Non-O-GlcNAcylatable mutation at PER(S942) elevates PER repressor activity by increasing PER-CLK interaction and promoting CLK dissociation at circadian promoters.

(A) Western blots showing reciprocal coimmunoprecipitations (coIPs) to examine PER-CLK binding in wper0; p{per(WT)-HA10HIS} and wper0; p{per(S942A)-HA10HIS} flies at the indicated time-points. Flies were entrained for 2 days in 12h:12h LD cycles and collected on LD3. Protein extracts from fly heads were directly analyzed (input) or immunoprecipitated with α-HA to detect PER-HA or α-CLK. Immune complexes were then subjected to immunoblotting to detect bait or interacting proteins. (B) Bar graphs showing quantification of reciprocal coIPs to examine PER-CLK binding from three biological replicates. Values for binding of interacting proteins are normalized to amount of bait detected in the IPs. Error bars indicate ± SEM. *P (C) Western blots showing results of coIP to evaluate PER-CLK interaction in S2 cells expressing pAc-per(WT)-6Xcmyc or pAc-per(S942A)-6Xcmyc with pAc-clk-V5 in the presence or absence of pMT-ogt-3XFLAG. Protein extracts from S2 cells were either directly analyzed by immunoblotting (input) or subjected to immunoprecipitation with α-V5 or resin conjugated with a non-specific antibody (α-HA) as negative control (no signal detected). Immune complexes were analyzed by α-V5 to detect CLK-V5 protein (bait) or by α-cmyc to assess interaction with PER-cmyc. (D) Bar graph shows quantification of PER normalized to IP-V5 (bait protein, CLK) from four independent experiments. Error bars indicate ± SEM. *P-value = 0.05, **P-value (E) ChIP assays using fly head extracts showing reduced CLK occupancy at per and tim promoters in wper0; p{per(S942A)-HA10HIS} as compared to wper0; p{per(WT)-HA10HIS} flies. Error bars indicate ± SEM (n = 3). *P-value P-value < 0.01.