ChIP-PET

In ChIA-PET, long-range chromatin interactions are captured by formaldehyde cross-linking. Sonicated DNA-protein complexes are enriched by chromatin immunoprecipitation (ChIP). Tethered DNA fragments in each of the chromatin complexes are connected with DNA linkers via proximity ligation, and Paired-End Tags (PETs) are extracted for sequencing. The resulting ChIA-PET sequences are mapped to reference genomes to reveal relationships between remote chromosomal regions brought together in close spatial proximity by protein factors (Fig. 1a; Supplementary Fig. 1).
ChIA-PET proximity ligation generates two types of ligation products: self-ligation of the same DNA fragments and inter-ligation between different DNA fragments. PET sequences derived from self-ligation products are mapped in the reference genome within a 3 Kb span, demarcating ChIP DNA fragments, similar to the standard ChIP-sequencing method3 (link),8 (link). Tethered DNA fragments in individual chromatin complexes can also ligate with each other, and the mapping results of such inter-ligation PET sequences would reveal if they are intrachromosomal (both tags of each PET are from the same chromosome) or interchromosomal (the tags are from different chromosomes). Singleton PETs are presumed experimental noise, and overlapping PET clusters are considered enriched putative binding sites or interaction events (Supplementary Fig. 2).
To test the ChIA-PET strategy, we constructed two ChIA-PET libraries from independent ERα ChIP-enriched oestrogen-treated MCF-7 chromatin preparations, and generated two replicate pilot datasets (IHM001H and IHM001N) using Roche/454 pyrosequencing. Our analysis showed that both ChIA-PET libraries produced comparable putative binding sites and interactions. To assess levels of false positive chromatin interactions, we created a negative control ChIP-PET library (IHM043) from the same ChIP sample, wherein the DNA was reverse cross-linked before proximity ligation. We also analyzed a previously reported cloning-based ChIP-PET library (SHC007)8 (link). Both libraries generated abundant binding sites but no interactions. As an additional control, we used IgG, which binds to chromatin nonspecifically, to perform a mock ChIA-PET analysis (IHM062), and only a few binding sites and interactions were identified (Table 1; Supplementary Figs. 2-3; Supplementary Text I).
In proximity ligation-based analyses including 3C, the level of non-specific chimeric DNA ligations between different chromatin complexes can be high and thus may confound data analysis. To address this, we designed linker nucleotide barcodes in the ChIA-PET method to specifically identify such chimeric ligation PETs in another ERα ChIA-PET replicate. Linker barcoding analysis suggests that chimeric ligations are random and do not overlap with each other to form false positive interactions (Table 1, Supplementary Fig. 4, and Supplementary Text II). A possible complication is that ChIP-enriched loci with more DNA fragments would result in proportionally higher chances of inter-ligations, leading to false positive interactions comprising randomly overlapping inter-ligation PETs among highly-enriched ChIP DNA fragments. Hence, we devised a statistical scheme to calculate such probabilities and neutralize the potential ChIP-enrichment bias (Supplementary Materials and Methods; validations in Supplementary Fig. 5).
Together, these libraries indicate that the prevalent chromatin interactions (Supplementary Figs. 2d-g) identified by ERα ChIA-PET data depend on proximity ligations of chromatin complexes, and not technical artifacts of ligations between random DNA fragments, nor mapping errors.

HiCCUPS will not run on an interaction matrix that is too sparse (less than 300m filtered reads), and so for our low cell number samples which were not sequenced as deeply, we used the Fit-HiC contact caller. We also processed our 25m cell libraries with Fit-HiC for the comparisons in Figure 1d and Supplementary Figure 2a.
Bin pairs of the interaction matrix with statistically significant contact signal were identified using Fit-HiC^{19 (link)}. Genome wide intra-chromosomal bin pairs were filtered for an interaction distance between 20kb and 2Mb, and default Fit-HiC settings were used to calculate false discovery rate (FDR) values for each bin-pair in a given HiChIP experiment. For the comparison of mESC experiments in Figure 1D, Smc1a HiChIP binpairs or Smc1a ChIA-PET high-confidence interactions^{13 (link)} were filtered for overlap with Smc1a ChIP-seq peaks. The appropriate FDR was selected for each HiChIP experiment to result in approximately 10,000 contacts per experiment.
GM12878 Smc1a HiChIP filtered PETs from the HiC-Pro pipeline were processed through stages 4 and 5 of the Mango^{17 (link)} pipeline to call significant interactions. Stage 4 applies MACS2¹⁸ to call peaks using PETs. Stage 5 modeled the background interactions by taking interaction distance and depth into consideration and used a binomial distribution to call significant interactions. With our Mango parameters of PETs >= 4 and a FDR cutoff of 10⁻⁴ we obtained 61395 significant interactions.
The Juicer pipeline’s HiCCUPS tool was used to identify loops^{5 (link),20 (link),21 (link)}. Filtered read-pairs from the HiC-Pro pipeline were converted into .hic format files and input into HiCCUPS. The same parameters used for GM12878 Hi-C^{5 (link),20 (link),21 (link)} were used on the HiChIP datasets as follows: hiccups -m 500 -r 5000,10000 -f 0.1,0.1 -p 4,2 -i 7,5 -d 20000,20000 HiCCUPS_output.txt