Wu, L.; Lane, K. A.; Muhammad, R.; Harrod, A.; Naughton, C.; Wang, H.; Musacchio, A.; Gilbert, N.; Alfieri, C.; Downs, J. A.

CENP-B, a centromeric protein known for its role in binding the B box sequence of centromeric DNA, has long been recognized as important, though not essential, for kinetochore attachment and chromosome segregation. Here, we identify an unexpected, non-centromeric role for CENP-B. We demonstrate that CENP-B binds to specific non-centromeric sites along chromosome arms, predominantly at promoters, and depletion of CENP-B leads to dysregulated gene expression. Binding is enriched in G2 phase cells and, importantly, occurs independently of the canonical B box motif. Instead, CENP-B binding in chromosome arms is defined by regions of negatively supercoiled DNA containing repetitive sequences, such as multiple CCAAT boxes, that are prone to forming secondary structures. Consistently, we find that CENP-B binds to hairpin DNA in vitro via its DNA binding domain. The chromosome arm binding pattern is conserved across cell types and is particularly prominent in the promoters of transcriptionally active replication-dependent histone genes. These findings reveal a previously unrecognized centromere-independent binding activity of CENP-B.