Circadian regulation of stereotypic chromatin conformations at enhancers.

ABSTRACT

Cooperation between the circadian transcription factor (TF) CLOCKBMAL1 and other TFs at cis-regulatory elements (CREs) is critical to daily rhythms of transcription. Yet, the modalities of this cooperation are unclear. Here, we analyzed the co-binding of multiple TFs on single DNA molecules in mouse liver using single molecule footprinting (SMF). We found that SMF reads clustered in stereotypic chromatin states that reflect distinguishable organization of TFs and nucleosomes, and that were remarkably conserved between all samples. DNA protection at CLOCKBMAL1 binding motif (E-box) varied between CREs, from E-boxes being solely bound by CLOCKBMAL1 to situations where other TFs competed with CLOCKBMAL1 for E-box binding. SMF also uncovered CLOCKBMAL1 cooperative binding at E-boxes separated by 250 bp, which structurally altered the CLOCKBMAL1-DNA interface. Importantly, we discovered multiple nucleosomes with E-boxes at entry/exit sites that were removed upon CLOCKBMAL1 DNA binding, thereby promoting the formation of open chromatin states that facilitate DNA binding of other TFs and that were associated with rhythmic transcription. These results demonstrate the utility of SMF for studying how CLOCKBMAL1 and other TFs regulate stereotypical chromatin states at CREs to promote transcription.