The one-carbon metabolism enzyme Ahcy is a redox sensor that modulates gene expression to protect against light stress-induced retinal degeneration

One-carbon metabolism influences the response to oxidative stress through antioxidant biosynthesis and the transcriptional status of the cell via epigenetic mechanisms. One-carbon metabolism is responsible for the production of the universal methyl donor, S-adenosylmethionine (SAM), which generates the potent methyltransferase inhibitor, S-adenosylhomocysteine (SAH). The essential metabolic enzyme, S-adenosylhomocysteinase (AHCY), functions within one-carbon metabolism and is the sole enzyme to catabolize the metabolite S-adenosylhomocysteine (SAH). Under oxidative stress conditions, AHCY becomes oxidized at a critical residue for its activity correlating with increased SAH abundance in the Drosophila head. Here, we profiled the photoreceptor transcriptome at a single time point after 3-hour blue light exposure relative to untreated controls in the Drosophila eye. We used RNAi expressed in all cells of the eye under longGMR-Gal4 to knockdown Ahcy and examined photoreceptor cells enriched using nuclei-immunoenrichment. As a control, we expressed non-specific RNAi against mCherry. To knockdown Ahcy in the Drosophila eye, we crossed longGMR-Gal4, Rh1-KASHGFP flies to UAS-RNAi flies targeting either Ahcy (y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC03222}attP40/CyO, Bloomington Stock #51477) or mCherry (control) in a cnbw background. We then collected male progeny, which were raised in 12-hour light: 12-hour dark conditions at 25C until 5 days post-eclosion. Flies treated with blue light were secured in a blacked-out box with strip LED lights surrounding the samples and were exposed to continuous blue light for three hours relative to untreated controls. 150 male flies were collected per sample (3 replicates). We affinity enriched photoreceptor nuclei for DNA isolation as previously described (Jauregui-Lozano et al., 2021, Genetics). CUT&RUN was performed on bead-bound nuclei as described in (Meers et al. 2019) using pAG-MNase fusion protein (Addgene plasmid #123461) purified using the Pierce cobalt kit (ThermoFisher, PierceTM His Protein Interaction Pull-Down Kit, Cat. #21227). The following antibodies were used for CUT&RUN with a 20-minute pAG-MNase activation time: anti-H3K4me3 (EpiCypher, 13-0060), anti-H3K27me3 (EpiCypher, 13-0055) and IgG (EpiCypher, 13-0042). CUT&RUN libraries were prepared using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina®. Libraries were sequenced on the Illumina NovaSeq X Plus platform. We removed three of samples (mCherryRNAi untreated H3K4me3 replicate 2, mCherryRNAi untreated H3K27me3 replicate 1, and AhcyRNAi untreated H3K4me3 replicate 2) after preliminary analysis due to poor data quality. Pair-ended reads were processed similarly as RNA-seq data, except we used bowtie2 (Langmead and Salzberg 2012) for alignment with the dovetail argument. Data is shown as CPM-normalized and subtracted for respective IgG control. Metaplots were generated using deepTools (Ramírez et al. 2016) based on custom GTF files represented unique used transcripts.