The lncRNA CTD-2566 J3.1 orchestrates oncogenic gene regulation via chromatin interactions in luminal breast cancer.

Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and genome architecture in cancer. By scaffolding chromatin-modifying protein complexes and mediating enhancer-promoter communication, lncRNAs can shape transcriptional programs that sustain tumor growth. In this study, we investigated the functional role of CTD-2566 J3.1, an antisense lncRNA located within the RAD51B locus that carries enhancer-associated chromatin marks, including H3K27ac and H3K4me1, in luminal breast cancer cells. Using chromatin conformation capture experiments (4C-seq), we identified recurrent long-range chromatin interactions linking the CTD-2566 J3.1 locus to regulatory elements within three distant genes with established roles in cancer biology: RAD51B, MAP3K9, and ACTN1. These genomic regions were enriched for estrogen receptor (ESR1) and FOXA1 binding sites, suggesting hormone-responsive regulation. Interestingly, in three-dimensional spheroids, these genes were consistently more expressed than in monolayer cultures, underscoring the importance of microenvironmental context for transcriptional regulation in breast cancer. Functionally, loss of CTD-2566 J3.1 impaired cell proliferation and invasion and increased endogenous DNA damage. Our findings establish CTD-2566 J3.1 as a multifunctional enhancer-associated lncRNA (e-lncRNA) that orchestrates key oncogenic processes in luminal breast cancer. Additionally, this work underscores the utility of three-dimensional culture systems to reveal context-specific lncRNA functions and highlights CTD-2566 J3.1 as a potential regulator and therapeutic target in breast cancer.