Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification [RNA-seq]

Adult stem cells must coordinate transcriptional programs with external cues to maintain tissue homeostasis. In the Drosophila midgut, intestinal stem cells (ISCs) generate enterocytes (ECs) or enteroendocrine (EE) cells through Notch-dependent fate decisions, but how chromatin regulators influence this balance remains unclear. In this study we identified the Trithorax gene (trx) as a key factor that safeguards ISC lineage fidelity. Trx depletion biases ISCs toward EE differentiation without affecting proliferation, a phenotype exacerbated during aging or DSS-induced damage. Transcriptomic and chromatin profiling revealed the homeobox transcription factor Ptx1 as a Trx-dependent target. Ptx1 knockdown phenocopies Trx loss, whereas Ptx1 overexpression reverts trx-RNAi-induced EE overproduction, establishing Ptx1 as a critical mediator of Trx function. These findings support a model in which Trx constrains the scute–prospero axis through Ptx1-mediated repression, thereby limiting inappropriate EE specification and maintaining ISC plasticity. Overall design: To identify genes regulated by Trx, we performed RNA-seq on FACS-sorted adult GFP? progenitor cells from control (esgtsGFP x w1118) and trx RNAi (esgtsGFP x UAS-trxRNAi BDSC#33703) female midguts under homeostatic (sucrose-fed) and regenerative (DSS-treated) conditions. We then performed gene expression profiling analysis using RNA extracted with miRNeasy Micro Kit and libraries were prepared with the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen).

成体干细胞需协调转录程序与外部信号，以维持组织稳态。在果蝇中肠中，肠干细胞（intestinal stem cells, ISCs）通过Notch依赖的命运决策，分化为肠细胞（enterocytes, ECs）或肠内分泌（enteroendocrine, EE）细胞，但染色质调控因子如何影响这一分化平衡仍不明晰。本研究鉴定出三胸基因（Trithorax gene, trx）是维持ISCs谱系保真度的关键因子。敲低trx会使ISCs偏向EE细胞分化，且不影响细胞增殖，该表型在衰老或葡聚糖硫酸钠（Dextran Sulfate Sodium, DSS）诱导的肠道损伤中会进一步加剧。转录组与染色质谱分析显示，同源框转录因子Ptx1是trx的依赖型靶基因。敲低Ptx1会模拟trx缺失的表型，而过表达Ptx1则可逆转trx-RNAi诱导的EE细胞过度生成，由此确定Ptx1是trx功能的关键介导因子。上述研究结果支持如下模型：trx通过Ptx1介导的基因抑制，调控scute–prospero信号轴，从而限制异常的EE细胞特化，并维持ISCs的可塑性。实验整体设计：为鉴定trx调控的靶基因，本研究在稳态（蔗糖喂养）与再生（DSS处理）两种条件下，分别从对照组（esgtsGFP × w1118）与trx RNAi组（esgtsGFP × UAS-trxRNAi BDSC#33703）的雌性果蝇中肠中，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）获取成体GFP⁺祖细胞，随后进行RNA测序。实验所用RNA通过miRNeasy Micro Kit提取，文库构建采用QuantSeq 3' mRNA-Seq Library Prep Kit（Lexogen）。