Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

Abstract

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.

Author Summary

Endogenous human centromeres are defined by large arrays of α-satellite DNA. A portion of each α-satellite array is assembled into CENP-A chromatin, the structural and functional platform for kinetochore formation. Most chromosomes are monocentric, meaning they have a single centromere. However, genome rearrangement can produce chromosomes with two centromeres (dicentrics). In most organisms, dicentrics typically break during cell division; however, dicentric human chromosomes can be stable in mitosis and meiosis. This stability reflects centromere inactivation, a poorly understood phenomenon in which one centromere is functionally silenced. To explore molecular and genomic events that occur at the time of dicentric formation, we describe a cell-based system to create dicentric human chromosomes and monitor their behavior after formation. Such dicentrics can experience several fates, including centromere inactivation, breakage, or maintaining two functional centromeres. Unexpectedly, we also find that dicentrics with large (<20Mb) inter-centromeric distances are stable through at least 20 cell divisions. Our results highlight similarities and differences in dicentric behavior between humans and model organisms, and they provide evidence for one mechanism of centromere inactivation by centromeric deletion in some dicentrics. The ability to create dicentric human chromosomes provides a system to test other mechanisms of centromere disassembly and dicentric chromosome stability.