Aryl Hydrocarbon Receptor (AHR) modulates glycosphingolipid production by transcriptional regulation of key genes involved in the de novo biosynthetic pathway

Authors

• S Majumder
• M Kono
• YT Lee
• C Byrnes
• RL Proia

Abstract

Sphingolipids are critical components of membranes and act as important signaling molecules. Therefore, it is not surprising that aberrant sphingolipid homeostasis has been implicated in diverse pathologic conditions, such as in aging, neurodegenerative and metabolic diseases and inflammation. However, a global approach to identify sphingolipid regulatory genes in human cells is lacking. Here we report development of a toxin-based genome-wide CRISPR screen to identify genes involved in sphingolipid synthesis. We also show that Aryl Hydrocarbon Receptor (AHR), a transcription factor, is a major regulator of de novo sphingolipid synthesis. Verotoxin (VT), an exotoxin, binds specifically to glycosphingolipids (globotriaosylceramide or GB3) on the cell surface. Cells lacking GB3 on the cell surface are resistant to VT. Upon binding to the cell surface, VT is endocytosed, blocks protein translation and induces apoptosis. In mammalian cells, biosynthesis of GB3 starts from sphingolipid precursors. We hypothesized that a genome wide knockout screen for VT resistance will identify novel regulators of sphingolipid/glycosphingolipid metabolism. We generated genome-wide CRISPR mediated knockout mutant pool by lentiviral delivery of human sgRNA(guide) library in HeLa cells, stably expressing Cas9. Resulting mutants were screened for VT resistance. Loss-of-functions of genes that confer toxin resistance were identified by deep sequencing amplicons of stably integrated sgRNA regions from the genomic DNA. Relative abundance of guides between untreated and treated groups showed a robust increase of 18% of the total guides in resistant cells. Some of the highest-ranking genes enriched in resistant cells include genes such as, A4GLT, B4GALT5 which are reported to be involved in GB3 synthesis and toxin resistance. Individual knockouts of 20 top ranking candidates were validated by toxin resistance and GB3 expression on its cell surface. Surprisingly AHR-KO, one of the top candidate, is not only resistant to the toxin but also express significantly less amount of GB3. This finding, along with the fact that sphingolipid biosynthesis plays a crucial role in toxin resistance, we hypothesized that AHR might be involved in transcriptional regulation of genes in the biosynthetic pathway. We show that AHR binds to the promoter of key genes in the pathway and transcriptionally modulate their expression.

Scientific Focus Area: Cell Biology

This page was last updated on Friday, March 26, 2021