H3K27 and H3K9 Methylation Mask Potential CTCF Binding Sites to Maintain 3D Genome Integrity

The three-dimensional (3D) genome structure is essential for gene regulation and various genomic functions. CTCF plays a key role in organizing Topologically Associated Domains (TADs) and promoter-enhancer loops, contributing to proper cell differentiation and development. Although CTCF binds the genome with high sequence specificity, its binding sites are dynamically regulated during development, and aberrant CTCF binding is linked to diseases such as cancer and neurological disorders, and aging. However, the mechanisms controlling CTCF binding remain unclear. Here, we investigated the role of repressive chromatin modifications in CTCF binding using H3K9 methyltransferase-deficient immortalized mouse embryonic fibroblasts (iMEFs) and H3K27 methyltransferase EZH1/2 inhibitor. We found that H3K9 and H3K27 methylation regulate CTCF binding at distinct genomic regions, and their simultaneous loss induces drastic changes in CTCF binding. These changes were associated with alterations in 3D genome architecture and gene expression, suggesting that repressive chromatin modifications preserve proper chromatin organization by preventing aberrant CTCF binding. Additionally, while CTCF binding sites repressed by H3K9 methylation were bound by CTCF in early mouse embryos, those repressed by both H3K9 and H3K27 methylation remained inaccessible, with early embryonic-specific H3K27 methylation forming at these sites. These findings implicate that H3K27 methylation prevents abnormal CTCF binding in early embryos, ensuring proper genome organization during development.