ATACseq of Polycomb KD ISCs [NGS2975]. ATACseq of Polycomb KD ISCs [NGS2975]

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 Pc-RNAi experiments, virgins of the ISC ts 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 sorted by Fluorescent Activated Cell Sorting (FACS) as we have previously described (Tauc et al., 2014). In short, midguts from flies of WDah; esg-Gal4, UAS-2xEYFP; Su(H)GBE-Gal80, 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, resuspended 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.

组织稳态依赖于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保障谱系决策的忠实执行，目前仍未得到充分阐明。本研究通过全基因组染色质可及性分析、转录组分析及单细胞RNA测序（single cell RNA-seq），针对衰老果蝇肠道内的干细胞内在变化展开探究，以解答上述科学问题。

本研究结果显示，老年果蝇的干细胞中，多梳蛋白（Polycomb, Pc）靶基因的启动子区域染色质开放状态发生差异改变，进而上调肠内分泌（enteroendocrine, EE）细胞特化相关基因的表达。与之相符的是，我们观察到衰老果蝇肠道内肠内分泌祖细胞群体的组成发生年龄相关性变化，同时衰老果蝇中已特化为肠内分泌细胞的肠道干细胞（intestinal stem cell, ISC）及其祖细胞的比例显著升高。

我们进一步证实，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。多梳蛋白可在维持干细胞基因表达的同时，抑制分化及特化相关基因的表达。本研究结果确认多梳家族蛋白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RNA干扰（Pc-RNAi）实验，将肠道干细胞温度敏感型果蝇品系（ISC ts fly line）的处女蝇与樱桃RNA干扰（cherry-RNAi）或Pc-RNAi（BL36070）品系的雄蝇进行杂交，于18℃条件下培养。子代果蝇羽化后4~7天，转移至29℃环境继续培养8~11天，随后进行解剖。按照我们此前报道的方法（Tauc等，2014），通过荧光激活细胞分选（Fluorescent Activated Cell Sorting, FACS）分离肠道干细胞。具体操作如下：取WDah; esg-Gal4, UAS-2xEYFP; Su(H)GBE-Gal80品系果蝇的中肠，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，随后于室温（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；期间每隔20~30分钟收集一次解离细胞，将细胞重悬于含1×PBS、1%BSA及2%胎牛血清（FBS）的溶液中并置于冰上，直至分选。细胞分选采用碧迪生物科学（BD Biosciences）FACSAria II流式细胞分选仪完成。