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.

组蛋白修饰对于以DNA为模板的生物学活动（如转录）具有关键调控作用。本研究第一部分采用CUT&RUN技术（CUT&RUN），对年轻（羽化后第10天，D10）与衰老（羽化后第40天，D40）果蝇的光感受器细胞中的H3K4me2/3、H3K36me3、H3K9me3及H3K27me3进行图谱分析，以解析衰老过程中组蛋白甲基化的全局变化特征。实验数据显示，与年轻果蝇相比，衰老果蝇光感受器细胞中的激活型与抑制型组蛋白修饰标记均呈现持续下调的趋势。这种组蛋白甲基化的全局下调现象，提示衰老果蝇光感受器细胞的甲基化潜能整体降低。

本研究第二部分中，我们针对对照（靶向mCherry的shRNA）以及两种H3K4甲基转移酶Trr和Trx分别构建了shRNA干扰体系，并采用CUT&RUN技术对成年果蝇光感受器细胞中的H3K4me1、H3K4me2及H3K4me3进行图谱分析。实验数据表明，在成年果蝇光感受器细胞中，敲低Trr与Trx的表达会显著降低H3K4me3的水平，并小幅下调H3K4me2与H3K4me1的表达量。

### 整体实验设计
果蝇饲养于标准果蝇培养基中，培养条件为25℃、12小时光照:12小时黑暗。按设定日龄（羽化后天数，D）±1天，且在ZT12±15分钟时段收集雄性果蝇。

#### 衰老组（D10/D40）CUT&RUN实验
我们收集了在光感受器细胞核膜上表达GFP的Rh1-Gal4>UAS-GFPKASH果蝇，分别在D10和D40两个时间点取样，设置4个生物学重复，每个重复包含400只果蝇。通过抗GFP包被的磁珠对光感受器细胞核进行免疫富集（NIE）（Jauregui-Lozano等，2021）。磁珠结合的光感受器细胞核的CUT&RUN实验操作参照Meers等（2019）的方法进行。本部分实验使用的抗体包括：抗H3K4me2/3抗体（Abcam #ab8580）、抗H3K36me3抗体（Abcam #ab9050）、抗H3K9me3抗体（Abcam #ab176916）、抗H3K27me3抗体（Epicypher #13-0055）以及抗IgG阴性对照抗体（Epicypher #13-0042）。加入CaCl₂后，pAG-MNase的激活与切割反应持续20分钟。

#### 基因敲低组CUT&RUN实验
我们将Rh1-Gal4>UAS-GFPKASH果蝇分别与对照（mCherry）RNAi、Trr RNAi（BDSC #36916）以及Trx RNAi（BDSC #33703）果蝇进行杂交，在羽化后第10天收集子代果蝇，设置3个生物学重复用于后续CUT&RUN实验。本部分实验使用的抗体包括：抗H3K4me1抗体（Abcam #ab8895）、抗H3K4me2抗体（Thermo Fisher #710796）、抗H3K4me3抗体（EpiCypher #13-0041）以及抗IgG阴性对照抗体（Epicypher #13-0042）。加入CaCl₂后，pAG-MNase的激活与切割反应持续30分钟。

CUT&RUN文库构建采用NEBNext® Ultra™ II DNA文库制备试剂盒（适配Illumina®测序平台）。基于免疫富集的CUT&RUN详细实验流程可访问：dx.doi.org/10.17504/protocols.io.kxygxp1d4l8j/v2 获取。