Identification of master regulators in goblet cells and Paneth cells using transcriptomics profiling of gut organoids and multi-layered networks

Background: Normal function of specific intestinal epithelial cell types, like the mucin producing goblet cells or the anti-microbial peptide producing Paneth cells, is key to the homeostasis of the gut. Dysfunction of these cells is often associated with severe gut pathologies, such as inflammatory bowel disease (IBD), including Crohns disease and ulcerative colitis. Although transcriptional signatures of intestinal cells have been identified, an integrated and cell type specific network analysis that identifies the key regulators with their disease relevance has been lacking. Method: In this study, we profiled the expression of mRNAs, microRNAs and long non-coding RNAs from mouse derived 3D intestinal organoids whose differentiation was directed towards Paneth cells or goblet cells. We generated cell type specific regulatory networks by integrating expression data into multi-layered networks of regulatory interactions. Results: By mapping cell type specific marker genes to the network, we were able to identify regulators potentially contributing to cell type specific functions. Among the seven putative master regulators (those targeting many of the markers), we identified four nuclear hormone receptors with links to IBD, immunity and autophagy: Vdr, Rxra, Nr1d1 and Nr3c1. We also found common regulators relevant in both Paneth and goblet cells that regulate different sets of cell type specific markers as a result of regulatory rewiring after differentiation. Conclusions: We describe an integrative organoid study that combines -omics data with multi-layered networks to study the regulatory landscapes of Paneth cells and goblet cells. Using the developed computational workflow, we generated cell type specific regulatory networks encompassing transcription factor, microRNA and long non-coding RNA regulation at the transcriptional and post-transcriptional level. Analysis of the networks uncovered potential cell type specific master regulators. Specific investigation of four of these regulators identified links to IBD and to cellular phenotypes associated with IBD pathology. Therefore, we demonstrate that application of our workflow in a cell type specific context can be used to disentangle multifactorial mechanisms of IBD and improve our understanding of disease pathomechanisms.