1280–1294 Nucleic Acids Research, 2018, Vol. 46, No. 3 Published online 14 December 2017 doi: 10.1093/nar/gkx1260 FANCD2 binding identifies conserved fragile sites at large transcribed genes in avian cells Constanze Pentzold1, Shiraz Ali Shah1, Niels Richard Hansen2, Benoˆıt Le Tallec3, Andaine Seguin-Orlando4,5, Michelle Debatisse6, Michael Lisby1,7 and Vibe H. Oestergaard1,* 1Department of Biology; University of Copenhagen; Copenhagen N 2200, Denmark, 2Department of Mathematical Sciences, University of Copenhagen, Copenhagen 2100, Denmark, 3Institut de Biologie de l’Ecole Normale Superieure´ (IBENS), CNRS-UMR8197 – Inserm U1024, Paris F-75005, France, 4Center for GeoGenetics, Natural Downloaded from https://academic.oup.com/nar/article/46/3/1280/4739372 by guest on 01 October 2021 History Museum of Denmark; University of Copenhagen; Copenhagen 1350, Denmark, 5Danish National High-throughput DNA Sequencing Centre, University of Copenhagen, Øster Farimagsgade 2D, Copenhagen K 1353, Denmark, 6Institut Gustave Roussy, UMR 8200, 94800 Villejuif, France and 7Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen N, Denmark Received September 27, 2017; Revised December 01, 2017; Editorial Decision December 05, 2017; Accepted December 05, 2017 ABSTRACT INTRODUCTION Common Chromosomal Fragile Sites (CFSs) are spe- During mitosis, a copy of the genome is passed on to each cific genomic regions prone to form breaks on of the two daughter cells. Mitosis constitutes a short win- metaphase chromosomes in response to replication dow of the entire cell cycle, but is of immense importance for genome integrity. In response to severe replication stress, stress. Moreover, CFSs are mutational hotspots in / cancer genomes, showing that the mutational mech- the G2 M checkpoint will normally arrest cells in G2 phase but under conditions of mild replication stress, certain re- anisms that operate at CFSs are highly active in gions in the genome escape checkpoint detection and en- cancer cells. Orthologs of human CFSs are found ter mitosis in an underreplicated state (1–5). This leads to in a number of other mammals, but the extent of the formation of microscopically visible breaks and gaps CFS conservation beyond the mammalian lineage is on metaphase chromosomes (6). Genomic regions prone to unclear. Characterization of CFSs from distantly re- form gaps and breaks in response to replication stress are lated organisms can provide new insight into the bi- named chromosomal fragile sites (6). Interestingly, chro- ology underlying CFSs. Here, we have mapped CFSs mosomal fragile sites are hotspots for large deletions and in an avian cell line. We find that, overall the most rearrangements in cancer genomes (7–10). The genome of significant CFSs coincide with extremely large con- healthy individuals contains two classes of chromosomal served genes, from which very long transcripts are fragile sites; (i) common fragile sites (CFSs) and (ii) early produced. However, no significant correlation be- replicating fragile sites (ERFSs) (8,11). Underreplicated re- gions that persist in mitosis pose a problem to disjunction tween any sequence characteristics and CFSs is of sister chromatids and accordingly CFSs can remain in- found. Moreover, we identified putative early repli- terlinked by ultrafine DNA bridges (UFBs) or chromatin cating fragile sites (ERFSs), which is a distinct class bridges during anaphase (3,12,13). of fragile sites and we developed a fluctuation anal- Since CFSs were first discovered, a variety of features ysis revealing high mutation rates at the CFS gene have been suggested to be involved in their breakage and PARK2, with deletions as the most prevalent muta- mutagenesis. Firstly, it has been proposed that specific DNA tion. Finally, we show that avian homologs of the hu- features, such as AT-richness or increased flexibility of the man CFS genes despite their fragility have resisted double helix could cause difficult-to-replicate secondary the general intron size reduction observed in birds structures (14–17). Secondly, CFSs are generally late repli- suggesting that CFSs have a conserved biological cating and this was suggested to make them specifically vul- function. nerable to replication stress (18). In line with this idea, it was also shown that some CFSs have a scarcity of replica- *To whom correspondence should be addressed. Tel: +45 3533 0444; Fax: +45 3532 2128; Email: [email protected] Present address: Constanze Pentzold, Institute of Human Genetics, Jena University Hospital, DE-07747 Jena, Germany. C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2018, Vol. 46, No. 3 1281 tion origins (19,20) and consequently they cannot respond Generation of targeting constructs for FANCD2, PARK2 and to replication stress by firing backup origins, which would OVAL make these genomic regions more prone to enter mitosis The construct for GS-tagging (protein G-streptavidin bind- in an underreplicated state (11). Consistently, CFS fragility ing peptide/tandem affinity purification tag) of endogenous varies from one cell type to another, implying that epige- chicken FANCD2 (FANCD2, Gene ID: 415935) was gener- netic features rather than the DNA sequence per se is caus- ated by first, amplifying the 3 homology arm using primers ing fragility (9). Finally, many CFSs coincide with large VO300 and VO301; and the 5 homology arm using the transcribed genes, where potential collisions between repli- primers VO302 and VO303. Next, the 3’ homology arm cation and transcription machineries can delay replication was cloned into pBluescript SK+ (Agilent Technologies) as (21–23). ERFSs, on the other hand, are early replicating, a SpeI/NotI fragment. Then, a sequence encoding an N- gene dense regions, where high transcription activity of sev- terminal GS-tag (31) was inserted as a BamHI/XbaI frag- eral genes may sensitize the region to replication stress (8). ment. Thereafter, the 5 arm was inserted as a SalI/BamHI FANCD2 has an important role in CFS maintenance fragment and finally, the resistance cassette was inserted as Downloaded from https://academic.oup.com/nar/article/46/3/1280/4739372 by guest on 01 October 2021 (24,25) and localizes to CFSs during mitosis in human a BamHI fragment. The GS targeting construct was lin- cells even in the absence of breakage (12,13). FANCD2 earized with NotI before transfection. prevents replication fork stalling at the AT-rich cores of The BSR, PURO,andNEO resistance genes were excised FRA16D (housing the WWOX gene) and FRA6E (hous- from pLOX-BSR, pLOX-PURO and pLOX-NEO, respec- ing the PARK2 gene), and specific examination of FRA16D tively (32) using BamHI. also shows that FANCD2 deficiency leads to accumula- For assembly of the PARK2 (PARK2, Gene ID: 421577) tion of RNA-DNA hybrids at this CFS (25). Moreover, knock-in construct (pCP2), the 5 and 3 homology arms FANCD2 is a key component of the Fanconi Anemia path- were PCR amplified using primer pairs CP43-CP44 and way that protects cells against genotoxic agents such as CP45-CP46, respectively. Then they were individually sub- DNA interstrand cross-linkers and aldehydes (26). cloned into pCR® 2.1-TOPO® TA vector (Invitrogen) in Traditionally, CFSs have been defined and mapped cy- the listed order using restriction sites KpnI–NotI or NotI– togenetically (11,27) and are mainly characterized in hu- XhoI, respectively. The HyTK cassette was excised by NotI mans, other primates and mice (9,22,28–30). Here, we per- from the original vector (kindly provided by Christine Farr) formed ChIP-seq of FANCD2 from cells subjected to repli- (33). The PARK2 targeting construct was linearized with cation stress to map fragile sites genome-wide in the avian Eam1105I (AhdI) before transfection. cell line DT40. By cytogenetic analyses using fluorescence in The vector for targeting the chicken ovalbumin gene lo- situ hybridization (FISH) we confirm that the most signif- cus 2 (OVAL, Gene ID: 396058) was assembled by first icant peaks from our ChIP-seq are bona fide CFSs. More- subcloning the 3 homology arm in the XhoI/KpnI site over, we identify a number of gene-dense highly expressed of pBlueScript SK+ (Fermentas GmbH). The 5 homology regions likely corresponding to DT40 ERFSs. Additionally, arm was subcloned in the NotI/SpeI site of pLOX-PURO we have developed a fluctuation assay to estimate the site- (32). Thereafter, the 3 and 5 homology arms were assem- specific mutation rate at a CFS. bled into one vector by subcloning a KpnI/ClaI fragment This study provides a genome-wide map of CFSs and pu- to generate pML27. The HyTK counterselection gene was tative ERFSs, plotted together with replication timing and XhoI-adapted by PCR, using the original vector (33)as transcription level as well as other features suggested to con- template. To finally insert the HyTK counterselection gene tribute to CFS fragility. We do not find significant correla- between the OVAL homology arms, the adapted HyTK gene tion with any of the tested sequence features formerly sug- was cloned into the XhoI site of pML27, resulting in the fi- gested to induce fragility, but our results support that tran- nal targeting vector pCP9. The OVAL targeting construct scription is involved in fragility at both CFSs and ERFSs. was linearized with Eam1105I (AhdI) before transfection. Specifically, transcribed genes over 500 kb always coincide Plasmids and primers used in this study are listed in sup- with CFSs. Moreover, we report the conservation of long in- plemental methods, respectively. tron sizes in CFSs within the two major branches of the am- niotes – the synapsids to become mammals and the saurop- Chromatin immunoprecipitation followed by massively paral- sids to become birds and reptiles.