Identifying the impact of H3K4 methylation on the circadian clock in aging Drosophila photoreceptors

To synchronize with environmental stimuli, predominantly light, the circadian clock directs the physiological and behavioral cycles with a periodicity of approximately 24 hours. The circadian clock is progressively dysregulated during aging, correlating with the development of many neurodegenerative diseases. Circadian disruption in the eye affects retinal development and accelerates photoreceptor degeneration during aging, which implies a protective effect of the functional circadian clock on photoreceptor neurons. At the core of the molecular clock, Clock (CLK) and Cycle (CYC), a pair of transcription activators, regulate the rhythmic transcription of output genes, including a substantial fraction of phototransduction genes. In the aging photoreceptors, CLK:CYC exhibit differential activity while the mechanisms that contribute to this change and its impact on downstream gene expression are poorly understood. H3K4me3 is a ubiquitous chromatin modification that is present at actively transcribed gene promoters. By profiling nuclear transcriptome throughout the 24h day, we identified that knockdown of any of the H3K4 methyltransferase significantly alter the rhythmic gene expression, and collectively, H3K4 methyltransferases (Set1, Trr and Trx) contribute to 90% of the rhythmic transcriptome in Drosophila photoreceptors. In addition, all of the core clock genes showed decreased expression amplitude in any of the KD conditions, indicating H3K4 methylation elevates clock-controlled transcription activation. Given that a global decrease of H3K4me3 is observed in the aging eye, our study favors the scenario where decreased H3K4me3 levels during aging reduce clock-controlled transcription. Overall design: To knockdown H3K4 methyltransferase in Drosophila photoreceptors, we cross Rh1-Gal4 flies with UAS-RNAi lines, and use F1 progenies for RNA-seq. mCherry knockdown is used as a negative control. F1 flies are raised in 12-hour light: 12-hour dark incubators to Day 10. 400 males are collected every 4 hours spanning the 24- hour day (from ZT0 to ZT20) with a replicate of three each time point. Photoreceptor cells are extracted using nuclear-immuno enrichment (NIE) technique developed in our lab (Jauregui-Lozano et al., 2021, Genetics). Nuclear RNA is purified using ZYMO Direct-zol RNA Microprep Kits (#R2061). RNA library is prepared using Ovation® SoLo RNA-Seq System (#0502-32). mCherry and Set1 KD cDNA libraries are split into 3 laned and sequenced on HiSeq PE150. Trr and Trx KD cDNA libraries are sequenced using NovaSeq X Plus PE150.