Decoding 3D enhancer architecture identifies hierarchical oncogenic regulatory programs in prostate cancer

Aberrations in non‑coding DNA regions, particularly those located in regulatory elements, are increasingly implicated as a hallmark of prostate cancer. Yet, mapping enhancers that underpin tumor‑specific transcription remains challenging. Here, we developed an integrative workflow to prioritize prostate cancer‑specific enhancers (PSEs) by analyzing 204 H3K27ac ChIP-seq datasets from prostate tumor and normal tissues, alongside prostate cell lines. We connected the differentially activated enhancer landscape to three‑dimensional (3D) chromatin organization in prostate cancer by identifying key oncogenic topologically associating domains (TADs) from Hi-C datasets. We selected a previously uncharacterized but high-priority locus at chr6q24.1 for in-depth study.  To define cancer-specific 3D architecture at chr6q24.1, we generated Region‑Capture Micro‑C (RCMC) maps at nucleosome resolution in RWPE‑1 (normal) and 22Rv1 (cancer) cells. This revealed highly nested enhancer-promoter (E-P) interactions, which we termed multi-connected enhancer hubs, that were prominent in cancer but absent in normal, exceeding the sensitivity of conventional Hi-C for enhancer-centered contacts. CRISPR/Cas9 deletion of individual enhancers across the locus, followed by multi‑omic profiling, revealed two distinct enhancer classes. Central PSEs (cPSEs) function as core regulatory organizers, whose deletions weakened activities of other PSEs, collapsed locus-wide chromatin interactions, reduced target gene expression, and impaired cancer cell proliferation without broadly altering CTCF/cohesin architecture. In contrast, redundant PSEs (rPSEs) are buffered by neighboring rescuing enhancers to preserve transcription via compensatory rewiring that strengthens alternative E-P contacts. Our data also suggest that these two classes of enhancer behaviors are associated with differential activity of FOXA1, a pioneer transcription factor in prostate cancer. Together, our study revealed cancer-specific, multi-connected enhancer hubs essential for prostate tumorigenesis and uncovered the regulatory hierarchy of enhancers, providing a framework to functionally characterize and validate oncogenic non-coding DNA regions that sustain prostate cancer phenotypes. This study advances our understanding of non-coding regulatory regions and offers future opportunities for developing novel precision epigenome-based clinical interventions for prostate cancer.