Nucleolar Expansion Drives 3D Genome Reorganization and Transcriptional Repression in Cancer

The nucleolus plays a pivotal role in organizing the three-dimensional chromatin architecture through the sequestration and regulation of heterochromatin, yet how such structure remodeling contributes to tumorigenesis remains poorly understood. Analysis of TCGA data revealed widespread upregulation of nucleolar components across multiple human cancers, indicating nucleolar expansion during tumor progression, which was further validated by immunofluorescent imaging. To investigate this phenomenon, we developed Nucleo-seq and Nucleo-C, a pair of highly sensitive methods for high-resolution profiling of nucleolus‑associated chromatins (NADs) and their three-dimensional (3D) interactions from low-input isolated nucleoli. Applying these approaches to normal (MCF10A) and cancer (MDA-MB-231) breast cell lines, we uncovered global architectural alterations: cancer cells exhibited increased NAD signal intensity and enhanced 3D connectivity within NADs, closely correlated with nucleolar enlargement. This NAD reorganization was strongly associated with transcriptional repression of NAD-resident genes, which results in silencing of key tumor-suppressive pathways, including cell adhesion, negative regulation of cell proliferation, and Wnt signaling. Our findings establish a novel paradigm in which nucleolar architectural expansion in cancer drives NAD reorganization and gene dysregulation, offering new 3D genome-related biomarkers for cancer prognosis and therapeutic intervention.