The biogenesis of extrachromosomal circular DNA is associated with enhancer-promoter dynamics and chromatin architecture.

Extrachromosomal circular DNAs (eccDNAs) have been discovered in various species and play a significant role in cancer development. However, our understanding of their chromosomal biogenesis is limited. Here, we identified a correlation between eccDNA generation and the density of transcription regulatory elements, such as enhancers and promoters. We find that the regulators of enhancer-promoter interactions, including RAD21, LDB1 and YY1, may contribute to eccDNA production, with only a modest effect from transcriptional activity itself. The BRD4 inhibitor JQ1 reduces eccDNA production, while the HDAC inhibitor TSA induces an increase of eccDNAs. This TSA-induced effect can be rescued by the TSA antagonist, ITSA. The generation of eccDNA positively correlates with the density of R loops. Furthermore, large eccDNA breakpoints are found to be coordinated with TAD boundaries and inclined to align at enhancers and promoters. Our study suggests that eccDNAs are more frequently detected from regulatory regions in the genome, consistent with their generation being associated with enhancer-promoter dynamics.