Profiling of RNA Pol II occupancy in Drosophila neural stem cell populations using TaDa (Targeted DamID). Drosophila melanogaster

Cell-type specific transcriptional profiling is key to understanding cell fate specification and function. In order to achieve this it has been necessary, to date, to isolate specific cell types from complex tissues. We have developed 'TaDa', a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation or affinity purification. The method is simple, sensitive, highly reproducible and is in principle transferable to any model system. Here we show that TaDa can be used to identify transcribed genes in a cell-type specific manner. We profile the genome-wide binding of RNA polymerase II (Pol II) in adjacent, clonally related neural stem cells in intact Drosophila brains. Our data reveal the activity of non-canonical metabolic pathways in proliferating neuroepithelial cells, and highlight a possible role for the retinal determination gene regulatory network in patterning neural stem cell fates. We also identify temporal differences in the activity of signalling pathways that control neuroepithelial cell fate by profiling Pol II occupancy at two different stages of brain development. Using RNA Pol II TaDa to profile, in a cell-type specific manner, the transcriptional state of neuroepithelial cells at two stages of larval brain development. Closely related asymmetrically dividing neural stem cells (neuroblasts) were also profiled, in order to compare the transcriptomes of two different types of neural stem cells. Overall design: 3 biological relicates were performed for 3rd instar neuroepithelial cells (with one dye-swap). 2 biological relicates were performed for 3rd instar neuroblasts (with dye-swap). 2 biological relicates were performed for 1st instar neuroepithelial cells (with dye-swap). As additional supporting evidence for the Pol II TaDa technique, 2 biological relicates were performed for 3rd instar salivary glands (with dye-swap) in order to compare with previous Pol II-ChIP data for this tissue [PMID 22821985].

细胞类型特异性转录组分析是解析细胞命运特化与功能的核心手段。迄今为止，要达成此类研究目标，需从复杂组织中分离特定细胞类型。我们开发了TaDa技术，可无需细胞分离即可实现细胞特异性分析。TaDa无需细胞分选、固定或亲和纯化，即可完成DNA或染色质结合蛋白的全基因组分析。该方法操作简便、灵敏度优异、重复性极佳，理论上可推广至任意模型系统。本研究证实，TaDa可通过细胞类型特异性的方式鉴定转录基因。我们对完整果蝇大脑中相邻的克隆相关神经干细胞的RNA聚合酶II（Pol II）全基因组结合谱进行了分析。研究数据揭示了增殖性神经上皮细胞中非经典代谢通路的活性，并突显了视网膜决定基因调控网络在塑造神经干细胞命运中可能发挥的作用。此外，我们通过分析大脑发育两个不同阶段的Pol II结合占据情况，鉴定出调控神经上皮细胞命运的信号通路活性存在时序差异。本研究利用RNA Pol II TaDa技术，以细胞类型特异性的方式分析了幼虫大脑发育两个阶段神经上皮细胞的转录状态。我们同时对密切相关的不对称分裂神经干细胞（成神经细胞，neuroblasts）进行了分析，以比较两种不同类型神经干细胞的转录组。实验整体设计如下：针对三龄期神经上皮细胞开展3次生物学重复（含1次染料交换实验）；针对三龄期成神经细胞开展2次生物学重复（含染料交换实验）；针对一龄期神经上皮细胞开展2次生物学重复（含染料交换实验）。作为Pol II TaDa技术的额外验证证据，我们针对三龄期唾液腺开展2次生物学重复（含染料交换实验），以便与该组织已发表的Pol II染色质免疫沉淀（ChIP）数据进行对比[PMID 22821985]。