Characterization of Human Ectocentromeric Sites

Abstract Centromeres are composed of DNA repeats within chromosomes’ primary constriction. CENP-B is the only centromeric protein known to bind a specific motif, the CENP-B box, promoting kinetochore stability. We recently uncovered degenerate CENP-B binding motifs outside centromeres, whose position and orientation defines chromosome specific banding patterns. Here, we leveraged telomere-to-telomere assemblies to map conservation of these ectocentromeric sequences (ECS) across hundreds of haplotypes. We found strong negative selection acting on their occurrence along chromosome arms, implying functional constraints incompatible with stochastic drift. We classified four categories: (i) ECSs that lack CENP-B binding (∼84%); (ii) ECSs bound by CENP-B (∼10%); (iii) ECSs near CENP-B-enriched accessible chromatin (∼6%); (iv) we further identified ∼700 CENP-B binding sites outside centromeres without CENP-B boxes. Integrating chromatin conformation capture (HiC), neocentromeres and meiotic recombination mapping with CENP-B CUT&RUN, methylation and ATAC-seq data, we found heterogenous functionalities driven by distance-dependent enrichment and local contacts of boxes in inverted orientation on the same strand, analogous to ALU repeats affecting topological folding. CENP-B knockdown significantly reduced neighboring gene expression, revealing a moonlighting regulatory role outside centromeres. Our findings characterizes human ectocentromeric sites as evolutionarily constrained and functionally heterogeneous elements along chromosome arms with context-dependent roles in chromatin state. Graphical Abstract Ectocentromeric sites exhibit heterogeneous CENP-B occupancy and context-dependent chromatin functions. Ectocentromeric sequences (ECSs) along chromosome arms fall into four categories: (i) CENP-B box motifs alone, lacking protein binding, embedded within repressed or boundary chromatin and contributing to TAD organization. Motifs with opposite orientation (forward and reverse complement) paired on the same strand may further promote self-complementary chromatin contacts analogous to Alu inverted repeats, reshaping topology with long-range looping contacts; (ii/iii) ECSs bound by CENP-B protein, associated with specific open chromatin state downstream of H3K27me3-marked compacted chromatin that modulate local accessibility and gene expression; and (iv) CENP-B binding peaks lacking a canonical box motif, located proximal to transcription start sites and linked to active gene expression. Together, ectocentromeric sites represent functionally heterogeneous elements with context-dependent roles spanning chromosome architecture, chromatin state, and transcription regulation.