Shin, K.-J.; Kang, J.; Kim, A.

CTCF-binding sites are frequently found at the boundaries of chromatin domains enriched with specific histone modifications in vertebrate genomes, where they function as barriers that limit the spreading of these modifications. However, their role in domains marked by H3K9me2 or H3K9me3 remains poorly understood. In this study, we analyzed CTCF-binding sites and the distribution of H3K9me2 and H3K9me3 around them in human K562 cells, and investigated the spreading of these modifications following CTCF depletion. Genome-wide analysis revealed that certain CTCF-binding sites are located at the boundaries of chromatin domains enriched with H3K9me2 or H3K9me3. Upon loss of CTCF binding, both modifications spread to neighboring chromatin at over half of the boundaries delimiting highly and distinctly enriched domains. Boundaries where such spreading did not occur were flanked by chromatin in a more active state compared to those where spreading occurred. Gene transcription was significantly reduced in neighboring regions where H3K9me2 or H3K9me3 had spread. Further comprehensive analysis revealed that this transcriptional reduction was specifically associated with the spread of H3K9me2. These findings suggest that CTCF-binding sites demarcate chromatin domains enriched with H3K9me2 or H3K9me3 and act as barriers to restrict the spread of these modifications in mammalian cells. Notably, the spread of H3K9me2 appears to play a specific role in transcriptional repression.