The Early Apoptotic DNA Fragmentation Targets a Small Number of Specific Open Chromatin Regions Miriam Di Filippo, Giorgio Bernardi* Laboratory of Molecular Evolution, Stazione Zoologica Anton Dohrn, Naples, Italy Abstract We report here that early apoptotic DNA fragmentation, as obtained by using an entirely new approach, is the result of an attack at a small number of specific open chromatin regions of interphase nuclei. This was demonstrated as follows: (i) chicken liver was excised and kept in sterile tubes for 1 to 3 hours at 37uC; (ii) this induced apoptosis (possibly because of oxygen deprivation), as shown by the electrophoretic nucleosomal ladder produced by DNA preparations; (iii) low molecular-weight DNA fragments (,200 bp) were cloned, sequenced, and shown to derive predominantly from genes and surrounding 100 kb regions; (iv) a few hundred cuts were produced, very often involving the same chromosomal sites; (v) at comparable DNA degradation levels, micrococcal nuclease (MNase) also showed a general preference for genes and surrounding regions, but MNase cuts were located at sites that were quite distinct from, and less specific than, those cut by apoptosis. In conclusion, the approach presented here, which is the mildest and least intrusive approach, identifies a preferred accessibility landscape in interphase chromatin. Citation: Di Filippo M, Bernardi G (2009) The Early Apoptotic DNA Fragmentation Targets a Small Number of Specific Open Chromatin Regions. PLoS ONE 4(4): e5010. doi:10.1371/journal.pone.0005010 Editor: Rodolfo Aramayo, Texas A&M University, United States of America Received January 16, 2009; Accepted March 2, 2009; Published April 6, 2009 Copyright: 2009 Di Filippo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The financing of the experiments was covered by Stazione Zoologica Anton Dohrn. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bernardi@szn.it Introduction Chromatin structure plays a fundamental role in the regulation of gene expression (see [1,2] for reviews). A classical approach to investigate chromatin structure since the work of Hewish and Burgoyne [3] is to study the regions that are accessible to DNA- degrading enzymes. This approach was widely used on interphase nuclei whose isolation, however, potentially affects chromatin structure. Here we investigated the targeting of cuts early in DNA fragmentation by apoptosis of chicken liver cells and discovered that a small number of specific open chromatin regions were attacked with strong preference. Apoptosis, or programmed cell death, is a highly ordered mechanism of cell elimination which is involved in many processes such as embryogenesis, metamorphosis and tissue homeostasis [4,5]. At the molecular level, a degradation of proteins and DNA is observed [6,7]. The latter involves several endogenous DNases (see [8,9] for reviews), that break DNA into high molecular weight (HMW) fragments ranging from 50 to 300 kb (as analyzed by pulse-field electrophoresis [10]). These molecules are subsequently further degraded to low molecular weight (LMW) oligonucleo- some-sized fragments. It is generally accepted that the endonu- clease activity(ies) responsible for the generation of the large fragments is different from that catalyzing internucleosomal DNA fragmentation [11] and that the two stages of HMW and LMW DNA fragmentation are discrete processes that can be separated experimentally (see [12] for a review). The cleavage in the internucleosomal regions generates DNA fragments having sizes corresponding to multiples of 180–200 bp, giving rise to a typical ‘‘ladder’’ (when analyzed by conventional gel electrophoresis [6,11]), similar to that obtained after micrococcal nuclease (MNase) digestion [13]. While both DNA degradations preferen- tially cut genes and their surroundings, most remarkably apoptotic cuts are quite distinct from, and more specific than, MNase cuts. This indicates a very different accessibility of open chromatin in apoptotic cells and isolated nuclei. Results Apoptotic DNA ladder formation Apoptosis was induced by keeping excised chicken liver in sterile tubes at 37uC. DNA degradation was investigated just after the first appearance of the nucleosomal ladder when the vast majority of DNA was still present as a very high molecular weight band and some DNA had not even entered the gel (Figure 1A). A similar pattern of DNA degradation was produced when isolated chicken liver nuclei were briefly digested with micrococcal nuclease (MNase; Figure 1A). Expectedly, no degradation was observed before the addition of the enzyme, as already observed in previous work from other laboratories. GC level of cloned fragments In order to obtain further information on the apoptotic LMW DNA fragments, we cloned and sequenced 370 fragments having a size of ,200 bp. The average GC level of the cloned fragments, 46.8%, was higher than the average GC level of the chicken genome, 41%, but not quite as high as the average GC level of the small DNA fragments as directly extracted from the gel (49.6% GC). This result was in all likelihood due to a cloning bias against the GC-richest DNA fragments. When the distribution of the PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5010