Mapping 3D genome architecture through in situ DNase Hi-C

Abstract

With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves ∼4 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.

Figure 1. A schematic overview of in situ DNase Hi-C

First, fixed cells are lysed and digested with the endonuclease DNase I in the presence of divalent manganese—yielding double stranded breaks. Nuclei are then immobilized on carboxylated paramagnetic beads (i.e. ‘AMPure’ beads) to purify intact nuclei and remove free digested DNA fragments. Chromatin is then end-repaired and dA-tailed in situ, and a biotinylated ‘bridge adaptor’ containing a half BamHI site is ligated onto free chromatin ends. Nuclei are then subjected to phosphorylation and in situ proximity ligation, after which DNA is purified and fragments containing ligation junctions are enriched for via streptavidin beads and on-bead Illumina library prep (optionally following sonication).

Figure 2. Nuclei remain intact during the in situ DNase Hi-C protocol

a.) Purified supernatant DNA (see Box 1) from 6 different steps of the DNase Hi-C protocol. Minimal DNA is purified after each enzymatic purification, compared to a large amount of DNA, taken from 5% of the total gDNA yield following nuclear lysis. b.) Phase contrast micrograph (20X magnification) of GM12878 nuclei bound to beads, following proximity ligation (Step 46). Nuclei are highlighted using black arrows, and an example of a clump of carboxylated beads, which are found scattered across the image, is shown circled in white, with an accompanying white arrow.

Figure 3. Digestion quality controls throughout the in situ DNase Hi-C protocol

a.) A typical digestion pattern for DNase I-digested fixed chromatin prior to proximity ligation, run on a 6% TBE-PAGE gel. b.) Example of the BamH1 quality control experiment performed on GM12878 in situ DNase Hi-C libraries; in this example, BamH1 shifts the in situ DNase Hi-C library by digesting the reconstituted BamH1 site that forms following proximity ligation of the biotinylated bridge adaptors. Crucially, digestion with another 6-cutter (EcoR1), does not recapitulate this pattern, proving that the BamH1 digestion is specific to proximity ligated fragments. All reactions were run on one 6% TBE-PAGE gel.

Figure 4. In situ DNase Hi-C results for the mouse embryonic kidney Patski cell line

a.) In situ DNase Hi-C reads (950,206 downsampled reads from data published in Deng, Ma et al (using the mouse Patski cell line, rather than GM12878) demonstrate an enrichment for long-range (i.e. > 1 kb) intrachromosomal read pairs expected of Hi-C libraries. b.) Expected breakdown of mate orientations for read pairs in in situ DNase Hi-C data. For intrafragment distances > 1 kb, a roughly 25% split should be observed for each orientation class. c.) Normalized heat map generated from data published in Deng, Ma et al (GEO Accession: GSE68992) for mouse chromosome 18 at 100 kb resolution. The dataset used to generate this heatmap contained 60,666,200 uniquely mapped, high-quality read pairs.