Distal cis-regulatory elements in 3’ UTRs orchestrate gene expression via single-gene domain architecture in Brassica rapa

Cis-regulatory elements (CREs) are fundamental to precise gene regulation. Although distal CREs located in intergenic regions have been extensively studied, the functional landscape of those residing in 3′ untranslated regions (UTRs) remains largely unexplored. In this study, we generate comprehensive epigenomic profiles of Brassica rapa leaves at two developmental stages (rosette and reproductive), integrating ATAC-seq, CUT&Tag for five histone modifications (H3K4me3, H3K27ac, H3K27me3, H3K36me3, H3K9me2), and BL-Hi-C data. We identify 6362 CREs within 3’ UTRs, defined as accessible chromatin regions within 0.5 Kb of transcription termination sites. These CREs within 3’ UTRs are functionally classified by histone marks: H3K27ac-marked, H3K27me3-marked, and bivalent CREs. Using BL-Hi-C at resolutions up to 100 bp, we uncover that these CREs within 3’ UTRs contribute to the formation of single-gene domains. Comparative analysis between developmental stages reveals that distal CREs orchestrate stage-specific gene expression by modulating chromatin interaction, a mechanism exemplified by the flowering regulator BrFLC2. The transcriptional activation activity of the distal CRE of BrFLC2 was verified by dual-luciferase reporter assays. Together, our findings establish 3’ UTR-embedded distal CREs as critical regulatory elements that shape gene expression through single-gene domain architecture in B. rapa.