Proteomic Insights into Circadian Transcription Regulation: Novel E-box Interactors Revealed by Proximity Labeling

Abstract Circadian clocks (∼24h) are responsible for daily physiological, metabolic and behavioral changes. Central to these oscillations is the regulation of gene transcription. Previous research has identified clock protein complexes that interact with the transcriptional machinery to orchestrate circadian transcription, but technological constraints have limited the identification of de novo proteins. Here we use a novel genomic locus-specific quantitative proteomics approach to provide a new perspective on time-of-day-dependent protein binding at a critical chromatin locus involved in circadian transcription - the E-box. Using proximity labeling proteomics at the E-box of the clock-controlled Dbp gene in mouse fibroblasts, we identified 69 proteins at this locus at the time of BMAL1 binding. This method successfully enriched BMAL1 as well as HDAC3 and HISTONE H2A.V/Z, known circadian regulators. New E-box proteins include the MINK1 kinase and the XPO7 transporter, whose depletion in human U-2 OS cells results in disrupted circadian rhythms suggesting a role in the circadian transcriptional machinery. Overall, our approach uncovers novel circadian modulators and provides a new strategy to obtain a complete temporal picture of circadian transcriptional regulation.