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.

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.