Computational protocol: Mining for novel candidate clock genes in the circadian regulatory network

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Protocol publication

[…] The rhythm generation in the circadian clock is a result of a TTFL, as described earlier. Within the TTFL, clock genes acting as transcription factors mutually regulate each other. Thus, it is likely that putative clock genes are transcriptionally regulated by known clock transcription factors (TFs), such as BMAL1, CLOCK, NPAS2, E4BP4, ROR α, REV-ERB α and REV-ERB β. Therefore, we searched for ChIP-seq binding sites for each clock TF in the vicinity of the genes in the master list. Moreover, when time-resolved binding of the TF factor was measured in a study, we restricted our attention further to only those ChIP-seq binding sites that show circadian (cycling) binding of the TF.Koike et al. [] provide time-resolved genome-wide ChIP-seq binding sampled every four hours over one day in mouse liver for the TFs BMAL1, CLOCK, NPAS, CRY1, CRY2, PER1 and PER2 (the list of ChIP-seq peaks and associated genes for each TF were provided in Table S2 of []). It is worth noting that PERs and CRYs do not possess DNA binding domains, but we still retain the data as we believe that they might represent regulation by a complex composed of these important circadian proteins. We used the false discovery rates (FDR) for circadian binding from the original study to filter genes with circadian TF binding using a cutoff of 0.05.Rey et al. [] similarly performed a detailed study of the genome-wide binding of activator BMAL1 in the mouse liver sampled every four hours over one day (the list of ChIP-seq peaks and associated genes for each TF were provided in Text S2 of []). As before, we restricted our choice to gene with circadian BMAL1 binding with a FDR<0.05, based on a Fisher test for periodicity as suggested by the authors of the study.Cho et al. [] compared the cistromes of both isoforms of the nuclear receptor repressor REV-ERB (α and β) at a single time point (Zeitgeber time (ZT) 8) and against the cistrome of BMAL1. The raw ChIP-seq (data accessible at NCBI GEO database [–], accession GSE34019) was processed using the TFTargetCaller [settings: Ouyang, TFAS and closestGene methods] package in R [] to produce an annotated list of ChIP-seq peaks. Although they discovered significant overlap between the binding sites of both isoforms, we retained the genes proximal to binding sites of each isoform, separately.Bugge et al. [] and Feng et al. [], in related studies, investigated the role of nuclear-receptor repressors REV-ERB α and REV-ERB β in metabolism in the liver. They measured binding using ChIP-seq of both TFs at two time points, 12 hours apart. As before, we processed the raw ChIP-seq data (NCBI GEO database [], accession GSE36375 and GSE26345, respectively) using the TFTargetCaller [settings: Ouyang, TFAS and closestGene methods] package in R [] to produce an annotated list of ChIP-seq peaks. Since circadian binding cannot be reliably determined from two time points, we pooled binding sites from both time points for both TFs.Fang et al. [], recently, measured binding of the D-box repressor E4BP4 (NFIL3) and nuclear receptor activator (ROR α) in the mouse liver at ZT22 as part of a study on identifying functional circadian ChIP-seq binding sites. We processed their raw data (data accessible at NCBI GEO database [], accession GSE59486) using HOMER (v4.7.2) [] for peak-calling and assigning peaks to the nearest gene. […]

Pipeline specifications

Software tools Cistrome, HOMER
Application ChIP-seq analysis
Diseases Neoplasms, Peripheral Nervous System Diseases