Abstract
All animals have circadian rhythms which are driven by a core molecular clock. CLOCK (CLK) is one of the core clock components which binds to genes to activate transcription. In this study, we performed chromatin immunoprecipitation (ChIP) of CLK, and RNA extraction from both Drosophila heads and brains to see whether CLK binding is tissue specific and to examine the expression levels of the mRNA isoforms controlled by CLK. There are around 1,500 CLK direct targets discovered in Drosophila, but less than 5% of these genes produce cycling mRNAs. One possible explanation is that CLK could bind and drive the transcription on different genes in different tissues. To test this question, we tried to develop a chromatin IP assay from brains. Although we could generate fragmented chromatin of approximately the correct size, we were unable to detect a CLK ChIP signal above background. To circumvent this problem, we ablated the eyes using GMR-hid and performed whole head chromatin IPs. These experiments revealed potential tissue specific CLK transcriptional targets. To validate these and test the possibility of isoform specific CLK transcriptional activation, I isolated RNA from brains and heads and used qPCR to measure the expression levels of different isoforms in these tissues at six timepoints throughout the day. We then further modified the experiment by using GMR-hid CLK-V5 flies instead of brains to reduce the workload. We found that several genes that are direct targets in the eye are not only expressed in the eye but also in other tissues in the head.