TRITHORAX-dependent arginine methylation of HSP68 mediates circadian repression by PERIOD in the monarch butterfly

Transcriptional repression drives feedback loops that are central to the generation of circadian (∼24-h) rhythms. In mammals, circadian repression of circadian locomotor output cycles kaput, and brain and muscle ARNT-like 1 (CLOCK:BMAL1)-mediated transcription is provided by a complex formed by PERIOD (PER) and CRYPTOCHROME (CRY) proteins. PER initiates transcriptional repression by binding CLK:BMAL1, which ultimately results in their removal from DNA. Although PER's ability to repress transcription is widely recognized, how PER binding triggers repression by removing CLK:BMAL1 from DNA is not known. Here, we use the monarch butterfly as a model system to address this problem because it harbors a simplified version of the CLK:BMAL1-activated circadian clock present in mammals. We report that an intact CLOCK mouse exon 19 homologous region (CLKe19r) and the histone methyltransferase TRITHORAX (TRX) are both necessary for monarch CLK:BMAL1-mediated transcriptional activation, CLK-PER interaction, and PER repression. Our results show that TRX catalytic activity is essential for CLK-PER interaction and PER repression via the methylation of a single arginine methylation site (R45) on heat shock protein 68 (HSP68). Our study reveals TRX and HSP68 as essential links between circadian activation and PER-mediated repression and suggests a potential conserved clock function for HSPs in eukaryotes.