CTCF-binding sites demarcate chromatin domains enriched with H3K9me2 or H3K9me3 and restrict the spreading of these histone modifications in human cells

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, the role of CTCF-binding sites in chromatin 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 these sites 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 boundaries delimiting highly and distinctly enriched domains. Boundaries where such spreading did not occur (non-spreading boundaries) were flanked by regions with a more active chromatin state compared to those where spreading occurred (spreading boundaries). 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 rather than H3K9me3. These findings suggest that CTCF-binding sites demarcate chromatin domains enriched with H3K9me2 or H3K9me3 and act as barriers to the propagation of these modifications in mammalian cells. Notably, the spread of H3K9me2 appears to play a specific role in transcriptional repression.