Extended Data Figure 8: Moonshiner function can be bypassed by directly connecting Deadlock to Trf2.

a, Experimental scheme used to recruit GFP or TRF2 to DNA upstream of sequences of interest to test for stimulation of Luciferase transcription. Bar diagram shows fold changes in reporter activity upon tethering of TRF2 versus GFP to wild-type or mutant Histone 1 core promoter or to random piRNA cluster fragments (error bars, s.e.m.; n = 5 biological replicates; *P <0.05 based on two-tailed paired t-tests). b, Firefly luciferase values underlying the relative activities shown in a. Firefly luciferase activity was normalized to Renilla luciferase activity (transfection and viability control) upon tethering of TRF2 versus GFP to wild-type or mutant Histone 1 core promoter or to ten random piRNA cluster fragments (error bars, s.d. of five biological replicates each with six technical replicates. c, Confocal images showing localization of LAP–Moonshiner and Rhino in germline nuclei of ovaries depleted for indicated factors (scale bar, 5 μm). d, Western blot showing levels of LAP–Moonshiner in ovaries where the indicated factors were depleted in the germline via sh-lines (ATP synthase serves as loading control). e, Confocal images showing localization of germline-expressed LAP–TRF2 and endogenous Rhino in control ovaries (top) or in ovaries expressing the Deadlock–GFP-nanobody fusion protein (bottom) (scale bar, 5 μm). The TRF2 accumulations in wild-type nuclei do not overlap with Rhino foci and instead are reported to be TRF2 accumulations at the repetitive histone loci³¹. We note that TRF2 accumulation at Rhino foci is not visible in wild-type cells, most probably as the levels of this protein are too high to detect this local enrichment, which depends on Moonshiner (a protein expressed at only low levels). f, Representative images of DAPI-stained embryos (inverted monochromatic) assessed for progress of early embryogenesis. Left: two images of normal embryo development at the blastoderm stage (top) and at the extended germband stage (after gastrulation; bottom). Right: a typical moonshiner mutant embryo arrested early in development (no distinct nuclei are visible; the lower image displays the top image at increased brightness). g, Percentages of embryos with the indicated genotype displaying successful hatching. h, Relative levels of steady-state transposon mRNAs underlying the panel displayed in Fig. 5d. Bars show mean levels relative to those measured in moon^−/− samples. Error bars, s.d. of three biological replicates. *P < 0.05 from two-tailed t-tests for difference to moonshiner full mutant samples. i, Levels of piRNAs map** uniquely to the indicated clusters (grey, Rhino-independent; black, Rhino-dependent) in the indicated genotypes (values are normalized to the wild-type control levels). j, The log₂(fold changes) in levels of piRNAs map** antisense to transposons are plotted relative to levels in moonshiner mutants. The green boxes highlight the set of transposons for which mutation of moonshiner results in decreased antisense piRNAs (n = 111; transposons with fewer than 100 antisense piRNAs per million were removed from the analyses).

Source data