Fig 6. The HMR C-terminus is required for HMR physiological function in D. melanogaster.

(A) Schematic representation of Hmr genotype in fly stocks used for complementation assays. Hmr null mutants (Df(1)Hmr ^-, here referred to as Hmr^ko) are complemented with wild type (Hmr⁺) or mutant (Hmr^dC and Hmr²) transgenic alleles inserted in the 3^rd chromosome. (B) Schematic representation of crosses performed for complementation assays. F1 siblings are compared: control animals (no Hmr transgene: Df(1)Hmr ^-; +/+) and test animals (with Hmr transgene: Df(1)Hmr ^-;Hmr*/+). Hmr* refers to any transgene used in this work. (C) Truncation of the HMR C-terminus abrogates HP1a-proximal localization of HMR in vivo. Representative immunofluorescence images of stage 7 follicle cells in ovarioles from Hmr mutant D. melanogaster females complemented with either of the Hmr transgenic alleles. Insets represent zoom into one representative cell for anti-HMR (a), anti-HP1a (b) and merge of the two channels (c). Size bars indicate 5 μm. (D) Defective retrotransposons silencing in Hmr^dC and Hmr². RT-qPCR analysis measuring mRNA abundance for the telomeric retrotransposons HeT-A and TART in female ovaries. Heterozygous genotypes (Hmr^ko; Hmr*/+) were compared with the respective non-complemented siblings (Hmr^ko; +/+). Y-axis: log₂ fold-change of the mean (complemented/non-complemented) after normalization to a housekeeping gene (18s-rRNA). Error bars represent standard error of the mean (n = 3). Welch t-test was used for pairwise comparisons with Hmr⁺ as a reference group and FDR for multiple testing adjustment (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E) HMR C-terminus is required for female fertility. Fertility defects are not complemented by Hmr^dC: heterozygous females (Hmr^ko; Hmr*/+) were compared with the respective non-complemented siblings (Hmr^ko; +/+). Number of adult offspring per female mother assessed in a time course (females aged 5–10, 10–15 and 15–20 days). Wilcoxon rank sum test was used for pairwise comparisons with Hmr⁺ as a reference group and FDR for multiple testing adjustment (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). For details about fertility assays refer to S4 Table.