Abstract
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 1α 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 β-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 β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.
Highlights
Cellular signaling systems have evolved complex circuitry involving multiple layers of regulation, making their comprehensive characterization a major challenge
We identified many of the known core pathway components and found a new requirement for the transcription factor AP-4 (TFAP4)
Since WNT reporter fluorescence is a continuous readout, in contrast to digital readouts such as cell viability or the presence or absence of a phenotype, it enabled us to enrich for cells with enhanced or reduced signaling phenotypes by fluorescence activated cell sorting (FACS) with complete flexibility on the stringency of selection
Summary
Cellular signaling systems have evolved complex circuitry involving multiple layers of regulation, making their comprehensive characterization a major challenge. Forward genetics in model organisms has been a general and unbiased way to identify new components in signaling pathways and to map their connectivity. Since signaling pathways have often diverged between humans and these simpler model systems, their analysis in human cells becomes an important goal. Our ability to identify the best therapeutic strategy or to predict the effectiveness of drugs targeting
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