Cell. Mol. Life Sci. (2014) 71:4519–4544 DOI 10.1007/s00018-014-1717-x Cellular and Molecular Life Sciences MULTI-AUTHOR REVIEW Are common fragile sites merely structural domains or highly organized ‘‘functional’’ units susceptible to oncogenic stress? Alexandros G. Georgakilas • Petros Tsantoulis • Athanassios Kotsinas • Ioannis Michalopoulos • Paul Townsend • Vassilis G. Gorgoulis Received: 28 August 2014 / Accepted: 28 August 2014 / Published online: 20 September 2014 Ó The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Common fragile sites (CFSs) are regions of within CFSs. We propose that CFSs are not only sus- the genome with a predisposition to DNA double-strand ceptible structural domains, but highly organized breaks in response to intrinsic (oncogenic) or extrinsic ‘‘functional’’ entities that when targeted, severe reper- replication stress. CFS breakage is a common feature in cussion for cell homeostasis occurs. carcinogenesis from its earliest stages. Given that a number of oncogenes and tumor suppressors are located Keywords Fragile sites Á miRNAs Á Genomic instability Á within CFSs, a question that emerges is whether fragility DNA elements Á DNA repair Á Carcinogenesis in these regions is only a structural ‘‘passive’’ incident or an event with a profound biological effect. Furthermore, there is sparse evidence that other elements, like non- Introduction coding RNAs, are positioned with them. By analyzing data from various libraries, like miRbase and ENCODE, Activated oncogenes are a key feature of cancer devel- we show a prevalence of various cancer-related genes, opment from its earliest stages [1]. One of their major miRNAs, and regulatory binding sites, such as CTCF effects is the induction of DNA damage via replication stress (RS) [2]. Specifically, oncogene-induced DNA replication stress (OIRS) leads to the formation of DNA A. G. Georgakilas, P. Tsantoulis, and A. Kotsinas contributed equally double-strand breaks (DSBs), due to replication forks to this manuscript. A. G. Georgakilas P. Townsend Á V. G. Gorgoulis Physics Department, School of Applied Mathematical and Faculty Institute for Cancer Sciences, University of Manchester, Physical Sciences, National Technical University of Athens Manchester Academic Health Science Centre, Manchester M13 (NTUA), Zografou, 15780 Athens, Greece 9WL, UK e-mail: [email protected] P. Tsantoulis Division of Oncology, Geneva University Hospitals, P. Townsend Á V. G. Gorgoulis 1211 Geneva, Switzerland Manchester Centre for Cellular Metabolism, University of Manchester, Manchester Academic Health Science Centre, A. Kotsinas Á V. G. Gorgoulis Manchester M13 9WL, UK Molecular Carcinogenesis Group, Department of Histology and Embryology, V. G. Gorgoulis School of Medicine, University of Athens, Biomedical Research Foundation of the Academy of Athens, 4 11527 Athens, Greece Soranou Efessiou, 11527 Athens, Greece I. Michalopoulos V. G. Gorgoulis (&) Computational Biology and Medicine, Department of Histology-Embryology, Medical School, National Center of Systems Biology, Biomedical Research Kapodistrian University of Athens, 75 Mikras Asias Str., Goudi, Foundation of the Academy of Athens, 11527 Athens, Greece 4 Soranou Efessiou, 11527 Athens, Greece e-mail: [email protected] 123 4520 A. G. Georgakilas et al. (RFs) collapse, fueling genomic instability (GI) [2, 3]. In Oncogenes/Chemical inhibitors early precancerous lesions, the collapse of DNA RFs occurs preferentially at specific loci termed fragile sites Replications Stress (FSs) [4]. As a result, FSs exhibit breaks, gaps, and rearrangements, collectively termed FSs expression. This is due to the activation of pathways responsible for fork Fragile sites collapse resolution and completion of DNA replication, which involve recombinogenic processes and DNA DSBs DSBs production [2]. Chromosome breaks An important issue is whether the instability mani- Genomic instability fested at these sites has any wider biological impact on cancer development. As further discussed in the manu- Fragile sites script, although FSs are heterogeneous in their expression patterns, they possess unique features that make them vulnerable to structural destabilization under RS conditions. Are these regions simply prone to DNA damage due to their intrinsic characteristics, conferring Fragile sites only to GI? Sparse evidence indicates that FSs enclose ? ? genes and non-coding RNAs, like microRNAs (miRs), • genes while their expression could be epigenetically modulated ? ? • non-coding RNA by histones, implying that they are regions of the gen- ? ome of a higher organization level (Fig. 1)[5–9]. • binding elements Important bioinformatic resources are currently available • histone marks and can be exploited to define potential topological associations between CFSs and these elements. Notably, Fig. 1 CFSs are not only vulnerable structural domains but may also the miRbase is constantly expanding while the ENCODE be functional units of the genome that are sensitive to replication project [10] has deposited information on a vast range of stress. CFS’s stability is affected by replications stress (RS). During binding elements and genomic modifications, including cancer development, they are affected from the earliest precancerous histone marks (like H3K79me2, H3K9ac, H3K4me3, and lesions due to oncogene-induced replication stress (OIRS). Breakage at CFSs (broken red rectangle) may not only confer to genomic H3K27ac) that have a prominent influence on the instability (GI) (dashed black rectangle), but could also have wider expression process of the genome. As the pattern of biological implications by affecting elements located within them instability at FSs in human tumors is variable, suggesting (question mark). DSBs DNA double-strand breaks that it also depends on the cell type, this further com- plicates the role of FSs in malignancy. Last but not least, Heterogeneity of fragile sites if such sites contribute to cancer development, why have they not been evolutionary selected for elimination? FSs have been assigned in two classes, defined as rare Could there be a higher reason that makes them at the fragile sites (RFSs) and common fragile sites (CFSs). Rare same time vulnerable ‘‘units’’ of the genome with FSs are mainly induced by folate deficiency, correspond to potentially meaningful function? Attempting to address dinucleotide or trinucleotide repeats, usually CGGn, and are these questions, in the current work we conducted an found in less than 5 % of the human population and in extensive review on the nature of their heterogeneity that specific families [11]. Their fragility is due to expansions of accounts for their preferential instability. Next, by the micro- or mini-satellites sequences that they contain and applying bioinformatic tools on data from the latest in some cases are responsible for inherited diseases [11]. miRbase and the ENCODE project, we reveal that these Therefore, RFSs will not be further discussed in this work. sites are enriched in various (coding and non-coding) Historically, CFSs were recognized as recurrent hotspots elements, such as cancer-related genes, miRs, and of double-stranded DNA breaks in cultured lymphocytes binding elements, as well as specific variations in his- from healthy individuals [12]. They are present in all tone modifications (Fig. 1). Based on these findings, we individuals, are part of the normal chromosomes, but propose that these sites may represent unique ‘‘func- exhibit different frequencies of expression in a population tional’’ units of the genome that may have a complex (reviewed in [13, 14]). CFSs are typically vulnerable to role upon OIRS with implications both in normal cell extrinsic replication stress, most notably to aphidicolin survival and cancer progression. (APH), an inhibitor of DNA polymerase a, d and e, but are 123 Are common fragile sites merely structural domains 4521 otherwise quiescent under normal conditions. This obser- rat, and many mammals [27, 28], and across kingdoms, vation has been gradually broadened to include breakage such as in the yeast S. cerevisiae [29]. Evolutionary con- patterns resulting from various replication inhibitors, such servation could argue in favor of a meaningful function if as nucleotide analogs (5-azacytidine, bromodeoxyuridine) CFSs are considered outliers compared with the overall or antitumor antibiotics (distamycin) and RS resulting from fragility of the genome in general. Nevertheless, variation folate deficiency. Dietary and environmental factors like between individuals can be significant. In a study of 20 caffeine, cigarette smoke, and hypoxia may also enhance normal adults [30], only FRA3B and FRA16D were found FS expression [15]. Recently, the induction of stress during to be fragile in all individuals, and only 42 % of CFSs (19 the early S phase in B lymphocytes by hydroxyurea has of 45 identified) were present in the majority of individu- been found to provoke DNA damage in a distinct pattern, als. In the earliest studies, less than 20 CFSs would explain corresponding to a new class of ‘‘early replicating fragile more than 80 % of gaps and breaks [12]. A similar distri- sites’’ (ERFSs) [16]. They occur primarily in early repli- bution was found in a population study of Deer mice, cating DNA, close to replication origins, and are mainly where high-frequency CFSs constituted approximately situated in actively transcribed gene clusters (coding 26 % of the population total breaks and 38 % of CFSs were