Characterizing age-dependent changes in histone methylation and determine the role of Trr and Trx in histone methylation in adult fly photoreceptors
Histone modifications are important for DNA-templated activities, such as transcription. In the first part of this study, we profiled H3K4me2/3, H3K36me3. H3K9me3 and H3K27me3in young (D10) and old (D40) fly photoreceptors using CUT&RUN to characterize global changes in histone methylation during aging. Our data show that both active and repressive marks exhibit consistently decreased levels in photoreceptors cells of old flies compared to young flies. This global decrease in histone methylation suggests an overall reduction in methylation potential in old photoreceptors. In the second part of the same manuscript, we expressed shRNA against mCherry (control), Trr and Trx, two H3K4 methyltransferases, and profile H3K4me1, H3K4me2, and H3K4me3 using CUT&RUN. Our data show that the knockdown of Trr and Trx leads to pronounced decrease in H3K4me3 levels and modest decrease in H3K4me2 and H3K4me1 in adult photoreceptors. Overall design: Drosophila were maintained on standard fly food at 25°C under 12-h:12-h light:dark conditions. Male flies were collected at indicated age (days post-eclosion, D) ± 1 day and ZT12 ± 15 minutes. For D10/D40 CUT&RUN, Rh1-Gal4>UAS-GFPKASH flies that express GFP on the photoreceptor nuclei membrane were collected on either D10 or D40 in quadruplicates, 400 flies per replicate. Photoreceptor nuclei were immuno-enriched (NIE) by anti-GFP coated magnetic beads (Jauregui-Lozano et al., 2021). CUT&RUN was performed on bead-bound photoreceptor nuclei as described in (Meers et al., 2019). Antibodies used are: anti-H3K4me2/3 (Abcam #ab8580), anti-H3K36me3 (Abcam #ab9050), anti-H3K9me3 (Abcam #ab176916) and anti-H3K27me3 (Epicypher #13-0055) and anti-IgG negative control (Epicypher #13-0042). pAG-MNase activation/ cleavage was performed for 20 minutes upon the addition of CaCl2.  For knockdown CUT&RUN, Rh1-Gal4>UAS-GFPKASH were crossed to either control (mCherry) RNAi, Trr RNAi (BDSC #36916), or Trx RNAi (BDSC #33703) and progenies were collected on D10 in triplicates for CUT&RUN. Antibodies used are: anti-H3K4me1 (Abcam #ab8895), anti-H3K4me2 Thermo Fisher #710796), anti-H3K4me3 (EpiCypher #13-0041) and anti-IgG negative control (Epicypher #13-0042). pAG-MNase activation/ cleavage was performed for 30 minutes. CUT&RUN libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® kit. Detailed protocols for NIE-based CUT&RUN are available: dx.doi.org/10.17504/protocols.io.kxygxp1d4l8j/v2.