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.
Highlights
Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes
The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis
Because the level of cell-surface Gb3 on HeLa cells correlates with their sensitivity to the toxin [23, 24], we reasoned that selection of Shiga toxin-resistant HeLa cells after transduction triaosylceramide; GeCKO, genome-scale CRISPR/Cas9 knockout; KO, knockout; MOI, multiplicity of infection; qPCR, quantitative PCR; S1P, sphingosine-1-phosphate; SPT, serine palmitoyltransferase; sgRNA, single guide RNA
Summary
Shiga toxin binds to the cell-surface trisaccharide glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes cell death after uptake as a result of toxin-mediated protein synthesis inhibition (20 –22). Compared with control HeLa cells, SPTLC1 KO HeLa cells were resistant to Shiga toxin treatment (Fig. 1B) and expressed significantly decreased cellsurface Gb3 (Fig. 1D), indicating that Shiga toxin resistance was due to the loss of Gb3 receptor caused by the deletion of SPTLC1 (Fig. 1A) Based on these results, we established a genome-wide lossof-function screen using Shiga toxin selection to identify genes that positively regulate membrane SL levels. The 20 top-ranking genes included SPTSSA, UGCG, B4GALT5, SPTLC2, A4GALT, and SPTLC1 These biosynthetic genes, along with CERS2, showed relatively high positive log -fold changes in the toxin-treated cells compared with untreated cells (Fig. 1F, inset), which is consistent with previous reports [25, 26] and our finding that the SL biosynthetic pathway is critical for Shiga toxin sensitivity in HeLa cells (Fig. 1B). AHR, a transcription factor with broad physiological functions (14 –19), showed high enrichment comparable with that observed for the SL biosynthetic genes (Fig. 1F)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
