Site-Specific Recombination with Inverted Target Sites: A

| INVESTIGATION

Site-Speciﬁc Recombination with Inverted Target Sites: A Cautionary Tale of Dicentric and Acentric Chromosomes

Simon W. A. Titen,* Makenna T. B. Johnson,† Mario Capecchi,* and Kent G. Golic†,1 *Eccles Institute of Human Genetics, School of Medicine and †School of Biological Sciences, University of Utah, Salt Lake City, Utah 84112 ORCID ID: 0000-0002-0495-0084 (K.G.G.)

ABSTRACT Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

KEYWORDS Cre; dicentric; Drosophila; FLP; FRT; lox; mouse; recombinase; sister chromatid; site-speciﬁc recombination

ITE-specific recombinases have become essential tools for Weissman and Pan 2015; Pontes-Quero et al. 2017; Germani Sbiological experimentation. The FLP-FRT and the Cre-lox et al. 2018). systems have been used to great effect in a number of differ- The FRT and lox ttarget sites are both asymmetrical, and ent species. Genes or gene segments may be arranged in a FLP- or Cre-mediated recombination generates orientation- variety of configurations with FRT or lox target sites to acti- specific outcomes. Intramolecular recombination between vate or inactivate genes at different developmental stages in sites in the same (direct) orientation excises the material be- animal and plant systems. By using such methods, cells and tween the sites, along with one target site (Figure 1A). Intra- their descendants may be marked to study the fates of mutant molecular recombination between sites that are inverted cells or to trace cell lineage. Additionally, recombination be- results in an inversion of the material between them (Figure tween widely separated target sites on the same chromosome, 1B). Intermolecular recombination may also occur, and the or between homologous or nonhomologous chromosomes, consequence of recombination between sister chromatids is has been used to engineer a variety of chromosome rearrange- often overlooked. Recombination between target sites at iden- ments (Golic and Lindquist 1989; Golic 1991; Lakso et al. tical locations on sister chromatids is typically without effect 1992; Qin et al. 1994; Ramírez-Solis et al. 1995; Smith et al. (Figure 1C). Recombination between target sites at different 1995; Golic and Golic 1996; Gilbertson 2003; Zong et al. 2005; locations, though, does produce genetic changes that may be Wang et al. 2010; Lee 2013; Gierut et al. 2014; Hubbard 2014; significant. When two target sites are in the same orientation, recombination between staggered sites generates complemen- tary duplications and deletions (Figure 1D). Recombination Copyright © 2020 by the Genetics Society of America doi: https://doi.org/10.1534/genetics.120.303394 between inverted target sites on sister chromatids will gener- Manuscript received May 28, 2020; accepted for publication June 22, 2020; published ate a dicentric and an acentric chromosome (Figure 1E). If the Early Online June 25, 2020. 1Corresponding author: School of Biological Sciences, University of Utah, 257 South inverted target sites are close to one another, this can be quite 1400 East, Room 201, Salt Lake City, UT 84112. E-mail: [email protected] frequent. These outcomes are reversed if the target sites are on

Genetics, Vol. 215, 923–930 August 2020 923 Figure 1 Intramolecular and intermolecular site- speciﬁc recombination. Schematic representations of recombination between recombinase targets on the same chromatid or on sister chromatids. (A) Intramolecular recombination between targets in direct orientation results in excision of the material between the targets as an extrachromosomal circle. (B) Intramolecular recombination between inverted repeats inverts the orientation of the DNA between the target sites. (C) Intermolecular recombination between targets at the same location on sister chromatids results in equal exchange. (D) Exchange between targets in direct orientation at different sites on sisters results in duplication and deletion of intervening DNA. (E) Recombination between inverted target sites on sisters produces a dicentric chromosome and an acentric chromosome. Chromatids, are indicated as lines, target sites as arrowheads, and centromeres as ﬁlled circles.

opposite sides of the centromere, but since the frequency of (Lewandoski and Martin 1997). In the latter case, when recombination is strongly dependent on proximity (Golic and inverted lox sites were placed on the mouse Y chromosome, Golic 1996), this is of much less concern. and males with this Y were mated to females with a b-actin- In mitosis, a dicentric chromosome can break, leading to Cre gene, almost all XY zygotes developed as XO females, breakage-fusion-bridge cycles, chromosomal and segmental owing to loss of the lox-bearing chromosome during embry- aneuploidy, and cell death. In cells that do not die, continuing onic development. problems with chromosome segregation and aneuploidy can Since those examples, there have been a number of studies cause aberrant behavior. Falco et al. (1982) were the ﬁrst to that have utilized FLP- or Cre-mediated recombination be- recognize the problem created by inversely oriented recom- tween inverted target sites to cause dicentric chromosome binase target sites within a chromosome. They saw that chro- formation (Ahmad and Golic 1998, 1999; Grégoire and Kmita mosomal integration of the 2m plasmid of Saccharomyces 2008; Otsuji et al. 2008; Titen and Golic 2008, 2010; Tada cerevisiae, which carries inverted FRTs and encodes the FLP et al. 2009; Jones et al. 2010; Zhu et al. 2010, 2012; Kurzhals recombinase, caused chromosome instability, which they at- et al. 2011; Sato et al. 2017; Thomas et al. 2017). Other tributed to dicentric/acentric formation. Subsequently, this studies have identiﬁed cell death as a consequence of recom- event was directly demonstrated in Drosophila melanogaster bination between inverted target sites without directly pin- (Golic 1994), and inferentially shown in mice with Cre-lox pointing the cause (Eckrich et al. 2019).

Figure 2 Dicentric chromosomes in Drosophila larval neuroblasts. (A) The normal D. melanogaster karyotype. (B) Dicentric and acentric chromosomes formed by un- equal sister chromatid exchange using a FlipFlop construct inserted at 37B on chromosome 2. Nor- mal chromosomes are indicated in blue; dicentric (Dc) and acentric (Ac) products indicated in red. (C) Dicentric/acentric chromosomes produced using a FlipFlop construct at 82E on chromosome 3.

924 S. W. A. Titen Table 1 Dicentric chromosome frequencies with FlipFlop insertions Hours after Dicentric/ heat shock Number of acentric Normal Total Location (hr) brains karyotype karyotype metaphases

2L, 37B 4–5 5 67 (46%) 80 147 23 2 9 (20%) 37 46 3R, 82E 4–5 7 132 (63%) 76 208 23 3 21 (30%) 49 70

In spite of these many examples of inverted target sites causing dicentric chromosome formation in the presence of the recombinase, their use for gene switching and lineage tracing continues, apparently without recognition that dicentric chromosomes are formed. Flex and FlipFlox constructs, used for gene switching in mice, rely upon Cre- mediated inversion (Schnütgen et al. 2003; Xin 2005). The polylox system for barcoding cells and tracing cell lineage in mice relies on both direct and inverted lox repeats (Pei et al. 2017). The Confetti method for cell marking, and other methods based on Brainbow 2.0 or 2.1, also utilize inverted target sites (Livet et al. 2007; Snippert et al. 2010; Richier and Salecker 2014; de Roo et al. 2019). In flies, Flybow, FlpStop, Flip-Flop and other implementations have used inverted target sites to alter gene expression during development (Hadjieconomou et al. 2011; Chin et al. 2014; Fisher et al. 2017; Nagarkar-Jaiswal et al. 2017; Williams et al. 2019). In yeast, the SCRAMBLE system for rearranging chromosomes uses engineered sym- metrical lox sites that may recombine in either direction (Dymond et al. 2011; Annaluru et al. 2014; Wu et al. 2018). Studies that use these constructs will be impacted by the Figure 3 Dicentric chromosomes produced in mouse cells. Following death of cells with broken chromosomes, or by the persis- exposure to Cre, asymmetric recombination between inverted loxP tence of cells with aneuploidy and continuing genome in- sitestargetedtoalocusnear(A)theendofchromosome7,or(B) stability, possibly leading to misinterpretation or invalid near the centromere of chromosome 15, would result in a dicentric conclusions. Our purpose here is to raise awareness of the chromosome and an acentric chromosome fragment. As indicated, the recombinant chromosome products are expected to be significantly fact that dicentric chromosomes are formed when inverted different in size. recombinase target sites areusedineukaryotes,and that this event can be very frequent. This outcome should be considered when planning experiments with such shock, or 23 hr after heat shock. Neuroblast chromosomes constructs. were prepared as described (Fanti and Pimpinelli 2004) and then stained with 49,6-diamidino-2-phenylindole (DAPI). Materials and Methods Multiple brains were scored, and each clear mitotic figure was scored as showing acentric/dicentric chromosomes or Dicentric chromosomes in Drosophila melanogaster unaffected normal chromosomes. Nuclei in which all chro- Flies were maintained on standard medium at 25°. The 70FLP mosomes were not visible or could not be distinguished were gene has been previously described (Golic et al. 1997). Flip- not scored. Flop stocks were obtained from the Bloomington, IN, Generation of inverted loxP transgenic mice Drosophila Stock Center. Female y w 70FLP3F; Sb/TM6, Ubx flies were crossed to male yw; Mi{FlipFlop} Nedd8 [MI13776- The HPRTCre constitutive and germline Cre expression lines FF.PT-EGFP]/CyO (Bloomington 76596) or male yw; Mi{FlipFlop} were obtained from Jackson Laboratories. The mouse line Cdep[MI12769-FF.PT-EGFP] (Bloomington 76595). Parental bearing inverted loxP sites targeted to the middle of chromo- flies were removed from the vials after 7 days and vials were some 15, at the Ext1 locus, is described in Jones et al. (2010). placed into a circulating water bath for 1 hr at 38° to induce The mouse line bearing inverted loxP sites targeted to the FLP. Larval brains were dissected either 4–5 hr after heat distal tip of chromosome 7, to the Fgf4 locus, is described in

Dicentric Chromosomes 925 Figure 4 Evidence of Cre-mediated dicentric and acentric chromosome formation in mouse cells. Pic- tured here are mitotic figures of cultured cells transfected with a Cre expression vector or purified Cre protein. The targeted chromosomes were visualized with DAPI (A, C, E, and G), or DAPI plus whole chromosome paints (B, D, F, and H) specific for either chromosome 15 (A, B, E, and F) or 7 (C, D, G, and H). Exposure to Cre for a short duration (24 hr) results in large acentric fragments (A and B) or short acentric fragments and a large dicentric chromosome (C and D). Longer exposure to Cre results in evidence of further chromosome damage, such as smaller than expected chromosomes (E and F) or extraordinarily long chromosomes (G and H). Arrows indicate acentric fragments and arrowheads indicate dicentric chromosomes.

Moon et al. (2000), except that the loxP site in the 59 UTR is Mouse metaphase chromosome analysis inverted with respect the other loxP site.Themouselinewith For preparation of mitotic figures, mouse embryonic fibro- inverted loxP sites and the tetracycline-repressible tdTomato blasts (MEF) and embryonic stem (ES) cell lines cells were transgene targeted to the tip of chromosome 19 was generated grown to 50–70% confluence in Dulbecco’s modified Eagle’s using homology arms with sequence similarity to the region medium (DMEM) supplemented with Na pyruvate, nones- distal to the last exon of the coding region of Grk5.Briefly, th is construct contains a minimal CMV promoter immediately up- sential amino acids (NEAA), and 10% fetal bovine serum stream of seven copies of the tetracycline operon operator se- (FBS) for MEFs, or DMEM supplemented with Na pyruvate quence immediately upstream of the coding sequence of a and NEAA and 15% FBS and leukemia inhibitory factor (LIF) tandem dimer of the tomato fluorescent protein and a polyade- for ES cells. MEFs were synchronized by double block with nylation sequence. Downstream of tomato are the two inverted 50 ng/ml methotrexate and 1 mM thymidine, and then loxP sites followed by a CAG promoter driving expression of the arrested in metaphase with 1 mg/ml colcemid. ES cells were fusion protein consisting of TetR fused to the strong transcrip- arrested at metaphase with colcemid at 100 ng/ml. (De- tional silencing Kruppel-associated box domain (Tet-KRAB). tailed protocol available upon request.)

926 S. W. A. Titen Table 2 Dicentric chromosome production in mouse cells Insertion Normal Dicentric Acentric N optical site karyotype chromosome chromosome >2n fields chr. 7 40 15 73 16 100 chr. 15 40 19 23 19 29 Cells with inverted loxP targets, inserted on chromosome 7 or chromosome 15, were exposed to Cre protein and scored for dicentric or acentric figures, or polyploidy (.2n), in metaphase. Chromosome-specific paints were used to recognize chromosome 7 or 15

CreTAT treatment Figure 5 Dicentric bridge formation in early embryos. Mouse embryos Rapidly dividing cells were grown to 50–70% confluence. carrying two inverted loxP sites on chromosome 7 with maternal Cre and Cells were then washed with PBS and incubated in serum- Histone-GFP were used to make time-lapse movies of the early cleavage divisions. (A and B) are images of two successive timepoints during ana- m TAT free medium supplemented with 4 M Cre protein for phase showing persistence of a dicentric chromosome during mitotic 1hrat37°. anaphase (arrow). Data availability statement Strains and plasmids are available upon request. The authors telomere adjacent on chromosome 7 within the coding re- affirm that all data necessary for confirming the conclusions of gion of fgf4, and MEFs with inverted loxP sites near the the article are represented fully within the article. centromere of chromosome 15 within the coding region of ext1. The ES cells were transfected with Cre recombinase expression vector and the MEFs were treated with the Cre- Results TAT protein on plates. In either case, we would expect to see As a practical demonstration of dicentric chromosome forma- splitting of the targeted chromosome into acentric and di- tion, we obtained flies carrying an insertion of the Flip-Flop centric portions (Figure 3). Following incubation in the construct, either on chromosome 2 or chromosome 3. The presence of the Cre recombinase we observed dicentric Flip-Flop cassette was developed as a method for switch- and acentric chromosome formation (Figure 4, A–Dand ing between GFP and mCherry labeling of gene traps in Drosophila (Nagarkar-Jaiswal et al. 2017). Inverted FRTs were placed so that inversion of the flanked segment switches between the two markers. To test whether dicentric chromosomes would form after expression of the FLP recombinase, we generated progeny that contained the Flip-Flop construct and a heat shock inducible FLP gene (70FLP). We heat shocked larvae for 1hrat38° and then examined neuroblast mitotic spreads from third instar larvae (Figure 2). With a Flip-Flop insertion on 2L,63%ofmetaphasesshowed dicentric/acentric chromosome 2 figures at 4–5hrafterheatshock.On3R, 46% of chromosome 3 figures showed the same. At 23 hr after heat shock, cells showing dicentric/acentric figures were approximately half as frequent as the earlier time point, owing, no doubt, to the arrest and death of cells suf- fering from aneuploidy and broken chromosomes during the intervening period (Table 1; Titen and Golic (2008)). Figure 6 Marking cells after loss of acentric chromosome. (A) Sche- These results demonstrate that FLP recombinase does not matic representation of the transgene targeted to the tip of benignly switch markers within the Flip-Flop cassette, but chromosome 19. The transgene encodes a minimal CMV promoter (PCMV), the tetracycline operator sequence (tetO7), and the also causes dicentric chromosome formation. Our previous tdTomato coding sequence, centromere proximal to inverted loxP workshowsthatthisisfrequently,butnotinevitably,fol- sites; a strong CAG promoter (PCAG) controlling expression of the lowed by apoptosis (Golic 1994; Titen and Golic 2008; TetR-KRAB repressor gene lies distal to the loxP sites. Loss of the Kurzhals et al. 2011). PCAG-tetR-KRAB portion relieves repression of tdTomato expression, fl In the mouse, there is also ample evidence that the use of resulting in cells that uoresce red. Early embryos carrying this construct, and maternally deposited Creprotein,wereexaminedfortdTo- inverted lox sites produces dicentric chromosomes. As a di- mato fluorescence. Nuclei are visualized with histone-GFP (green). rect demonstration of this, we generated ES cells carrying a Cells that have lost the TetR gene express tdTomato (red): two cells targeted insertion of a construct with inverted loxP sites in (B) and 6cellsin(C).

Dicentric Chromosomes 927 Table 2). Upon extended culture we observed further chro- part of development of the central nervous system, in this mosome rearrangements, polyploidy, and evidence of experiment it may instead be a consequence of the forma- breakage-fusion-bridge cycles (Figure 4, E–H). We also di- tion of dicentric and acentric chromosomes. In this context, rectly visualized dicentric chromosome formation in live it is worth noting that cells which experience dicentric/ embryos carrying the inverted loxP sites on the tip of chro- acentric formation can divide several times to produce a mosome 7 and an HPRTCRE transgene—a constitutive Cre clone before they die. Such cells can even differentiate into allele whose expression results in maternal deposition of adult tissues, though they are less successful than wildtype the Cre protein (Figure 5). cells (Golic 1994; Otsuji et al. 2008; Titen and Golic 2008; As a second test of dicentric/acentric formation in live Tada et al. 2009; Zhu et al. 2010; Kurzhals et al. 2011). embryos, we generated mice carrying a construct with the Our results, and those from other groups, make it Tet Repressor (TetR) gene distal to inverted loxP sites inserted exceedingly clear that the use of inverted target sites for in the subtelomeric region of chromosome 19. Proximal to a site-specific recombinase will frequently lead to the the loxP sites is a tetracycline-repressible reporter gene production of dicentric and acentric chromosomes, with encoding the red fluorescent protein tdTomato (Figure 6). significant effects on the behaviors and fates of cells in When dicentric/acentric chromosomes are formed by Cre which they occur. There are schemes that use directly activity, and the acentric portion carrying the TetR is lost, repeated target sites for gene switches and for lineage tdTomato can be expressed. We examined 2–32 cell early tracing (Nagy 2000; Kwan 2002; Zong et al. 2005; Gierut mouse embryos carrying this construct and HPRTCre. We ob- et al. 2014; Hubbard 2014; Richier and Salecker 2014; served tdTomato activation in a subset of cells, indicating that Weissman and Pan 2015; Muñoz-Jiménez et al. 2017; TetR had been lost (Figure 6). Pontes-Quero et al. 2017; Germani et al. 2018). These do not suffer the drawback of generating dicentric and acentric chromosomes and can be applied to many exper- Discussion imental situations. The occurrence of dicentric/acentric chromosomes will almost certainly confound the interpretation of experiments Acknowledgments that use inverted target sites. We have shown, here and previously, that generation of dicentric/acentric chromo- This work was supported by grants R01 GM065604 (K.G.G.) somes can be very frequent—in some cases nearly 100% and R01 MH093595 (M.C.) from the National Institutes of of cells that express FLP and carry inverted FRTscanexpe- Health (NIH), and by a grant from the Halt Cancer at X rience this event (Titen and Golic 2010; Kurzhals et al. Foundation (S.W.A.T.). M.T.B.J. was partially supported by 2017). These cells frequently, but not inevitably, undergo Training Grant T32GM007464 from the NIH. 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