FIG. 4.
Transrepressive action of dCBP at the pericentric locus requires dSir2. (A to D) Photomicrographs of the adult dorsal thorax from wt (A), UAS-dSir2/+; pnr-GAL4/+ (B), nej3/+; UAS-dSir2/+; pnr-GAL4/+ (C), and nej3/+; pnr-GAL4/+ (D) flies. (B) Ectopic expression of UAS-dSir2 with the pnr-GAL4 driver induced an alteration in the distribution of microchaetae (small bristles) and macrochaetae (large bristles), particularly the dorsocentrals and scutellars. (C) Expression of UAS-dSir2 with the pnr-GAL4 in nej3/+ mutants induced a stronger phenotype than that of either alone. (D) nej3/+ mutants with pnr-GAL4 did not show an alteration in the distribution of micro- and macrochaetae. The red dashed lines in panel A show the approximate area where the phenotype is located in panels A to D. (E to J) Polytene chromosomes from the third instar larvae of flies carrying a UAS-FLAG-dSir2 transgene with a BLK-GAL4 driver were dissected and stained with DAPI to visualize DNA (blue [E]) or with rat polyclonal anti-dCBP antibodies (red [F]) or rabbit polyclonal anti-FLAG antibodies (green [H]) to visualize dSir2 tagged with FLAG. (J) The regions in chromosomes showing the colocalization of dCBP and FLAG-dSir2 are indicated with open arrowheads. (K) dCBP physically interacts with both dSir2 and AR AF-1 in flies carrying the reporter gene at the pericentric region. Whole-cell protein extracts from flies expressing AR AF-1 in the eye by the use of a UAS-GAL4 system and carrying an ARE-GFP-white reporter in the pericentric heterochromatin (pericentric fly model) were immunoprecipitated (IP) with antibody against dCBP or control preimmune immunoglobulin G (IgG). Fractions (5%) of the input material (lower panels) and the precipitated proteins (upper panels) were analyzed by Western blotting using anti-dSir2 (dF-16), anti-AR (N-20), and anti-dCBP antibodies as indicated. IB, immunoblot. (L) Flies from the pericentric fly model line expressing ARAF-1 proteins and carrying an ARE-GFP-white reporter gene in the pericentric region were crossed to flies carrying UAS-dCBP alone or together with a dSir2 loss-of-function mutation (dSir205327a/+) as indicated. Overexpression of AR AF-1 in the eye disc of the third instar larvae was assessed by immunostaining using an anti-AR antibody (upper panels). The effect of dCBP or loss of function of dSir2 on AR AF-1-mediated transactivation was assessed by examination of GFP expression (middle panels). Merged images are shown in the lower panels. The effect of dSir205327a was further confirmed by treatment with nicotinamide (Sir2 inhibitor) (20 mM).