Apoptotic Attack on Chromatin Figure 5. Scheme of autolytic and MNase cuts within 100 kb. Maps of MNase and autolytic cuts (black and red arrows, respectively) and of genes (colored stretches) located at (and between) cuts. See Table S3 online for the origin and description of the genes presented in the Figure. doi:10.1371/journal.pone.0005010.g005 perfectly reproducible, we propose it as an alternative approach to induction by physical or chemical agents. Our approach has at least two implications. The first one is that oxigen deprivation most likely kills cells by apoptosis, a point of obvious interest in medicine. The second implication derives from the preference for the open chromatin around active genes and of the double-break nature of apoptotic cuts [22] . Indeed, this implies that the cell death sentence in all likelihood takes place at the very beginning of apoptosis, when the very first 50–300 kb fragments are produced (see Introduction). Chromatin structure and its accessibility to nucleases It is known from previous work that the GC-rich, gene-rich isochores are characterized by a more expanded (open) configu- ration than the GC-poor, gene-poor isochores in the interphase nucleus of human and chicken [23–25]. The present investigations go beyond this initial observation in that they provide information on the structure of open chromatin, as investigated in cells undergoing apoptosis and in isolated nuclei. When the open chromatin regions were explored at a nucleotide resolution, a number of features appeared to be shared by the two degradations. Indeed, both of them were shown to target the GC-rich, gene-rich regions of the genome, as well as repeats that are preferentially located in those regions (see Table 1). Incidentally, such preference is also true for DNase I hypersensitive sites as assessed in human isochores [26]. The minimal distances between sites from each degradation were found to be very large. On a first instance, this result can be understood on the account that in both cases an early phase of degradation was explored. Undoubtedly, additional sites could have been discovered if losses of GC-rich fragments had not been incurred because of cloning problems. However, the abundance of redundant clones, namely of clones having an identical chromosomal location, in both apoptotic and MNase degradation (but especially in the former one), indicates that the most preferred sites were clearly detected in both cases. Distances between cuts by the two degradations were found to be shorter than those within each set of sites, indicating a trend for a very broad ‘‘clustering’’, without however, a single coincidence of the two kinds of cuts. Distances generally remained, however, very large, 90% of the cuts being separated by more than 1 Mb on the average (see Figure 4). The distribution of sites in different isochore families was very unequal, cuts being exceedingly rare in GC-poor isochores, which are known to be in a very compact chromatin structure. In this case apparently the large 50–300 kb fragments are only rarely produced and are unavailable for the further degradation to oligonucleosomes.. The patterns of cuts caused by the two degradations are different. Actually, a majority (55%) of the two kinds of cuts concern different isochores. Moreover, there are remarkable differences in the microchromo- somes attacked by the two degradations (Figure S2). Since this is most unlikely due to the different specificities of the two enzymes, because these specificities are very low and largely similar, the most plausible explanation is that chromatin structures from tissues undergoing apoptosis and from isolated nuclei expose different open regions as sites available for enzymatic attack. Differences may be due to the procedure used in the preparation of nuclei, which includes the use of Ca2+, Mg2+, spermine and spermidine (two agents that strongly interact with DNA), as opposed to the much milder apoptotic degradation. In other words, what we suggest is that the most accessible chromatin regions woul be artefactually modified by the cations and the basic organic compounds, so presenting different regions of the open chromatin to micrococcal nuclease. On the other hand, in the latter case one cannot rule out the action of proteolysis which might also concern chromatin [27,28]. However, if this unlikely PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5010