Microbial Metabolites Orchestrate a Distinct Multi-Tiered Regulatory Network in the Intestinal Epithelium That Directs P-Glycoprotein Expression.

P-glycoprotein (P-gp) is a key component of the intestinal epithelium playing a pivotal role in removal of toxins and efflux of endocannabinoids to prevent excessive inflammation and sustain homeostasis. Recent studies revealed butyrate and secondary bile acids, produced by the intestinal microbiome, potentiate the induction of functional P-gp expression. We now aim to determine the molecular mechanism by which this functional microbiome output regulates P-gp. RNA sequencing of intestinal epithelial cells responding to butyrate and secondary bile acids in combination discovered a unique transcriptional program involving multiple pathways that converge on P-gp induction. Using shRNA knockdown and CRISPR/Cas9 knockout cell lines, as well as mouse models, we confirmed the RNA sequencing findings and discovered a role for intestinal HNF4α in P-gp regulation. These findings shed light on a sophisticated signaling network directed by intestinal microbial metabolites that orchestrate P-gp expression and highlight unappreciated connections between multiple pathways linked to colonic health. IMPORTANCE Preventing aberrant inflammation is essential to maintaining homeostasis in the mammalian intestine. Although P-glycoprotein (P-gp) expression in the intestine is critical for protecting the intestinal epithelium from toxins and damage due to neutrophil infiltration, its regulation in the intestine is poorly understood. Findings presented in our current study have now uncovered a sophisticated and heretofore unappreciated intracellular signaling network or "reactome" directed by intestinal microbial metabolites that orchestrate regulation of P-gp. Not only do we confirm the role of histone deacetylases (HDAC) inhibition and nuclear receptor activation in P-gp induction by butyrate and bile acids, but we also discovered new signaling pathways and transcription factors that are uniquely activated in response to the combination of microbial metabolites. Such findings shed new light into a multi-tiered network that maintains P-gp expression in the intestine in the context of the fluctuating commensal microbiome, to sustain a homeostatic tone in the absence of infection or insult.

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states.

The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development.