mini-INTACT reveals social isolation-induced epigenetic changes in Drosophila dopaminergic neurons

We introduce a more sensitive method for purifying specific cell-types from small numbers of fly heads for epigenomic analysis: a method we call 'mini-INTACT'. We modified the INTACT method in Drosophila [Henry G. L. et al., ?Nucleic Acids Research, 2012, 1–14] to enable purification of genetically tagged rare cell types from Drosophila brain requiring ~50-100 fold less material. We used male fruit flies as a model to examine the effects of social-isolation vs. social-enrichment on the epigenome and transcriptome of dopaminergic neurons defined by expression of the TH-GAL4 driver. Using ChIP-seq on mini-INTACT purified nuclei, RNA-seq and bioinformatic analysis, we identify regulation of the transcriptome and behavior by Activity Related Genes who's expression is induced by social enrichment. Overall design: Genome wide occupancy profiling for histone modification marks by high throughput sequencing. Two or three biological replicates were used for each condition. Corresponding input DNA was used as control (ChIP-seq). Expression profiling by high throughput sequencing on FACS purified dopaminergic neurons isolated from group-housed and single-housed fruit fly adult male brains. Three biological replicates were used for each condition (RNA-seq).

本研究开发了一种灵敏度更高的方法，可从少量果蝇头部中纯化特定细胞类型以用于表观基因组学分析，我们将该方法命名为‘mini-INTACT’。本研究对果蝇中的INTACT方法[Henry G. L.等，《核酸研究》，2012，1–14]进行了改良，使其可从果蝇大脑中纯化经遗传标记的稀有细胞类型，所需实验材料量仅为原方法的1/50~1/100。本研究以雄性果蝇为模型，探究了社会隔离与社会富集对由TH-GAL4驱动元件标记的多巴胺能神经元的表观组与转录组的影响。本研究通过对mini-INTACT纯化的细胞核进行ChIP测序（ChIP-seq）、RNA测序（RNA-seq）及生物信息学分析，鉴定出受社会富集诱导表达的活动相关基因（Activity Related Genes）对转录组与行为的调控作用。实验整体设计：通过高通量测序对组蛋白修饰标记进行全基因组结合位点分析。每组实验设置2或3次生物学重复，对应的输入DNA作为ChIP测序的对照样本。通过高通量测序对从群居饲养与单独饲养的成年雄性果蝇大脑中分离的、经荧光激活细胞分选术（FACS）纯化的多巴胺能神经元进行表达谱分析，每组实验设置3次生物学重复（RNA-seq）。