The PER2:BRCA1:POU2F1(OCT-1) ternary complex represents a multi-component scaffold model for circadian gene regulation

Abstract

The circadian clock component PER2 coordinates daily oscillations in gene expression across multiple tissues, yet its role in assembling multi-protein regulatory complexes remains incompletely understood. Here, we report that PER2 nucleates a ternary complex with the tumor suppressor BRCA1 and the transcription factor POU2F1(OCT-1) to impose circadian control on target gene promoters. Using bacterial two-hybrid screening, we identified BRCA1 as a novel PER2-interacting protein. Biochemical mapping revealed that PER2 engages BRCA1 through multiple discrete binding interfaces: PER2 spanning residues 356–574 and 683–872 interact with both the N-terminal (1–400) and C-terminal BRCT (1670–1863) domains of BRCA1. Structural modeling predicted 361 residue contacts between PER2 and BRCA1, substantially more than the 74 contacts predicted for PER2:POU2F1(OCT-1), indicating differential affinities that enable ordered complex assembly. Sequential pull-down assays demonstrated that PER2, BRCA1, and POU domain form a stable ternary complex in vitro, with POU2F1(OCT-1) serving as the DNA-binding platform. Electrophoretic mobility shift assays revealed that pre-assembly of PER2 with POU domain inhibits DNA binding, while BRCA1 is essential for stabilizing PER2 recruitment to DNA-bound POU2F1(OCT-1). Using ESR1 as a functional readout, we demonstrated that this ternary complex directly regulates promoter activity. Circadian transcriptome analysis revealed that Esr1 exhibits robust clock-dependent oscillations that are abolished in Per1/2 double-knockout mice, while Pou2f1 and Brca1 maintain constitutive expression. These findings establish PER2 as a circadian scaffold that assembles multivalent protein complexes to temporally gate transcription, providing mechanistic insight into how circadian disruption can influence target gene expression.