Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA

Abstract

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 artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells

“…Although additional studies are needed to determine whether genes inserted into the alphoid tetO -HAC may eventually be subject to silencing, our results and those obtained with other HACs (21,41) support the view that proximity to a functional kinetochore does not negatively affect gene expression. It is possible that, in the human genome, centromeric chromatin may be a privileged region for Pol II-transcribed genes, as previously shown for rice centromeres (43) and suggested by the resemblance of the centromeric chromatin profile to the downstream region of transcribed genes in human cells (39,44). Indirectly, this hypothesis is supported by our data on spreading of CENP-A chromatin to the 3′ end of NBS1 and transcriptional activity of genes in neocentromeres (reviewed in ref.…”

“…This indicates that the CENP-A chromatin domain extends to beyond the gene insertion site. CENP-A enrichment at the 3′ NBS1 sequence may be explained by the ability of CENP-A chromatin to spread onto noncentromeric DNA (16,39,40). The observed stable gene expression in the HAC suggests that close proximity of the CENP-A domain does not prevent expression of Pol II-transcribed genes.…”

“…During de novo HAC formation, input DNA is multimerized and then assembled into different types of chromatin, including arrays of CENP-A nucleosomes that are interspersed with those carrying different modifications of histone H3 (39). To better understand the chromatin structure of regions flanking the NBS1 gene in the alphoid tetO HAC, alphoid tetO -HAC/NBS1 was transferred back to human HT1080 cells (Fig.…”

Human centromere genomics: now it's personal

“…Changes in inner kinetochore protein abundance in human cancer cell lines have demonstrated that centromeric domains expand relative to the underlying satellite array (Sullivan et al 2011). Human artificial chromosome assays provide further support for establishment and dosagedependent spreading of kinetochore-binding proteins to adjacent DNA (Lam et al 2006). With increased dosage, it is likely that other optimal regions of the genome might acquire ectopic localization of inner kinetochore proteins, perhaps coupling epigenetic Sequence optimality and centromere identity.…”

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

“…Besides CENP-A, active centromeres display a specific profile of post-translational modifications on their associated histone H3-containing nucleosomes (Bergmann et al, 2011;Lam et al, 2006;Ribeiro et al, 2010;Sullivan and Karpen, 2004). This special chromatin, which has been termedx 'centrochromatin' (Sullivan and Karpen, 2004), suggests a functional link between the local chromatin environment and kinetochore function.…”