Fig. 1. DR-white system to induce single DSBs in euchromatin and facultative heterochromatin.

A Schematic of the DR-white system as previously established in²². I-SceI expression creates a DSB in the upstream white gene. Homologous recombination (HR) with the iwhite sequence (intra-chromosomally or with the sister chromatid) results in loss of the I-SceI cut site (−23bp). Imperfect non-homologous end joining (NHEJ) may result in indels at the DSB site. Perfect NHEJ or HR using the I-SceI sequence on the sister chromatid can recreate the I-SceI cut site. B Schematic of DR-white integration sites in five fly lines (2× in euchromatin [3eu_1, 3_eu2] and 3× in facultative heterochromatin [2fH_1, 3fH_1, 3fH_2]). Created in BioRender. Janssen, A. (2021) BioRender.com/h58g617. C ChIP-qPCR analysis for H3K27me3 at the DR-white locus (primers Fig. 1A). H3K27me3 levels were normalized using ubx gene primers (ubx has high H3K27me3 levels⁶³). Averages +SEM are shown for n = 5 biological replicates (except 3eu_1: n = 4). p-values: 3eu_1 = 0.0350, 3eu_2 = 0.0009, 2fH_1 = 0.0006, 3fH_1 = 0.0333, 3fH_2 = 0.0432. D Schematic of the hsp.I-SceI construct. The hsp70 promoter upstream of I-SceI can be activated by shifting larvae for one hour (1 h) to 37 °C. RT=room temperature. E Schematic of the ecDHFR-I-SceI system. Proteasomal degradation of ecDHFR-I-SceI can be blocked by adding trimethoprim (TMP). F Experimental set up for ChIP-qPCR experiments. 3rd instar DR-white larvae with and without hsp.I-SceI (control) were heat-shocked. Six hours later chromatin was subjected to ChIP-qPCR (primers Fig. 1A). Figure partly created in BioRender. Janssen, A. (2024) BioRender.com/n98i722. G ChIP-qPCR analysis for yH2Av in the absence (-) and presence (+) of hsp.I-SceI (as in F). Averages +SEM are shown for n = 4 (3eu_1, 3fH_2), n = 5 (3fH_1), or n = 9 (3eu_2, 2fH_1) biological replicates. H, I DR-white/ecDHFR-I-SceI larvae were fed TMP for 3-4 days. Repair products at the upstream white gene were amplified and analyzed using TIDE³⁷. Graphs show the total identified repair events in all PCR products (H) and the percentage of repair products with either HR (color) or NHEJ (gray) signatures (I). Bars indicate averages +SEM of n = 11 single larvae (biological replicates) (except 2fH_1: n = 9). If not shown, p-value (>0.05), (*) p-value ≤ 0.05, (***) p-value ≤ 0.001, ratio two-sided paired t-test (G), one-way ANOVA + Tukey’s multiple comparisons test (I). Source data are provided as Source Data file.