Dicentric chromosomes: unique models to study centromere function and inactivation.

Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell division. The molecular basis for centromere inactivation is not well understood, although studies in model organisms and in humans suggest that genomic and epigenetic mechanisms can be involved. Furthermore, constitutional dicentric chromosomes ascertained in patients presumably represent the most stable chromosomes, so the spectrum of dicentric fates, if it exists, is not entirely clear. Studies of engineered or induced dicentrics in budding yeast and plants have provided significant insight into the fate of dicentric chromosomes. And, more recently, studies have shown that dicentrics in humans can also undergo multiple fates after formation. Here, we discuss current experimental evidence from various organisms that has deepened our understanding of dicentric behavior and the intriguingly complex process of centromere inactivation.

Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes.

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 alpha-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 alpha-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.

Proliferation of Ty3/gypsy-like retrotransposons in hybrid sunflower taxa inferred from phylogenetic data.

BACKGROUND: Long terminal repeat (LTR) retrotransposons are a class of mobile genetic element capable of autonomous transposition via an RNA intermediate. Their large size and proliferative ability make them important contributors to genome size evolution, especially in plants, where they can reach exceptionally high copy numbers and contribute substantially to variation in genome size even among closely related taxa. Using a phylogenetic approach, we characterize dynamics of proliferation events of Ty3/gypsy-like LTR retrotransposons that led to massive genomic expansion in three Helianthus (sunflower) species of ancient hybrid origin. The three hybrid species are independently derived from the same two parental species, offering a unique opportunity to explore patterns of retrotransposon proliferation in light of reticulate evolutionary events in this species group. RESULTS: We demonstrate that Ty3/gypsy-like retrotransposons exist as multiple well supported sublineages in both the parental and hybrid derivative species and that the same element sublineage served as the source lineage of proliferation in each hybrid species' genome. This inference is based on patterns of species-specific element numerical abundance within different phylogenetic sublineages as well as through signals of proliferation events present in the distributions of element divergence values. Employing methods to date paralogous sequences within a genome, proliferation events in the hybrid species' genomes are estimated to have occurred approximately 0.5 to 1 million years ago. CONCLUSION: Proliferation of the same retrotransposon major sublineage in each hybrid species indicates that similar dynamics of element derepression and amplification likely occurred in each hybrid taxon during their formation. Temporal estimates of these proliferation events suggest an earlier origin for these hybrid species than previously supposed.

Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA). The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2) causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

Centromeres poised en pointe: CDKs put a hold on CENP-A assembly.

Eukaryotic centromeres are propagated by incorporation of the centromere-specific histone CENP-A into centromeric chromatin. Silva et al. (2012) now show that cyclin-dependent kinases (CDKs) hold the CENP-A assembly machinery in an inactive state until mitotic exit and entry into G1, at which time new CENP-A is loaded.

Epigenomics of centromere assembly and function.

The centromere is a complex chromosomal locus where the kinetochore is formed and microtubules attach during cell division. Centromere identity involves both genomic and sequence-independent (epigenetic) mechanisms. Current models for how centromeres are formed and, conversely, turned off have emerged from studies of unusual or engineered chromosomes, such as neocentromeres, artificial chromosomes, and dicentric chromosomes. Recent studies have highlighted the importance of unique chromatin marked by the histone H3 variant CENP-A, classical chromatin (heterochromatin and euchromatin), and transcription during centromere activation and inactivation. These advances have deepened our view of what defines a centromere and how it behaves in various genomic and chromatin contexts.