Fig 1: EcR but not USP is essential for recruitment of dMi-2 to ecdysone-activated genes.(a) Enrichment of predicted EcR–USP-binding sites in EIMRs. The histogram shows the distribution (sampled from 10,000 runs) of putative EcR–USP site number within the same number of randomly selected genomic regions with the same sizes as the experimentally determined EIMRs. The red line indicates the number of computationally identified EcR–USP-binding motifs within EIMRs (142; P<10-8). The P-value for the observed number of binding motifs was estimated from the normal distribution that can be approximated based on the displayed simulated distribution, that is, using mean and s.d. estimates from the simulated sample. (b) Western blot analysis of protein extracts of S2 cells treated with dsRNA directed against GFP (control), EcR or USP. Cells were either left untreated (−20HE) or were treated with ecdysone (+20HE). Antibodies used are shown on the right, molecular masses on the left. Tubulin served as a loading control. (c) dMi-2 binding to Br-C and vrille following EcR and USP depletion analysed by ChIP-qPCR. Chromatin was prepared from S2 cells that were first treated with dsRNA against GFP (control), EcR or USP and then either left untreated or were exposed to 1 μM ecdysone for 6 h as indicated. ChIP-qPCR was performed with dMi-2 antibody. Error bars denote s.d. of technical triplicates. Experiments were performed as biological triplicates. One representative example is shown.
Fig 2: dMi-2 forms a complex with EcR and competes with USP for binding to EcR.(a) dMi-2 and EcR interact. Nuclear extracts from untreated and ecdysone exposed (+20HE) S2 cells were immunoprecipitated with dMi-2 antibody, dp53 antibody or IgG as indicated on top (lanes 2–4 and 6–8). 1% input was loaded in lanes 1 and 5. Antibodies used for western blot analysis are indicated on the right, molecular masses on the left. (b) Sf9 cells were infected with recombinant baculoviruses directing the expression of dMi-2-FLAG, dMi-2 or EcR-FLAG as indicated on top. Extracts were immunoprecipitated with FLAG antibody and washed with high salt buffer. Immunoprecipitates were then analysed by SDS–PAGE and Coomassie staining. Lane 1: molecular weight marker. Lanes 2, 4 and 6: 500 ng protein, lanes 3, 5 and 7: 1 μg protein. (c) dMi-2 and USP bind EcR in a mutually exclusive manner. Sf9 cells that were either left untreated (left panels) or were exposed to ecdysone (+20HE, right panels) were infected with recombinant baculoviruses expressing dMi-2-FLAG, EcR or HA–USP as indicated on top. Extracts were analysed by western blot for expression of recombinant proteins (top panels). Extracts were immunoprecipitated with FLAG (FLAG IP, middle panels) or HA (HA IP, bottom panels) antibody and immunoprecipitates were analysed by western blot. Recombinant proteins detected by western blots are indicated on the right.
Fig 3: The effect of knockdown of EcR or USP on the structure of male accessory glands.To assess the effect of depletion of EcR or USP, accessory glands were analyzed either at the ultrastructural level (panels at the top). Depicted at the top are the electron micrographs of male accessory glands from (A) EcR control (B) EcR knockdown (C) USP control and (D) knockdown males. Accessory glands from EcR control, USP control and USP knockdown males show normal protein filamentous structures (labeled as f) throughout in their lumen. However, glands of EcR knockdown have extreme vacuolization (v) and lack filamentous structures in the lumen. A minimum of five tissues from each group was used for ultrastructural analysis.
Fig 4: EcR increases dMi-2-mediated nucleosome remodelling in vitro.(a) REA assays were carried out with a 32P-labelled mononucleosome substrate in the presence of recombinant dMi-2, EcR, USP and BSA as indicated on top. Reactions were stopped at four time points (2.5, 5, 10 and 20 min) and analysed by non-denaturing PAGE and autoradiography. The positions of the uncut DNA fragment and the cut DNA fragment (product of the remodelling reaction) are indicated on the right. The top panel shows REA assays carried out in the absence (−20HE), the bottom panel shows REA assays carried out in the presence of 1 μM ecdysone (+20HE). (b,c) REA assays were carried out in the presence of 110 nM dMi-2, 110 nM EcR, 445 nM USP and/or 745 nM BSA for 2.5, 5, 10 and 20 min as indicated. Panel (b) shows results obtained in the absence of ecdysone; panel (c) shows results obtained in the presence of 1 μM ecdysone. Bands containing cut and uncut DNA were quantified using the Science Lab Image Gauge (FUJIFILMS) software. The ratio of cut and total DNA (cut plus uncut) is plotted as ‘% cut DNA'. Curves were fitted using the GraphPad Prism software according to one-phase decay equation. Error bars represent s.e.m. and are derived from four (b) and three (c) independent experiments.
Fig 5: The effect of knockdown of different hormone receptors on the fertility of mated females.To identify the nuclear/hormone receptor involved in male fertility, 19 receptors were knocked down, individually, in accessory gland specific manner in the male reproductive tract and were allowed to mate with virgin females. Shown here is the number [Mean±Standard Error (SEM)] of progeny produced by females mated to knockdown or control males over a period of 10 days. Mates of met (Methoprene tolerant, a juvenile hormone receptor, **p<0.001) knockdown males produced significantly fewer progeny when compared to their controls and also those in strain background control (Jhe). Interestingly, females mated to EcR knockdown males failed to produce progeny (EcR; ***p<0.0001; Bonferroni corrected p value for significance is p<0.002). However, fertility of females mated to USP (p = 0.27) or the remaining 16 hormone receptor knockdown males was not significantly different from their respective controls. Number of females ranged from 15–45 depending on the hormone receptor analyzed.
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