High-resolution 3D chromatin maps reveal PTEN enhancer hubs bridged to the promoter through flanking anchors

Understanding the cis-regulatory architecture of tumor suppressor loci is crucial for elucidating the mechanisms of gene dysregulation in cancer and genetic syndromes. Despite its central role in multiple cancers and PTEN Hamartoma Tumor Syndrome (PHTS), the regulatory landscape of the PTEN locus remains largely uncharacterized. Using high-resolution Tiled-C and multi-omics profiling across 11 human cell lines, including two patient-derived models, we map a 3 Mb region surrounding the PTEN topologically associated domain (TAD). We identify seven chromatin hubs anchoring eleven enhancers, whose CRISPR interference substantially reduces PTEN expression. In these hubs, most enhancers preferentially interact with each other rather than the PTEN promoter, forming a spatially organized enhancer network. Notably, enhancers are not in direct contact with the promoter but are brought into proximity through associated flanking chromatin anchor points enriched in REX elements, enabling gene regulation by bridging distal regulatory elements. Lastly, while structural variants can disrupt TAD, PTEN mutations alone only slightly reinforce intra-TAD contacts without changing the structure. These findings provide the first high-resolution map of PTEN enhancer hubs, reveal new principles of enhancer cooperation and long-range gene regulation, and delineate candidate regions whose disruption may contribute to PHTS and PTEN-driven tumorigenesis.