Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1a uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling ß-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the ß-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.

由于细胞信号通路存在多层调控机制，此类调控往往仅在从不同层面探测通路或清除关键节点时才会显现，因此对其开展全面理解仍是一项颇具挑战性的工作。为解析经典WNT信号通路（canonical WNT signaling）的调控机制，本研究依托报告基因筛选体系，在单倍体人类细胞中完成了系统性正向遗传分析。通过对比针对WNT信号通路负调控、衰减调控与正调控因子，R-spondin依赖型信号通路介导因子，以及因肿瘤抑制基因腺瘤性结肠息肉病（adenomatous polyposis coli, APC）或酪蛋白激酶1α（casein kinase 1α, CK1α）缺失所诱导的组成型信号通路抑制因子的筛选结果，本研究在该通路的多数层面揭示了全新的调控特征。这些特征包括转录因子AP-4的必需调控作用、AXIN2的DAX结构域在调控β-连环蛋白（β-catenin）转录活性中的功能、糖基磷脂酰肌醇锚定生物合成与磷脂酰肌醇蛋白聚糖（glypicans）对R-spondin增强型WNT信号通路的贡献，以及当β-连环蛋白降解复合物的不同组分缺失时，两种不同的信号通路调控机制。本研究提出的概念与方法框架，可为其他信号通路系统的全面解析提供有力支撑。