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

Copyright: © 2010 Stimpson et al.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. Extrachromosomal 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.This work was supported by the Tumorzentrum Heidelberg/Mannheim grant (D.10026941)and by March of Dimes Research Foundation grant #1-FY06-377 and NIH R01 GM069514

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

<p>Abstract</p> <p>Background</p> <p>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 <it>Ty3/gypsy</it>-like LTR retrotransposons that led to massive genomic expansion in three <it>Helianthus </it>(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.</p> <p>Results</p> <p>We demonstrate that <it>Ty3/gypsy</it>-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.</p> <p>Conclusion</p> <p>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.</p

Disruption of TRF2 association, but not open or closed chromatin, at rDNA in the presence of dnTRF2.

<p>Chromatin from control/uninduced T19 cells and 30 hour dnTRF2-expressing cells was crosslinked with 1% PFA, sonicated to 100–700 bp, and immunoprecipitated with indicated antibodies: (A) H3K4me2, (B) H3K9me3, (C) UBF, and (D) TRF2. Each bar shows relative enrichment as percentage of input by ChIP-PCR. Actin and alpha satellite are control regions. alpha sat  =  alpha satellite (centromere), β-sat  =  distal beta satellite (located telomeric of rDNA), u18S  =  upstream of 18S rDNA region, 18S = 18S rDNA region, IGS  =  intergenic spacer region in rDNA repeat. Error bars show standard error of the mean. (*) indicates significant difference (<i>p</i><0.001) between control and dnTRF2 ChIP enrichment.</p

Timing of nucleolar protein disruption with increasing dnTRF2 expression.

<p>(A) Immunostaining of 3D-preserved whole nuclei with nucleolar protein fibrillarin (green) and Ki-67 (red) in control and 45 hour dnTRF2 nuclei show that nucleolar morphologies changes with increased dnTRF2 expression and telomere dysfunction. (B) Induced T19 (dnTRF2-expressing) cells were analyzed at intervals over a 24-hour period using immunofluorescence with antibodies specific to fibrillarin and FLAG (to detect FLAG-tagged dnTRF2 protein - red). Nucleolar changed from a normal spherical shape to less condensed structures resembling nucleolar necklaces. (C) Quantitation of the percent of nuclei showing visibly abnormal nucleolar staining over the timecourse. Abnormal morphology (decondensed, unraveled) of nucleoli (light grey) increased as dnTRF2 was expressed for longer periods. The number of nuclei examined at each timepoint is indicated at the top of each stacked bar. After dnTRF2 expression for 24 hours, there was a statistically significant increase (asterisk) in the proportion of abnormal nucleoli compared to control cells. Scale bars equal 5 micrometers, hr =  hour.</p

Non-random acrocentric fusion when telomeres are disrupted by various approaches.

<p>(A) Immunoblot of HT1080 whole cell lysates selected for empty vector or shTRF2 retroviral vector for 11 or 15 days with puromycin. Blot shows TRF2 protein as a doublet and β-actin as a loading control. (B) Acrocentric fusions are non-randomly induced in HT1080 cells expressing a retroviral shTRF2 construct for 10 days and 3 weeks. In addition, treatment of cells with the double-strand break inducer zeocin also results in high numbers of acrocentric fusions. asterisk (*) in graph legend denotes observations from dnTRF2 expression previously reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092432#pone.0092432-Stimpson1" target="_blank">[9]</a>.</p

Condensin localization decreases on acrocentric short arms in the presence of dnTRF2.

<p>Combined immunostaining-FISH for (A) SMC4 (green) and rDNA (red) on metaphase chromosomes from control and 36 hour dnTRF2-expressing cells. (B) The amount of SMC4 was quantitated by measuring arbitrary fluorescence along the length of the chromosomes and plotting signal intensity as a line plot. A line begins at the p arm (0 on x-axis) and extends to the telomere of the q arm (∼150+ on x-axis). (C) The extent of rDNA and SMC4 co-localization at chromatids of metaphase chromosomes is presented in graphical format. The number of individuals chromatids examined is indicated at the top of each bar. A significant reduction in SMC4 co-localization at rDNA was observed on metaphase chromatids from cells expressing dnTRF2 for 36 hours. Scale bars in (A) are 15 micrometers.</p

dnTRF2 expression correlates with rDNA repeat array dispersion.

<p>The rDNA arrays are located on the short arms of the 5 pairs of acrocentric chromosomes. FISH on RNase-treated nuclei hybridized with an rDNA (green) PAC probe showed that rDNA, normally appearing as multiple punctate foci in the nucleus, becomes more diffuse with increased dnTRF2 expression. The T19 (dnTRF2) cell line contains ∼18 acrocentric chromosomes. Multiple short arms normally converge in the nucleus, so each foci can contain more than rDNA regions from more than one acrocentric chromosome. With increased dnTRF2 expression and telomere dysfunction, the bright foci were reduced, instead appearing as dotted or beaded tracks of fluorescent signals stretching throughout the nucleus. Pseudo-colored and gray-scale single channel images for rDNA are shown below the merged images. Scale bars equal 5 micrometers.</p

DNA damage markers appear with increased dnTRF2 expression.

<p>(A) Immunoblot for TRF2, β-actin (loading control), and H2AX-p on whole cell lysates from UV-treated (∼20 J/m²), uninduced/0-hour, 12-hour, 24-hour, 48-hour, and 72-hour dnTRF2-expressing cells. (B) Graphical representation of protein levels measured by arbitrary fluorescence units normalized to β-actin showing increased dnTRF2 protein levels with longer induction periods up to 48 hours. H2AX-p levels increased by 72 hours. (C) Immunoblot for Chk2-p and β-actin showing appearance of phosphorylated Chk2 kinase after 24 hours of dnTRF2 expression.</p