Figure 7. Sumoylation inhibits NHR-25-dependent transcription in vivo. (A) NHR-25 binds to canonical SF-1 target sequences. Sequence of the wild-type (WT) and mutant (MUT) MIS and CYP11A1 binding sites used are shown on top. Bases altered in the MUT sequences are underlined. The annealed 26NR5RE oligonucleotides were incubated with combinations of the following: sumoylation enzymes (hUbc9+CeSMO-1) with or without hE1 enzyme, and NHR-25 DBD substrate. Recombinant hSENP1 SUMO protease was included to demonstrate that bands reflected sumoylated species. The corresponding proteins in the EMSA were detected by anti-MBP immunoblotting (input). The positions of unsumoylated and sumoylated NHR-25 DBD are indicated. (B) Animals carrying an 86NR5RE (WT)::NLS::36Venus transgene as an extrachromosomal array were generated. No Venus expression was detected in transgenic animals on vector RNAi (i and ii). The nematode body is outlined in (i), and the corresponding differential interference contrast (DIC) image of the same animal is provided (ii). Representative Venus expression in transgenic animals treated with
smo-1 RNAi (iii-vii). Expression was observed in seam cells at L4 (iii), in seam cells and
hyp7 at L3 (iv), in
hyp7 at L4 (v), in the AC and vulF at early L4 (vi), and in developing vulval cells at L3 (vii). Fluorescent and DIC images were merged in vi and vii. (C and D) Transgenic animals expressing the Venus reporter in at least one of the following tissues: seam cells,
hyp7, or vulval cells; were scored. Reduction of
nhr-25 function either by RNAi (C) or by
ku217 mutation (D) reduced the 86NR5RE (WT) reporter activity following
smo-1 RNAi. (n) number of animals scored. (E) Mutations (MUT) in NR5RE completely eliminated Venus expression following
smo-1 RNAi. DIC (i and iii) images corresponding to Venus fluorescence images (ii and iv, respectively) are provided. Positions of hypodermal nuclei (ii) and the developing vulva (iv) are outlined. (F) NHR-25::GFP is expressed in nuclei of seam cells and
hyp7 (i) and the developing vulva (ii), similar to 86NR5RE (WT)::NLS::36Venus reporter expression. All animals are positioned with the anterior to the left.