Genome wide analysis of heparan sulfate assembly

Abstract

It is well known that the arrangement and orientation of the sulfated sugar residues in heparan sulfate specify the location of distinct binding sites for protein ligands, and that binding regulates important biological processes including cell proliferation and development. Most of the enzymes involved in glycosaminoglycan biosynthesis have been cloned, purified and studied biochemically, but there is much less information regarding the regulatory mechanisms that control the spatial and temporal expression of heparan sulfate in cells and tissues. We have taken a two‐prong approach towards uncovering these control systems. In one approach, we examined the promoter regions of all genes involved in heparin/heparan sulfate assembly and uncovered a transcription factor binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR‐mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to a dramatic increase in expression of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin. Enhanced 3‐O‐sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition. Analysis of transcriptomics data showed an inverse correlation of expression of ZNF263 and HS3ST1 in mast cells and basophils compared to other (non‐heparin producing) immune cells, consistent with its role in regulation of anticoagulant heparin. In a second approach we developed a genome‐wide CRISPR/Cas9‐mediated screen. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human melanoma cell line. High‐throughput screening assays were adapted to identify lentiviral‐encoded sgRNAs that induce resistance to a cytotoxin whose action depends on HSPGs and that reduce binding of HS‐dependent ligands. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their involvement in the regulation of HS composition.Support or Funding InformationWe thank generous support from the National Institutes of Health (R01GM33063 to J.D.E.; R21CA199292 to J.D.E. and N.E.L., R35GM119850 to N.E.L, R21 HD089067 to P.L.S.M.G.), the Fondation Leducq (to C.K.G and P.L.S.M.G.), a fellowship award to R.J.W. (K12HL141956), and generous support from the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark (to N.E.L.).