Roughly half of the eukaryotic genome is under the control of a circadian clock at the level of transcript abundance, with rhythmic transcripts peaking at all possible phases of the day. However, the mechanism for phase regulation is not well understood. To determine how phase is regulated in Neurospora crassa, ChIP-seq was performed on the morning active transcription factor (TF) and core oscillator component, the White Collar Complex (WCC). The direct targets of WCC were found to be enriched for TFs. Further investigation of these first-tiered TFs revealed that they form a large and complex network made up of multiple feedback and feedforward loops. We hypothesized that this intricate TF network plays a role in phase regulation. To test this hypothesis, we focused our experiments on the network surrounding the first-tier TF, ADV-1. In preliminary data, we found that TF knockouts of CSP-1, CSP-2, SUB-1, and ZNF-21, altered the phase and/or period of ADV-1 protein rhythms. To confirm that these 4 TF knockouts, and not second site mutations, cause the changes in ADV-1 rhythms, genetic complementation will be performed. If the TF knockout causes the defect, then supplying the wild type gene back should rescue the ADV-1 rhythm back to wild type. This information will help us to validate which TFs in the network are necessary for generating proper phase control of ADV-1 protein rhythms
