Systematic morphological profiling of human gene and allele function reveals Hippo-NF-κB pathway connectivity

We hypothesized that human genes and disease-associated alleles might be systematically functionally annotated using morphological profiling of cDNA constructs, via a microscopy-based Cell Painting assay. Indeed, 50% of the 220 tested genes yielded detectable morphological profiles, which grouped into biologically meaningful gene clusters consistent with known functional annotation (e.g., the RAS-RAF-MEK-ERK cascade). We used novel subpopulation-based visualization methods to interpret the morphological changes for specific clusters. This unbiased morphologic map of gene function revealed TRAF2/C-REL negative regulation of YAP 1/WWTR1-responsive pathways. We confirmed this discovery of functional connectivity between the O_SCPCAPNF-C_SCPCAP{kappa}O_SCPCAPBC_SCPCAP pathway and Hippo pathway effectors at the transcriptional level, thereby expanding knowledge of these two signaling pathways that critically regulate tumor initiation and progression. We make the images and raw data publicly available, providing an initial morphological map of major biological pathways for future study.