TOC1 phosphorylation disproportionally enhances chromatin binding at rhythmic gene promoters

Protein phosphorylation is a key regulatory mechanism in circadian systems. TIMING OF CAB EXPRESSION 1 (TOC1) is a core transcriptional repressor in the plant circadian system that is phosphorylated near its N terminus. Phenotype testing of TOC1 phosphosite mutants shows incomplete rescue of the short period toc1 mutant. We establish that TOC1 phosphorylation (particularly at S175) is necessary for optimal interaction with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and PHYTOCHROME INTERACTING FACTOR 5 (PIF5) at the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter to down-regulate CCA1 expression. Expression of the closely related LATE ELONGATED HYPOCOTYL (LHY) also requires TOC1 but is independent of TOC1 phosphorylation, suggesting different TOC1-dependent gene repression mechanisms. We additionally show that TOC1 phosphorylation-dependent interactions at specific clock gene promoters selectively regulate these circadian system components more acutely than nonrhythmic genes. Our genome-wide analysis demonstrates that the TOC1 phosphostate is important for optimal chromatin presence and robust rhythmic gene expression.