SET2 mediated H3K36me3 couples chromatin regulation to transcription elongation in circadian period control
Circadian clocks depend on rhythmic transcription, but whether gene-body chromatin helps determine clock speed remains unclear. Here, an in vivo RNA interference screen in Drosophila identified the histone H3 lysine 36 methyltransferase SET2 as a regulator of circadian period. Set2 depletion in clock cells, including PDF-expressing pacemaker neurons, lengthened the free-running period without disrupting neuronal morphology. RNA sequencing and time-series RT-qPCR showed that Set2 loss preferentially reduced the transcriptional amplitude of the core clock repressors period, timeless and vrille. Consistently, PER and TIM protein accumulation was diminished in fly heads, and PER abundance was reduced in pacemaker neurons. Set2 depletion decreased H3K36me3 and elongation-associated Ser2-phosphorylated RNA polymerase II occupancy at core clock gene bodies, whereas expression of the H3.3K36M mutant phenocopied the period-lengthening effect. ATAC-seq further revealed time-dependent changes in chromatin accessibility at clock-associated loci. Restoring PER expression in PDF neurons rescued the long-period phenotype. Moreover, SETD2 depletion lengthened period and reduced PER2::LUC amplitude in human U2OS cells. These findings identify SET2-dependent H3K36me3 as a gene-body chromatin pathway that sustains circadian transcriptional amplitude and clock pace.