Onset of Chromatin Fragmentation in Chloroma Cell Apoptosis Is Highly Sensitive to UV and Begins at Non-B DNA Conformation

Abstract

UV irradiation induces programmed death, or apoptosis, in rat chloroleukaemia cells, a process characterized by typical cell morphology, fragmentation of chromatin into units of single and multiple nucleosomes, and transcriptional activation of LINE. Our study shows that this process is initiated by very low UV doses: exposure to one minimal erythema dose (MED, about 200 J/m2), at defined wavelengths ranging from 270 to 330 nm, is sufficient. By sequencing 100 randomly selected blunt-end chromatin fragments (single and multiple nucleosomes) we observed that at an early stage of apoptosis the fragmented DNA contains a four-fold excess of both long and short interspersed nuclear retroelements (LINEs and SINEs). A distinct sequence finding was also the observation that fragmented DNA is very rich in microsatellites, (CA)n dinucleotides in particular, and in long stretches of homopurine/homopyrimidine domains. The present results thus indicate that chromatin fragmentation in UV-induced apoptosis begins at non-random locations involving non-B DNA conformation, and that the onset of the suicide process in chloroleukaemia cells is surprisingly sensitive to UV exposure.

LINE L1 retrotransposable element is targeted during the initial stages of apoptotic DNA fragmentation

“…For the LINE L1 material, approximately three quarters contained an apoptotic scission event within the repeat sequence itself. Thus, although the percentage of these repetitive elements within the sequence library matches that found in the entire genome, they are preferentially found at the site of apoptotic cleavage, confirming the results of others [Luokkamaki et al, 1993;Moore et al, 1993;Fan and Smith, 1995]. In the specific case of those cloned sites containing L1 elements, it was found that a proportion of them was clustered within a 300-bp region of the second ORF (Fig.…”

“…These numbers are comparable to that reported in the literature for the genomic distribution of Alu and LINE elements [Kariya et al, 1987;Moran et al, 1999]. This would seem to be in contrast to other reports, in which apoptotic DNA fragments were reportedly enriched in repetitive DNA [Luokkamaki et al, 1993;Miore et al, 1993;Fan and Smith, 1995]. However, in this study we found that although the percentage of repetitive DNA within the entire cloned material was not elevated above normal levels, the location of the apoptotic cut site was biased toward both Alu and LINE locations.…”

“…Others have also concluded that apoptotic fragmentation occurs within regions of the genome linked to repetitive DNA sequences [Luokkamaki et al, 1993;Moore et al, 1993;Fan and Smith, 1995]. All of these studies, however, have used DNA isolated as oligonucleosomal fragments, where the orientation and context of the earliest DNA breaks are lost.…”

“…Here, apoptotic cleavage was observed at a specific location within the break-point cluster region of the MLL gene that includes a S/MAR. Several laboratories have reported the cloning and sequencing of random samples of apoptotic DNA fragments [Luokkamaki et al, 1993;Moore et al, 1993;Fan and Smith, 1995]. For the mammalian cells studied, these approaches were performed using only relatively short DNA fragments, derived from mononucleosomes and their oligomers.…”

The telomeric region is excluded from nucleosomal fragmentation during apoptosis, but the bulk nuclear chromatin is randomly degraded

“…Another approach to analyse the regional cleavage of chromosomal DNA during apoptosis was to clone and sequence the released oligonucleosomal DNA fragments. 27,30,31 Whereas the two former reports detected a relative accumulation of interspersed sequences (SINE and LINE) in the isolated fragments, Khodarev et al 27 observed a normal distribution of repetitive DNA within the entire cloned material. These authors observed, however, a preferential cleavage site in such repetitive sequences.…”