High-throughput capture of actively transcribed region-interacting sequences reveals an intricate promoter-centered regulatory network

Understanding the intricate relationship between three-dimensional chromatin structure and gene expression regulation is essential for cellular biology. However, current techniques are insufficient to capture regulatory elements functioning through three-dimensional chromatin structures. Here, we present high-throughput capture of actively transcribed region-interacting sequences (Hi-Coatis), a high-throughput method that seamlessly integrates the detection of active transcription signals with three-dimensional chromatin interaction studies. Hi-Coatis operates without antibodies or probes, enabling low-input cell experiments with high resolution and robustness, capturing more than 93% of expressed genes and over 60,000 regulatory loci in human cells. The repetitive/copy number variation (CNV) regions and the promoter regions of C2 genes, defined by the distribution patterns of Hi-Coatis signals, both exhibit strong regulatory element activity. Notably, in the Hemin-induced erythroid differentiation model of K562 cells, Hi-Coatis uncovers the potential for silent genes to transition to transcriptionally active states through the cooperative influence of specific transcription factors (e.g., CCCTC-binding factor [CTCF] and cohesin complex subunits Rad21 [RAD21]) and regulatory elements.