Regulation of ADV-1 Protein Rhythms by the Transcription Factor CSP-1 in Neurospora crassa

Abstract

The circadian clock regulates the rhythmic expression of roughly half of the eukaryotic genome at the level of mRNA abundance. However, little is known about how rhythmic phase is regulated (Delaunay & Laudet, 2002). Chromatin-immunoprecipitation (ChIP)-seq revealed a network of transcription factors (TFs) downstream of the core oscillator component, the White Collar Complex (WCC) (Smith, et al., 2010). We hypothesize this TF network is important in phase regulation of downstream clock-controlled genes (ccgs). To test this hypothesis, we investigated the role of a smaller TF network upstream of ADV-1, a direct TF target of the WCC, and assayed the effect of single TF deletions on ADV-1 rhythms. We found deletion of CSP-1, a TF within this network, resulted in an ~3 hr phase delay of ADV-1 protein rhythms, without affecting FRQ protein rhythmic accumulation. ChIP-seq also revealed two CSP-1 consensus binding motifs near the start of adv-1 transcription. If CSP-1 regulation of ADV-1 is through the aforementioned TF network, there will be little to no change from wildtype rhythms when the CSP-1 binding sites are deleted. However, if CSP-1 directly regulates ADV-1, then deletion of the binding sites will produce a phase delay similar to the single deletion of CSP-1. The binding site furthest upstream from the adv-1 start codon has been deleted and shown to not be solely responsible for ADV-1 phase regulation. However, the effect of the CSP-1 binding site nearest the adv-1 start remains to be deleted, and thus, the full nature of CSP-1 regulation of ADV-1 phase remains unknown.