molecular mechanisms controlling the diurnal oscillation of r-loop formation in arabidopsis thaliana

Abstract

The circadian clock is an endogenous timing mechanism that synchronizes biological processes with the 24-hour day and night cycles. Proper circadian function is essential for the rhythmic regulation of fundamental processes in plants, including among others, growth, development, and responses to stress. At its bases, the circadian function relies on the precise regulation of the rhythmic transcription of core oscillator genes. Active transcription also generates in many organisms the so-called R-loops, three-stranded nucleic acid structures composed of the DNA-RNA hybrid and the non-template single-stranded DNA. Based on the correlation of transcription with both the circadian system and R-loops, in this Doctoral Thesis, we aimed to uncover the potential diurnal regulation of R-loops and identify the molecular components and mechanisms underlying R-loop homeostasis in Arabidopsis thaliana. By using dot-blot assays, we found that indeed R-loops accumulated in a diurnal fashion with a significant enrichment during the night compared to the day. The photoreceptors (PHYTOCHROME B and CRYPTOCHROME 1 and 2), as well as morning- and evening-expressed clock components differentially contributed to the diurnal accumulation of R-loops, highlighting the intricate interplay between light signaling and the circadian clock in the control of R-loop homeostasis. R-loops also positively correlated with active transcription as assayed by RNA Polymerase II accumulation, pharmacological inhibition of transcription, chromatin compaction, and analyses of transcription-related mutant plants. We found that the component of the FACT (FAcilitates Chromatin Transcription) complex, SSRP1 (structure-specific recognition protein 1) also contributed to proper RNA Polymerase II accumulation, chromatin compaction, abundance of histone marks, and responses to DNA damage. SSRP1 was also responsible for the diurnal regulation of R-loops by reducing their accumulation predominantly during the day. Altogether, our studies establish a direct connection of SSRP1 function with transcription and the diurnal accumulation of R-loops. Our results open up possible new avenues for the manipulation of transcription and circadian regulation.

Date of Award	24 Jul 2024
Original language	English
Supervisor	Paloma Mas Martinez (Director)