OGT overexpression in clock neurons lengthens behavioral rhythms and disrupts PER-CLK interaction.

(A) Four independent transgenic fly lines expressing 3XFLAG-tagged ogt in tim-expressing neurons (TUG>FLAG-ogt) display lengthened periods in free-running locomotor activity rhythms as compared to their parental controls. Black and dark grey bars represent control parental lines. Light grey bars represent independent TUG>FLAG-ogt transgenic lines. Numbers above bars indicate percentage of flies displaying rhythmic activities. Asterisks above percentage denote statistical significance (***P-value TUG >FLAG-ogt as compared to their parental controls. Numbers within bars indicate sample size (number of flies). Error bars indicate ± SEM. (B) PER O-GlcNAcylation is enhanced in TUG>FLAG-ogt (line 4 is used for all subsequent experiments). Protein extracts from fly heads at the indicated time-points were either directly analyzed by immunoblotting (input) (Bottom two panels) or immunoprecipitated with α-PER (GP5620), followed by chemoenzymatic labeling with biotin. Samples were resolved on SDS-PAGE and analyzed by western blotting using α-streptavidin or α-PER (Top two panels). (C) Bar graph showing normalized O-GlcNAcylated PER in ogt overexpressor and control flies (n = 2). Error bars represent SEM. **P = 0.01; two tailed Student’s t test. (D) Western blots showing reduced PER-CLK binding in ogt overexpressor flies at ZT18 but not at ZT22 as compared to control flies. (E) Bar graph showing quantification of CLK immunoprecipitated by α-PER (GP5620) (n = 2). Error bars = SEM. **P = 0.01; two tailed Student’s t test.