CTCF

ChIA-PET

Transcription factor

Biology

Promoter

Gene

Enhancer

Bivalent chromatin

Gene expression

Nanopore sequencing

Genome

Chromatin remodeling

Human genome

ChIP-sequencing

DNA sequencing

Computational biology

Genetics

Scaffold/matrix attachment region

Chromatin

Chromosome conformation capture

Nature Biotechnology

High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background (‘synergies’). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure. High-order chromatin contacts are identified using a combination of 3C, nanopore sequencing and robust statistical analysis.

Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing