TOC1 phosphorylation disproportionally enhances chromatin binding at rhythmic gene promoters

Area of Science:

• Plant molecular biology
• Circadian biology
• Epigenetics

Background:

• Protein phosphorylation is a critical regulator of plant circadian systems.
• TIMING OF CAB EXPRESSION 1 (TOC1) is a central transcriptional repressor in the plant circadian clock.
• TOC1 is phosphorylated near its N terminus, influencing its function.

Purpose of the Study:

• To investigate the role of TOC1 phosphorylation in regulating circadian gene expression.
• To determine how TOC1 phosphorylation affects its interactions with other proteins at specific gene promoters.
• To elucidate the impact of TOC1 phosphorylation on chromatin presence and rhythmic gene expression genome-wide.

Main Methods:

• Phenotype testing of TOC1 phosphosite mutants.
• Analysis of TOC1 interactions with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and PHYTOCHROME INTERACTING FACTOR 5 (PIF5) at the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter.
• Genome-wide analysis of TOC1 phosphostate effects on chromatin presence and gene expression.

Main Results:

• TOC1 phosphorylation, particularly at S175, is essential for optimal interaction with FHY3 and PIF5 to repress CCA1 expression.
• TOC1 is required for LATE ELONGATED HYPOCOTYL (LHY) expression, but this regulation is independent of TOC1 phosphorylation.
• TOC1 phosphorylation-dependent interactions selectively and acutely regulate clock gene promoters compared to nonrhythmic genes.
• Genome-wide analysis confirmed TOC1 phosphostate importance for chromatin binding and robust rhythmic gene expression.

Conclusions:

• TOC1 phosphorylation is a key regulatory event for optimal plant circadian clock function.
• Specific phosphorylation sites on TOC1 mediate distinct regulatory mechanisms for different clock genes.
• Phosphorylation fine-tunes TOC1's role in chromatin association and rhythmic gene expression, ensuring circadian robustness.