Genome‐wide Analysis of Heparan Sulfate Biosynthesis

Abstract

Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and are involved in many important biological processes. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N‐ and O‐sulfated. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. A genome‐wide CRISPR/Cas9 screen was developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, high‐throughput screens were adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs and decrease binding of HS‐dependent ligands to the cell surface. Among the resulting targets, we identified well‐known HS biosynthesis enzymes as well as candidate genes with a previously unrecognized link to HS biosynthesis. The top overlapping hits from the screens were then characterized based on gene function and validated to regulate HS assembly. Overall, these studies demonstrate novel approaches to uncovering regulatory factors and provide a better understanding of the genetic regulation of HS biosynthesis. Furthermore, these studies lead us to new methods to manipulate heparan sulfate and its activities in other cellular processes that go awry in human diseases.Support or Funding InformationR01GM33063 (to J.E.), R21CA199292 (to J.E. and N.L.), R35GM119850 (to N.L.), and K12HL141956 (to R.W.)