Interstitial telomeric repeats-associated DNA breaks

Abstract

During a cell's lifespan, DNA break formation is a common event, associated with many processes, from replication to apoptosis. Most of DNA breaks are readily repaired, but some are meant to persist in time, such as the chromosome ends, protected by telomeres. Besides them, eukaryotic genomes comprise shorter stretches of interstitial telomeric repeats. We assumed that the latter may also be associated with the formation of DNA breaks meant to persist in time. In zebrafish and mouse embryos, cells containing numerous breakage foci were identified. These breaks were not associated with apoptosis or replication, nor did they seem to activate DNA damage response machinery. Unlike short-living, accidental sparse breaks, the ones we found seem to be closely associated, forming discrete break foci. A PCR-based method was developed, allowing specific amplification of DNA regions located between inverted telomeric repeats associated with breaks. The cloning and sequencing of such DNA fragments were found to denote some specificity in their distribution for different tissue types and development stages.

Keywords: Chromatin; DNA breaks; ETUNEL; development; interstitial telomeric repeats; mouse; zebrafish.

Figure 1.

ETUNEL staining of 4 dpf zebrafish and 11 dpf mice embryos. There is an uneven distribution of the cells with numerous DNA breakage foci within the body, these ones being particularly abundant in epithelia, but also present in the midbrain and forebrain (a). A rather intense ETUNEL signal can be observed in posterior lens, and is also detectable in ganglion cell layer (b). The ETUNEL signal shows a nonuniform distribution within nuclei, often forming small foci, from several to hundreds per nucleus(c). In mouse 11 dpf embryo head ETUNEL-positive cells are concentrated especially near the ventricular zone of the brain, in forming embryonic eyes and epithelia (d). Epithelial (e) as well as neural cells (f) show nonuniform nuclear labeling, with defined breakage foci.

Figure 2.

Double labeling of zebrafish 4dpf embryo cryosections for ETUNEL and apoptosis, proliferation and reparation markers. There is no evidence of colocalization between ETUNEL and PARP (a), caspase 3 (b), PCNA (c) and γH2AX (d) markers.

Figure 3.

In 4dpf zebrafish embryo cells the distribution of ETUNEL signal (green) is different from the one of SNF2 (red) chromatin remodeling marker (a). There is substantial colocalization between ETUNEL (green) and euchromatin marker H3K9-ac (red) (b), and virtually no overlapping with histone H3K9-Me3 (red), specific to heterochromatic regions (c). ETUNEL sites (green) are not located at sites of intense transcription, marked by RNA polymerase II (red) (d).

Figure 4.

Gel-electrophoresis of the amplified DNA fragments, selected by association of the free 3′ DNA ends with telomeric repeats on both sides, obtained by PCR with Term1-Term4 primer mixture. The distribution by size of the amplified DNA fragments from zebrafish hardroe (line 2,5), 4 dpf embryos (line 3,6) and adult fish (line 4,7) shows a general lowering of the molecular weight of the amplified fragments from germline to the embryonic stage and adult organism (a). There are also differences in the predominant fragment sizes of amplified fragments, comprised between DNA break-associated telomeric repeats for different zebrafish tissues, as seen for fin (line 1,7), gill (line 2,8), brain (line3,9), liver (line 4,10), muscle (line 5,11) and hardroe (line 6,12), also showing the highest molecular weight for germline and the lowest – for muscle (b). In both cases a specific signal appears after TdT treatment (a, line 2–4), (b, line 1–6), and there is almost no specific signal without TdT treatment (a, line 5–7), (b, line 7–12).

Figure 5.

Gel-electrophoresis of the amplified DNA fragments, selected by possible association of the free 3′ DNA ends with other inverted repeats, originated from 5 types of D.rerio transposons, obtained by PCR with Rep1-Rep5 primers. The distribution of the amplified fragments appears to be constant and not dependent on fish age, neither it is associated with DNA breaks, as there is no apparent difference between samples, treated or not treated with TdT: 1,5 dpf embryos (lanes 1,6), 4 dpf embryos (lanes 2,7), 21 dpf zebrafish (lanes 3,8), adult female (lanes 4,9) and adult male (lanes 5,10).