Abstract
Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.
Highlights
Extrachromosomal nuclear DNA consists of DNA molecules that reside in the cell nucleus and are derived from genomic DNA, but are not covalently linked to chromosomes
We report striking cell-to-cell variations in the number of extrachromosomal telomere-repeat (ECTR) DNA molecules in Alternative Lengthening of Telomeres (ALT) cells, we quantify the wide distribution of ECTR DNA lengths in these cells and we provide evidence that the large majority of ALT ECTR DNA molecules are composed of primarily G- or C-strand telomere-repeat DNA
We examined cells from GM847, VA13 and U2OS human ALT cell lines, which are commonly used in ALT studies, and find that they average 7- to 48-fold more ECTR DNA molecules per nucleus than primary and telomerasepositive cells (Figures 2B and 3A and B)
Summary
Extrachromosomal nuclear DNA consists of DNA molecules that reside in the cell nucleus and are derived from genomic DNA, but are not covalently linked to chromosomes. Extrachromosomal nuclear DNA has been detected in all human tissues tested to date, raising the possibility that they may be involved in fundamental biological processes [1,2]. These naturally occurring extrachromosomal DNA molecules range in length from 20 kb and are of diverse origin, including non-repetitive microDNAs as well as repetitive elements derived from satellite DNA and 5S ribosomal DNA [3,4]. The exact origin and mechanism of ECTR DNA production in human ALT cells is currently not well understood, the generation of circular ECTR DNA is dependent on several DNA repair proteins [13,14]
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