ChIP-Seq of V5-Clk in wildtype and tim promoter mutant Drosophila heads around the clock

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E box region and an upstream E box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. To examine the effect of promoter E-box deletions on Clk binding, we performed Clk-ChIP around the clock in wild-type and 126 mutant flies. Overall design: ChIP-seq was performed on yw;; WT dCLK-V5(wt) andyw; tim_up126; dCLK-V5. 3-5 day old flies were entrained in LD condition for at least 3 days, then collected every 4 hours throughout the day.