set2-RNA-SEQ in circadian clock of drosophila melanogaster
Circadian timekeeping is driven by self-sustained transcription-translation feedback loops (TTFLs) that generate rhythmic gene expression. Although promoter-associated epigenetic regulation has been extensively studied, the roles of transcription elongation and elongation-coupled chromatin modifications in circadian timing remain poorly understood. Here, we identify the conserved histone H3 lysine 36 methyltransferase SET2 as a critical epigenetic regulator of the Drosophila circadian clock. An in vivo RNAi-based genetic screen revealed that Set2 depletion in central pacemaker neurons lengthens the free-running circadian period. Integrated transcriptomic and ATAC-seq analyses further demonstrate that SET2 is required to maintain chromatin accessibility and transcriptional programs associated with circadian output pathways. Mechanistically, SET2 mediates histone H3 lysine 36 trimethylation (H3K36me3) deposition at core clock loci and supports productive serine 2-phosphorylated RNA polymerase II (Ser2P-RNA Pol II) elongation. Loss of Set2 reduces the rhythmic transcriptional amplitude of core circadian repressor genes and impairs PERIOD (PER) protein accumulation. Consistent with this mechanism, targeted transgenic restoration of PER expression in pacemaker neurons rescues the period-lengthening phenotype caused by Set2 depletion. Importantly, this regulatory role is evolutionarily conserved in mammalian cells, where depletion of the Set2 ortholog SETD2 lengthens the circadian period and reduces the amplitude of PER2::LUC reporter oscillations. Together, these findings establish SET2/SETD2-dependent transcription elongation and H3K36me3-coupled chromatin remodeling as a conserved fundamental mechanism that ensures the precision and robustness of circadian timekeeping.