RNAseq of Polycomb KD ISCs. 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）需要体细胞干细胞维持长期的谱系保真度。目前，体细胞干细胞的衰老相关变化是否以及如何影响谱系决策的忠实执行，仍不甚明确。本研究通过全基因组染色质开放程度分析（genome-wide chromatin accessibility）、转录组分析（transcriptome analysis）以及单细胞RNA测序（single cell RNA-seq），探究衰老果蝇（Drosophila）肠道内干细胞的内在变化，以此解答上述问题。

上述研究发现，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开放程度差异，导致肠内分泌（enteroendocrine, EE）细胞特化相关基因的表达上调。与之相符的是，我们观察到衰老肠道内肠内分泌祖细胞群的组成发生衰老相关变化，同时肠内分泌特化的肠道干细胞（intestinal stem cells, ISCs）及其祖细胞的比例在衰老果蝇中显著升高。

我们进一步证实，由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：多梳蛋白可在维持干细胞基因表达的同时，抑制分化与特化相关基因的表达。本研究结果表明，多梳家族蛋白是肠上皮（intestinal epithelium）内谱系同一性的核心调控因子，并揭示了染色质调控的衰老相关衰退对组织稳态的影响。

实验设计如下：在多梳蛋白（Polycomb, Pc）RNA干扰实验中，将ISCts果蝇品系的处女蝇与cherry-RNAi或Pc-RNAi（BL36070）雄蝇进行杂交，并在18℃下饲养。子代果蝇在羽化后4-7天被收集，并转移至29℃环境中饲养。果蝇在29℃下饲养8-11天后，进行解剖并分离肠道干细胞。

简言之，我们在1×PBS、1%牛血清白蛋白（bovine serum albumin, BSA）中解剖中肠，随后在室温（room temperature, RT）下用0.5%胰蛋白酶-EDTA溶液解离组织，解离时长不超过2小时；期间每20-30分钟定期收集解离细胞，重悬于1×PBS、1%BSA及2%胎牛血清（fetal bovine serum, FBS）中，并置于冰上直至分选。使用BD Biosciences FACSAria II流式细胞仪进行细胞分选。