RNAseq of Polycomb KD ISCs

Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single cell RNA-seq to explore stem cell intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified ISCs and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis. Overall design: For polycomb (Pc)-RNAi experiments, virgins of the ISCts fly line were crossed with cherry-RNAi or Pc-RNAi (BL36070) males and maintained at 18 °C. Progeny was collected and shifted to 29°C 4-7 days after eclosion. Flies were kept at 29 °C for 8-11 days at which point they were dissected and ISCs were isolated. In short, midguts were dissected in 1xPBS, 1% Bovine Serum Albumin (BSA) and dissociated in 0.5% Trypsin-EDTA solution for less than 2h at room temperature (RT), during which dissociated cells were collected periodically every 20-30min, re537 suspended in 1xPBS, 1%BSA and 2%FBS and kept on ice until sorting. A BD Biosciences FACSAria II flow cytometer cell sorter was used for cell sorting.

组织稳态（tissue homeostasis）需要体细胞干细胞的长期谱系保真度。体细胞干细胞的年龄相关变化是否以及如何影响谱系决策的忠实执行，目前尚不清楚。本研究采用全基因组染色质开放性分析、转录组分析以及单细胞RNA测序（single cell RNA-seq），探究衰老果蝇肠道内干细胞的固有变化，以解答上述问题。研究结果显示，老年果蝇的干细胞中，多梳蛋白（Polycomb，Pc）靶基因的启动子区域出现差异开放，最终导致肠内分泌（enteroendocrine，EE）细胞特化相关基因的表达上调。与此一致的是，我们观察到衰老肠道内肠内分泌细胞祖细胞群的组成发生年龄相关性变化，同时肠内分泌细胞特化的肠道干细胞（intestinal stem cells，ISCs）及其祖细胞的比例在衰老果蝇中显著升高。本研究进一步证实，多梳蛋白介导的染色质调控是果蝇肠道内肠内分泌细胞特化的关键决定因素：多梳蛋白对于维持干细胞基因的表达，同时抑制分化与特化相关基因的表达至关重要。本研究结果表明，多梳蛋白家族是肠道上皮细胞谱系身份的核心调控因子，并揭示了染色质调控的年龄相关性衰退对组织稳态的影响。

实验设计：针对多梳蛋白（Polycomb，Pc）RNA干扰（RNAi）实验，将肠道干细胞温度敏感型果蝇品系（ISCts）的处女蝇与樱桃蛋白RNA干扰（cherry-RNAi）或Pc-RNAi（BL36070）雄蝇进行杂交，于18℃条件下培养。羽化后4-7天收集子代果蝇，并转移至29℃环境中培养8-11天，随后对果蝇进行解剖并分离肠道干细胞。简言之，在含1×磷酸盐缓冲液（1×PBS）、1%牛血清白蛋白（Bovine Serum Albumin，BSA）的溶液中解剖中肠，随后将其置于0.5%胰蛋白酶-乙二胺四乙酸（Trypsin-EDTA）溶液中于室温（room temperature，RT）下解离不足2小时，期间每隔20-30分钟收集一次解离后的细胞，重悬于含1×PBS、1%BSA及2%胎牛血清（Fetal Bovine Serum，FBS）的溶液中并置于冰上以待分选。使用BD Biosciences FACSAria II流式细胞仪进行细胞分选。