Sumoylation and neddylation are opposing forces in stratified epithelial homeostasis

Summary: Abstract Body: Squamous epithelium stratification involves a coordinated, irreversible differentiation process, that modulates the expression of approximately 3,500 genes. To investigate the less understood post-translational protein modifications governing this process, we employed DIA-MS to map proteome-wide changes during epidermal differentiation. K-εGG-peptide enrichment identified modified peptides in progenitor and differentiated states. A single-cell perturb RNA-seq screen revealed unexpected opposing roles of two ubiquitin-like (UBL) conjugation pathways: Sumoylation was essential for differentiation, while neddylation maintained the undifferentiated progenitor state. These findings were validated through genetic and pharmacologic interventions across multiple stratified epithelia. Epidermal SUMO2 and NEDD8 UBL conditional knockout mice exhibited corresponding perturbations in epidermal homeostasis, with scRNA-seq revealing shifts in epithelial subpopulations and differentiation states. Further investigation of neddylation's role in homeostasis involved examining both canonical modifications and non-canonical mechanisms. DIA-MS analysis of primary keratinocytes treated with various inhibitors revealed overlapping and independent proteome alterations. A genome-wide CRISPR suppressor screen identified 616 genes (FDR<0.05) involved in non-canonical dependencies of neddylation, including pathway components, transcription factors, and epigenetic regulators. This study identifies Nedd8 and Sumo2 as key regulators of epithelial homeostasis through both canonical and non-canonical protein modifications, proposing a model where UBLs exert opposing impacts on the differentiation process. Marten C. Winge¹, Leandra V. Jackrazi¹, Douglas Porter¹, Lisa Ko¹, Robin M. Meyers¹, Spencer H. Cha¹, Konnie Q. Guo¹, Weili Miao¹, Smarajit Mondal¹, David L. Reynolds¹, Luca Ducoli¹, Brian Zarnegar¹, Paul Khavari¹ 1. Dermatology, Stanford University, Stanford, CA, United States. Epidermal Structure and Barrier Function