Xu, Y.; Loucks, H.; Menendez, J.; Ryabov, F.; Lucas, J. K.; Cechova, M.; Morina, L.; Xu, E.; Dubocanin, D.; Chittenden, C.; Asri, M.; Violich, I.; Ortiz, C.; Gardner, J. M. V.; Hillaker, T.; O'Rourke, S.; McNulty, B.; Potapova, T. A.; Mitchell, M. W.; Schwartz, J. P.; Straight, A. F.; Gerton, J. L.; Timp, W.; Alexandrov, I. A.; Altemose, N.; Miga, K. H.

Centromeres ensure proper chromosome segregation during cell division, yet the organization and regulation of centromeric chromatin within satellite DNA arrays remain incompletely understood. Here, we leverage the complete diploid human genome benchmark (T2T-HG002) to provide a detailed study of centromeric sequence and chromatin architecture on individual haplotypes. Using adaptive-sampling-enriched, ultra-long-read DiMeLo-seq, we achieve single-molecule chromatin profiling across all centromeres, revealing that along single chromatin fibers, CENP-A, the histone variant specifying centromere identity, forms multiple discrete subdomains within hypomethylated centromere dip regions (CDRs) that are flanked by H3K9me3-enriched heterochromatin. Despite underlying sequence variation, CDRs localize to sequence-homogeneous domains and maintain relatively balanced CENP-A dosage and aggregate length across all chromosomes and between haplotypes. Further, we show that bidirectional changes to centromeric and pericentromeric DNA methylation are accompanied by changes to centromeric chromatin architecture. In passaged cells with centromeric hypomethylation, subdomain boundaries are eroded, and adjacent CENP-A domains tend to merge and expand. Conversely, in pluripotent stem cells with centromeric hypermethylation, CDRs are fundamentally reorganized, such that discrete hypomethylated domains are frequently consolidated into broader contiguous tracts. These methylation-associated CDR restructuring events suggest that DNA methylation acts as a principal regulator of human centromere organization, with implications for understanding centromere plasticity, epigenetic inheritance, and chromosomal instability in development and disease.