Somatic Recombination Drives Frequent Loss of Heterozygosity in Aging Stem Cells

LaFave and Sekelsky, 2009 LaFave M.C.

Sekelsky J. Mitotic recombination: why? when? how? where?. Malkova and Ira, 2013 Malkova A.

Ira G. Break-induced replication: functions and molecular mechanism. Figure 1 Somatic Recombination Contributes Strongly to Frequent Spontaneous Loss of Heterozygosity in Aging Fly Midguts Show full caption (A) Inactivation of GAL80 leads to Gal4-dependent GFP expression. A GAL80 transgene at position 1E was used in females (XX) and males (XY). (B) Spontaneous GFP+ clones from an aged female midgut. (C) Frequency of GFP LOH events in young (0 to 1 week) and old (5 to 6 weeks) female (GAL80/+) and male (GAL80/Y) midguts. (D) Chromosomal locations of LOH markers Notch (N), O-fut1, and neur. (E) Notch LOH events in aged N55e11/+ midguts were identified by staining for Delta (Dl, cytoplasmic red) and Prospero (Pros, nuclear red). (F) A female N55e11/+ midgut with numerous LOH events. (G) Frequency of LOH events in young and aged female midguts heterozygous for N, N55e11/ FM7 (balancer chromosome), O-fut1, and neur. (H) N55e11 at position 3C and GAL80 at position 5B. (I) An LOH clone in an aged N55e11/GAL80 midgut with N phenotype and GAL80 inactivation. (J) Spontaneous neur LOH clone in an aged female neurIF65/+ midgut. (K) Spontaneous O-fut1 LOH clone in an aged O-fut14R6/+ midgut. ∗p < 0.05, ∗∗∗∗p < 0.0001; n.s., not significant (Fisher’s exact test, two-tailed). See also LOH clones are outlined in yellow. Scale bars: 50 μm.p < 0.05,p < 0.0001; n.s., not significant (Fisher’s exact test, two-tailed). See also Figure S1 and Table S1 To study somatic genome instability in adult intestinal stem cells, we first assessed the frequency of spontaneous inactivation of a single copy transgene inserted on the X chromosome at position 1E. The inactivation of the Gal4 repressor GAL80 in ISCs leads to heritable, clonal, Gal4-driven GFP expression ( Figure 1 A). GFP-positive single cells as well as clonal GAL80 inactivation events were readily detected in the midguts of old females and males, while very rarely present in young animals ( Figures 1 B and 1C). For further analysis, we focused on the clonal GAL80 inactivation events having stem cell origin. Such somatic gene inactivation or LOH could occur via mitotic recombination, point mutation, gene deletion or epigenetic silencing. Recombination-based mechanisms of LOH such as crossing over, gene conversion (), or break-induced replication (BIR) () require a homologous chromosome. Therefore, we compared the frequency of GAL80 inactivation events in females (two Xs) versus males (one X and one Y). Female midguts consistently had a higher frequency of at least one clonal GAL80 inactivation event than male midguts. This increased over time from 12.7% in “young” (0 to 1 week; n = 158) to 68.5% in “aged” females (5 to 6 week; n = 181; Figure 1 C). Multiple GFP-positive GAL80 inactivation clones suggestive of several independent events were detected ( Figures 1 B and 1C). In contrast, males had no detectable clonal GAL80 inactivation events in young flies (n = 114) and 2% in aged flies (n = 153; Figure 1 C).

If the observed GAL80 inactivation was due to homologous recombination-based mechanisms, LOH frequencies should dependent on the position of the marker gene on the chromosome arm: a distal gene would be more frequently exchanged during crossover events or copied via gene conversion or BIR. Consistent with that, the frequency of clonal GFP expression varied with the chromosomal location of the GAL80. 75% (n = 72) of aged females with GAL80 at position 5B, 18 Mb from the centromere, had at least one clonal GAL80 inactivation event, whereas only 40% (n = 75) did when GAL80 was at position 19E, 2.5 Mb from the centromere ( Table S1 ). Interestingly, the frequency of LOH at 19E is higher than predicted from meiotic recombination maps, which could suggest that additional factors influence GAL80 inactivation somatically. Of note, GAL80 position had no effect on the frequency of its inactivation in aged male midguts. Thus, a majority of the GAL80 gene inactivation events did not occur in the absence of a homologous chromosome and varied with chromosomal location.

55e11/+) present an overall wild-type midgut appearance. In contrast, midguts in which homozygous mutant N55e11/ N55e11 stem cells are genetically induced or express N RNAi ( Micchelli and Perrimon, 2006 Micchelli C.A.

Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Ohlstein and Spradling, 2006 Ohlstein B.

Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. 55e11/+ genetic background to assess spontaneous LOH (55e11 is a recessive lethal allele and is located on the X chromosome, only female flies could be assessed. In young flies, only rare LOH events were observed (1.4% of midguts, n = 145; To explore further the possible mechanisms and in vivo impact of somatic mutations in ISCs, we exploited the phenotype of inactivation of Notch signaling components. Female flies heterozygous for a null allele of Notch (N/+) present an overall wild-type midgut appearance. In contrast, midguts in which homozygous mutant N/ Nstem cells are genetically induced or express N RNAi ( Figure S1 ) produce hyperplastic clusters of excess ISCs and enteroendocrine cells (EEs) that fail to properly differentiate (). We therefore used the N/+ genetic background to assess spontaneous LOH ( Figures 1 D–1I). As Nis a recessive lethal allele and is located on the X chromosome, only female flies could be assessed. In young flies, only rare LOH events were observed (1.4% of midguts, n = 145; Figure 1 G). In contrast, 80.9% of midguts of aged flies (n = 262) contained at least one LOH cluster ( Figures 1 E–1G). This was not observed in wild-type females (n = 519). Interestingly, the majority of guts (67.7%) had more than one event, with up to 25 clusters in a single gut detected ( Figure 1 F), similar to that observed for GAL80 LOH ( Figure 1 C).

55e11 allele and a GAL80 transgene inserted close to the Notch locus (position 5B) on the homologous chromosome ( In order to test whether the LOH events were due to somatic homologous recombination, we then analyzed flies heterozygous for the Nallele and a GAL80 transgene inserted close to the Notch locus (position 5B) on the homologous chromosome ( Figure 1 H). We observed that in aged midguts 93.9% of LOH clones (n = 132) having the Notch phenotype were GFP positive, indicating concomitant inactivation of GAL80 ( Figure 1 I). This suggests that the majority of LOH events arise due to a recombination event initiated along the chromosome arm between the centromere and the GAL80 insertion site. The small fraction of LOH events (6.1%, n = 132) that displayed the Notch phenotype and were GFP negative could have arisen from recombination initiated within the 2-megabase region between the Notch locus and the GAL80 or from recombination-independent gene-inactivating events.