Figure 5: Pericentromeric heterochromatin instability in Tctp mutants.

(a) Suppression of w^m4 PEV phenotype by Tctp^h59/+(white arrowhead). (Histogram) Quantification of eye pigment (n=20 heads, mean±s.d.). Scale bar, 200 μm. (b–f) Defective gene silencing and pericentromeric heterochromatin instability in Tctp^EY/h59 mutants. (b) Effects of Tctp on expression of rDNA genes. An en-Gal4 was used to express UAS-transgenes only in the posterior compartment of wing disc (dotted GFP-positive region). Fibrillarin (Fib) antibody staining for nucleolus is pseudo-colored in gray. (Top) en>GFP discs show similar Fib levels in both anterior and posterior compartments. (Middle) The reduced Fib level and nucleolar size in Tctp-overexpressing cells of the posterior compartment. (Bottom) The increased Fib level and nucleolar size in Tctp-knockdown cells of the posterior compartment. Scale bars, 50 μm. (c) Disrupted nucleolar structures in SG cells from Tctp^EY/h59. n=30 SGs (mean±s.d.). Scale bars, 10 μm. (d) Elevated Fib levels in Tctp^EY/h59 larvae. Tctp^RES (Tctp^EY/h59 with Tctp^Ge30-2) shows normal level of Fib. Anti-H3, a loading control for western blotting (mean±s.d.). NS (P=0.5). (e) Increased Pre-rRNA transcript from nucleolar rDNA loci in Tctp^EY/h59 (QPCR). Tctp^RES rescued Tctp^EY/h59 (mean±s.d.). n.s. (P=0.5). (f) Increased eccDNA formation in the repeated genomic sequences such as rDNA and stellate genes of Tctp^EY/h59. su(var)3-9^1/2, a positive control. QPCR was shown as a fold change relative to wild-type (mean±s.d.). A representative gel image is shown. NS (P=0.1). (g–k) Reduced level of H3K9me2 or HP1a heterochromatic silencing mark in Tctp mutants. (g) (Top) High levels of silencing marks in SG nucleus from wild-type (w¹¹¹⁸). (Right-most) The enlarged image of a nucleus marked by an arrowhead. (Middle) Reduced levels of silencing mark in Tctp^EY/h59. (Bottom) Tctp^Ge30-2rescues Tctp^EY/h59 from reduced levels of silencing marks. Scale bar, 50 μm. (Histograms) Quantified silencing mark levels (n=10–14 SGs, mean±s.d.). NS (P=0.1). (h,i) Reduced H3K9me2 levels in Tctp^h59 null mitotic clones (GFP-negative, arrows) within the developing wing disc. The boxed region is enlarged in i. Scale bar, 50 μm. (j) QPCR in SG tissues. Reduced su(var)3-9 transcription in Tctp^EY/h59 (mean±s.d.). NS (P=0.3). (k) Western blot on SG tissues. The levels of both H3K9me2 and HP1a in Tctp^EY/h59 are reduced compared with wild-type.