A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels

Abstract

Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.

Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation

“…Experiments to determine MAG levels in lymphocyte cultures obtained from the blood of the patients yielded very low cDNA copy numbers, so we were not able to confirm this finding in our patients. Recently, AHR was found to bind and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis (Majumder et al, 2020). Thus analysis of genes related to lipid metabolism would constitute a logical next step in our studies.…”

“…AHR knockout mice (AHR-KO) were shown to possess an impaired optic nerve myelin sheath and exhibited modifications in lipid composition as well as in the expression of myelin-associated glycoprotein (MAG) (Juricek et al, 2017;Shackleford et al, 2018). Supporting the involvement of lipids in this pathology, AHR was recently shown to activate the promoter for a small subunit of the serine palmitoyltransferase, the first and rate-limiting step in sphingolipid synthesis, in both mice and HeLa cells (Majumder et al, 2020).…”

Sphingolipids in Type 1 Diabetes: Focus on Beta-Cells

“…Recently a new mechanism regulating the expression of SPT has been described [106]. Using genome-wide CRISPR/Cas9 screening it was demonstrated that AHR (aryl hydrocarbon receptor) binds and activates the gene promoter of SPT [106].…”

“…Recently a new mechanism regulating the expression of SPT has been described [106]. Using genome-wide CRISPR/Cas9 screening it was demonstrated that AHR (aryl hydrocarbon receptor) binds and activates the gene promoter of SPT [106]. Moreover, tissues from AHR KO mice were characterized by reduced expression of several other key genes in the SL biosynthetic pathway and decreased the SL content [106].…”

“…Using genome-wide CRISPR/Cas9 screening it was demonstrated that AHR (aryl hydrocarbon receptor) binds and activates the gene promoter of SPT [106]. Moreover, tissues from AHR KO mice were characterized by reduced expression of several other key genes in the SL biosynthetic pathway and decreased the SL content [106]. Interestingly, AHR is a transcription factor that has been recently shown to link the diet and gut microbiome alterations with islet autoimmunity [107], yielding the possibility that the observed activation of AHR in islets during T1DM development could stimulate the SPT expression and increase de novo SL biosynthesis in beta-cells.…”