specific for chromatin interactions bound to the protein of interest. The use of sonication reduces some noise, and the further use of ChIP reduces the noise levels still further.

Figure 5. ChIA-PET from different experimental approaches

Here we show expected and actual experimental work from performing paired end sequencing on proximity-ligated chromatin prepared by different experimental approaches: that of enzymatic fragmentation (left), sonicated fragmentation (center) and sonicated fragmentation with the addition of ChIP enrichment (right). The use of sonication reduces some noise, and the further use of ChIP reduces the noise levels still further, allowing for the identification of true chromatin interaction signals. In addition, the use of ChIP ChIA-PET gives specific information regarding the TFBS and chromatin interactions between TFBS.