Identification of CYCLE targets that contribute diverse features of circadian rhythms in the mosquito Culex pipiens.

Culex pipiens demonstrates robust circadian rhythms in adult eclosion, flight activity, mating, and development. These rhythmic patterns are believed to be controlled by the endogenous light-entrainable circadian clock that consists of positive and negative regulators working in a transcription-translation feedback loop. Moreover, these mosquitoes undergo seasonal diapause in exposure to the short photoperiod of late summer or early fall. However, the exact genetic and cellular mechanism behind the clock gene-mediated activity pattern, seasonal time measurement, and subsequent diapause initiation still need to be unraveled. To determine the possible linkage between clock genes and downstream processes, here we employed ChIP-sequencing to identify the direct targets of one of the core clock proteins, Cycle (CYC). The nearest genes with peaks mapping to their 1Kb upstream region of the transcription start site were extracted and scanned for consensus E box sequences, resulting in a dataset comprising the target genes possibly regulated by CYC. Based on the highest fold enrichment and functional relevance, we identified genes relating to five gene categories of potential interest, including peptide/receptors, neurotransmission, olfaction, immunity, and reproductive growth. Of these, we validated fourteen genes with ChIP-qPCR and qRT-PCR. These genes showed a significantly high expression in dusk compared to dawn in concert with the activity level of the CYC transcription factor and are thus strong candidates for mediating circadian rhythmicity and possibly regulating seasonal shifts in mosquito reproductive activity.