Human centromeres are specialized chromatin domains containing the centromeric histone H3 variant CENP-A. CENP-A nucleosomes are interspersed with nucleosomes containing histone H3 dimethylated at lysine 4, distinguishing centromeric chromatin (CEN chromatin) from flanking heterochromatin that is defined by H3 lysine 9 methylation. To understand the relationship between chromatin organization and the genomic structure of human centromeres, we compared molecular profiles of three endogenous human centromeres, defined by uninterrupted higher-order α-satellite DNA, with human artificial chromosomes that contain discontinuous blocks of higher-order α-satellite DNA and noncentromeric DNA. The underlying sequence did not correlate with chromatin states, because both higher-order α-satellite DNA and noncentromeric DNA were enriched for modifications that define CEN chromatin, euchromatin, and heterochromatin. Human artificial chromosomes were also organized into distinct domains. CENP-A and heterochromatin were assembled over noncentromeric DNA, including the gene blasticidin, into nonoverlapping domains. Blasticidin transcripts were enriched at sites of CENP-A binding but not at H3 methylated at lysine 9, indicating that formation of CEN chromatin within a repetitive DNA environment does not preclude gene expression. Finally, we tested the role of centric heterochromatin as a centromeric boundary by increasing CENP-A dosage to expand the CEN domain. In response, H3 lysine 9 dimethylation, but not trimethylation, was markedly decreased at all centromeres examined. We propose that human centromere regions normally exist in a dynamic state in which a regional boundary, defined by H3 lysine 9 dimethylation, separates CEN chromatin from constitutive heterochromatin.

Human centromeres contain multi-megabase-sized arrays of alpha satellite DNA, a family of satellite DNA repeats based on a tandemly arranged 171bp monomer. The centromere-specific histone protein CENP-A is assembled on alpha satellite DNA within the primary constriction, but does not extend along its entire length. CENP-A domains are estimated to extend over 1500-2000kb of alpha satellite DNA. However, these estimates do not take into account inter-individual variation in alpha satellite array sizes on homologous chromosomes and among different chromosomes. We defined the genomic distance of CENP-A chromatin on human chromosomes X and Y from different individuals. CENP-A chromatin occupied different genomic intervals on different chromosomes, but despite inter-chromosomal and inter-individual array size variation, the ratio of CENP-A to total alpha satellite DNA size remained consistent. Changes in the ratio of alpha satellite array size to CENP-A domain size were observed when CENP-A was over-expressed and when primary cells were transformed by disrupting interactions between the tumor suppressor protein Rb and chromatin. Our data support a model for centromeric domain organization in which the genomic limits of CENP-A chromatin varies on different human chromosomes, and imply that alpha satellite array size is a more prominent predictor of CENP-A incorporation than chromosome size. In addition, our results also suggest that cancer transformation and amounts of centromeric heterochromatin have notable effects on the amount of alpha satellite that is associated with CENP-A chromatin.