Figure 6: Defective gene silencing and genome stability in Tctp mutants are ameliorated by brm mutation.

(a) Genetic interaction between Tctp and Brm complex subunit genes. (Top panels) w^m4 eye color PEV phenotype in the wild-type background (+/+) and the effects of mutations in Brm complex genes as indicated. lid¹⁰⁴²⁴ is a PEV enhancer control. (Bottom panels) Tctp^h59/+ heterozygous mutation strongly suppresses the w^m4 PEV (asterisk in the first panel). The effects of Tctp^h59/+ is significantly suppressed by brm^2/+ mutation (black arrow). Likewise, mor¹/+ (white arrowhead) and bap170^Δ65/+;bap180^Δ86/+ double mutations (PBAP components, white arrow) suppress the Tctp^h59/+ effect. In contrast, snr¹⁰¹³¹⁹/+ mutation in an inhibitory subunit gene (black arrowhead) or osa²/+ (BAP component) does not suppress Tctp^h59/+. Scale bar, 200 μm. (Histogram) Quantification of eye pigment (Mean±s.d. n=20 heads). (b,c) Increased Fib levels in Tctp mutants. (b) Increased Fib level in the posterior compartment of en>Tctp-i flies (arrowhead) is suppressed by Brm-knockdown (arrow). Scale bars, 50 μm. (c) Increased Fib level in Tctp^EY/h59 whole body. Fib (nucleolus marker) and H3 (loading control) antibodies were used for western blotting or immunostaining. (d,e) QPCR on Pre-rRNA transcription and eccDNA formation. Additional heterozygous brm² mutation in Tctp^EY/h59 ameliorates defective gene silencing in rDNA loci from SG cell nucleoli (d) and larval eccDNA formation in repetitive sequences (e). Normalized DNA levels are shown as fold changes relative to the wild-type level (mean±s.d.). A representative gel image is shown. (f) Abnormal nucleolar morphology in Tctp^EY/h59 are improved by brm²/+ mutation. Scale bars, 10μm. (g) Shortened life span in adult Tctp^EY/h59 males (red) relative to wild-type (blue; log-rank test, P<0.0001) is ameliorated by reducing Brm level (brm^2/+,Tctp^EY/h59; green; log-rank test, P<0.0001). Adult flies were incubated at 29 °C.