RTF1 enhances CLK occupancy and histone methylation at key circadian clock pacemaker gene loci

Area of Science:

• Chronobiology
• Molecular Biology
• Genetics

Background:

• Circadian clocks govern daily physiological rhythms via transcriptional-translational feedback loops.
• CLOCK is a key transcription factor, but its regulation of clock genes involves coregulatory factors and histone modifications.

Purpose of the Study:

• To identify novel regulators of the circadian transcriptional machinery.
• To investigate the role of the Drosophila polymerase-associated factor 1 complex (Paf1C) in circadian clock function.

Main Methods:

• RNA interference (RNAi) to knock down Paf1C subunits in Drosophila pacemaker neurons.
• Locomotor activity assays to assess circadian period and amplitude.
• Western blotting and quantitative PCR to measure protein and gene expression.
• Chromatin immunoprecipitation (ChIP) to analyze protein occupancy and histone modifications.
• Co-immunoprecipitation to study protein interactions.
• Cell culture experiments in human U2OS cells.

Main Results:

• Knockdown of Paf1C subunits, particularly RTF1, in Drosophila pacemaker neurons extended the circadian locomotion period and dampened rhythm amplitude.
• RTF1 downregulation reduced PERIOD (PER) protein levels by decreasing per transcription.
• Overexpression of per rescued the circadian defects caused by RTF1 knockdown.
• RTF1 physically interacts with CLOCK, enhancing its occupancy at per and other clock gene promoters.
• The SET1 methyltransferase complexes with CLK and RTF1, increasing H3K4me3 levels at the per/tim promoter.
• Human RTF1 interacts with BMAL1/CLOCK, suggesting conserved function in mammals.

Conclusions:

• RTF1 is a novel regulator of Drosophila circadian rhythms.
• RTF1 modulates circadian gene expression by enhancing CLOCK binding and promoting H3K4me3 modification at target promoters.
• The mechanism involving RTF1, CLOCK, and histone modification is potentially conserved in mammals.