ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis Ryan J. Weissa,1, Philipp N. Spahnb,1, Alejandro Gómez Toledoa, Austin W. T. Chiangb, Benjamin P. Kellmanb,JingLia, Christopher Bennerc, Christopher K. Glassa,c,PhilipL.S.M.Gordtsc,d,NathanE.Lewisb,d,e,2, and Jeffrey D. Eskoa,d,2,3 aDepartment of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093-0687; bDepartment of Pediatrics, University of California San Diego, La Jolla, CA 92093-0760; cDepartment of Medicine, University of California San Diego, La Jolla, CA 92093-0687; dGlycobiology Research and Training Center, University of California San Diego, La Jolla, CA 92093-0687; and eDepartment of Bioengineering, University of California San Diego, La Jolla, CA 92093-0687 Edited by Tadatsugu Taniguchi, University of Tokyo, Meguro-ku, Japan, and approved March 9, 2020 (received for review December 2, 2019) Heparin is the most widely prescribed biopharmaceutical in pro- inactivate thrombin and Factor Xa, which accounts for its potent duction globally. Its potent anticoagulant activity and safety makes anticoagulant activity (4). it the drug of choice for preventing deep vein thrombosis and In 2008, the US Food and Drug Administration issued a major pulmonary embolism. In 2008, adulterated material was intro- recall of pharmaceutical heparin due to contamination of the duced into the heparin supply chain, resulting in several hundred raw heparin stock imported from China. This crisis prompted deaths and demonstrating the need for alternate sources of heparin. new guidelines for monitoring the purity of heparin, but the Heparin is a fractionated form of heparan sulfate derived from feedstock remains vulnerable to natural variation, susceptibility animal sources, predominantly from connective tissue mast cells in of the pig population to infectious agents, and potential con- pig mucosa. While the enzymes involved in heparin biosynthesis tamination (5). Thus, finding an alternative source of heparin is are identical to those for heparan sulfate, the factors regulating important. While HS is produced by all animal cells, anticoag- these enzymes are not understood. Examination of the promoter ulant heparin is biosynthesized and stored primarily in connective- regions of all genes involved in heparin/heparan sulfate assembly tissue type mast cells, a type of granulocyte derived from myeloid ZNF263 uncovered a transcription factor-binding motif for , a C2H2 stem cells. The factors that regulate the enzymes and give rise to zinc finger protein. CRISPR-mediated targeting and siRNA knock- anticoagulant heparin are unknown. Defining these factors could down of ZNF263 in mammalian cell lines and human primary cells BIOCHEMISTRY HS3ST1 facilitate the production of bioengineered heparin in typical cells led to dramatically increased expression levels of , a key used in the biopharmaceutical industry, such as Chinese hamster enzyme involved in imparting anticoagulant activity to heparin, ovary cells (6). and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed In this study, we analyzed the expression and promoter se- in cells. Enhanced 3-O-sulfation increased binding to antithrombin, quences of the enzymes involved in HS and heparin assembly which enhanced Factor Xa inhibition, and binding of neuropilin-1. and identified ZNF263, a C2H2 zinc finger transcription factor Analysis of transcriptomics data showed distinctively low expres- ZNF263 – (TF) with binding sites in 45% of the relevant genes. CRISPR- sion of in mast cells compared with other (non heparin- ZNF263 producing) immune cells. These findings demonstrate a novel mediated targeting and siRNA-mediated knockdown of regulatory factor in heparan sulfate modification that could fur- in human cell lines and primary cells revealed that ZNF263 is a ther advance the possibility of bioengineering anticoagulant heparin in cultured cells. Significance heparin | zinc-finger transcription factor | heparan sulfate 3-O-sulfotransferases | Heparin is the most widely prescribed biopharmaceutical world- anticoagulant | Factor X wide due to its potent anticoagulant activity. Therapeutic heparin is sourced primarily from porcine entrails and bovine lung. Mast eparin is one of the most frequently prescribed medications cells appear to be the primary cell type that produces heparin, Hin the United States, administered to more than 12 million but all cells make a related polysaccharide, heparan sulfate. Un- patients per year to prevent thrombosis during surgery and to derstanding the factors that regulate the production of antico- treat thromboembolism (1, 2). A member of a family of linear agulant heparin would provide tools for bioengineering heparin. Here we provide evidence that key sulfotransferases in heparin/ polysaccharides known as glycosaminoglycans, heparin consists heparan sulfate production are under repression by the zinc fin- of alternating glucosamine and uronic acids that are heteroge- ger protein ZNF263. neously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues specify distinct protein-binding sites Author contributions: R.J.W., P.N.S., A.G.T., P.L.S.M.G., N.E.L., and J.D.E. designed re- that endow heparin with its various biological activities, including search; R.J.W., P.N.S., A.G.T., A.W.T.C., B.P.K., and J.L. performed research; C.B. and C.K.G. contributed new reagents/analytic tools; R.J.W., P.N.S., A.G.T., A.W.T.C., B.P.K., anticoagulant activity. Heparin shares a common biosynthetic J.L., P.L.S.M.G., N.E.L., and J.D.E. analyzed data; and R.J.W., P.N.S., A.G.T., P.L.S.M.G., pathway with heparan sulfate (HS), which is less sulfated than N.E.L., and J.D.E. wrote the paper. heparin, generally lacks anticoagulant activity, and is expressed Competing interest statement: The University of California San Diego and J.D.E. have a on the cell surface and in the extracellular matrix of all mam- financial interest in TEGA Therapeutics, Inc. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its malian cells (3). conflict of interest policies. The biosynthesis of heparin and HS is a nontemplated process, This article is a PNAS Direct Submission. driven by the concerted activity of a large family of enzymes Published under the PNAS license. A localized to the Golgi and endoplasmic reticulum (Fig. 1 ). A 1R.J.W. and P.N.S. contributed equally to this work. key reaction in heparin biosynthesis is the addition of a sulfate 2N.E.L. and J.D.E. contributed equally to this work. N group to the C3 position of an -sulfoglucosamine residue within 3To whom correspondence may be addressed. Email: [email protected]. a sulfated pentasaccharide sequence, which confers high-affinity This article contains supporting information online at https://www.pnas.org/lookup/suppl/ binding to antithrombin. Binding leads to a conformational change doi:10.1073/pnas.1920880117/-/DCSupplemental. in antithrombin and ∼1,000-fold enhancement in its capacity to www.pnas.org/cgi/doi/10.1073/pnas.1920880117 PNAS Latest Articles | 1of7 Downloaded by guest on September 28, 2021 FAM20B A HS6ST1 PXYLP1 B C HS6ST2 EXTL2 B4GALT7 10 Binding Motifs (HS genes) HS2ST1 HS6ST3 GLCE EXTL3 B3GAT3 gD/Nrp1 Antithrombin 9 ZNF263 ZNF263 ue 6S 6S 6S 6S 2P l NANOG 8 OSR1 -va NSNS2S NS NS 2S NS n NFIL3 3S 3S p 7 NANOG EXT1 B3GALT6 NRF1 NDST1 SMAD3 HS3ST6 NDST4 EXTL2 HS3ST3B1 HS3ST3A1 HS2ST1 GLCE NDST1 EXTL3 HS3ST5 HS3ST4 HS3ST2 HS3ST1 NDST3 NDST2 EXT2 EXT1 HS3ST1 EXT2 HS6ST3 HS6ST2 HS6ST1 HS3ST2 HS3ST4 NDST2 XYLT1/2 6 NRF1 HS3ST3A1 HS3ST5 HS3ST5 NDST3 -log10 OSR1 HS3ST3B1 HS3ST6 NDST4 5 NFIL3 SMAD3 GlcN GlcNAc GlcA IdoA Gal Xyl 4 S = sulfate P = phosphate 0204060 % Input Genes D Binding Motifs (Other GAG genes) ZNF263 NANOG OSR1 NFIL3 NRF1 SMAD3 DSEL SDC4 CHPF CHSY3 CHSY1 DSE SRGN NCAN CSPG4 A CHST13 CHPF2 CSGALNACT2 NRP1 SDC1 GPC1 COL18A1 CD47 AGRN HPSE B3G B4GAL SPOCK3 LEPRE1 PTPRZ1 VCAN SLC35D2 UGDH SLC35B2 X UST CHST14 CHST7 CHST15 CHST12 CSGALNACT1 SDC3 SDC2 HSPG2 GPC3 CD44 TGFBR3 SULF2 SULF1 EPYC BCAN IM ENTPD4 BPNT1 CD74 PRG4 SPOCK2 SPOCK1 SRGN BGN DCN ESM1 THBD PTPRZ1 SLC35A3 SLC35D1 SLC35B1 SLC35A2 SLC35B4 GALE UXS1 SLC26A2 SLC26A1 SLC35B3 PAPSS2 P CHST CHST3 GPC6 GPC5 GPC4 GPC2 B3GA XY COL9A2 F AM20B APSS1 GC1 Y P L L AD1 T1 T2 A T3 L 1 T7 T6 1 Nucleotide Metabolism Chondroitin/Dermatan Sulfate Chondroitin/Dermatan Sulfate Heparan Sulfate HS Linkage Proteoglycans Biosynthesis Proteoglycans Processing Region Fig. 1. Heparin/HS structure, assembly, and regulation. (A) HS and heparin assemble while attached via a linkage tetrasaccharide to a core protein of a proteoglycan. NDSTs, HS2ST, HS3STs, and HS6STs install sulfate groups at specific sites along the HS/heparin chain, and an epimerase (GLCE) converts D-glucuronic acid to L-iduronic acid. The chain is rendered according the Symbol Nomenclature for Glycans (47). The gray and yellow oval shapes depict protein-binding sites for gD/NRP1 and antithrombin, respectively. Input genes for HOMER are indicated in bold red font. (B) HOMER motif enrichment analysis revealing TFs with predicted binding motifs in a set of HS biosynthesis genes. (C) Heatmap showing the presence (red) or absence (gray) of TF-binding motifs (y-axis) in regulatory regions of HS biosynthesis genes (x-axis), as predicted by HOMER. (D) Heatmap showing the presence (red) or absence (gray) of the same TF-binding motifs in a list of genes involved in nucleotide sugar and sulfate metabolism, CS/DS proteoglycans, CS/DS biosynthesis, HS proteoglycans, extracellular HS processing enzymes, and enzymes that generate the common linkage tetrasaccharide in heparin/HS and CS/DS. transcriptional repressor, and its inactivation or silencing en- sulfate/dermatan sulfate (CS/DS) biosynthesis. Intriguingly, a hanced mRNA expression of HS3ST1 and HS3ST3A1, enzymes motif associated with ZNF263 gained the greatest statistical involved in the formation of binding sites for antithrombin and significance, with predicted binding sites present on 45% of the neuropilin-1 (NRP1) and glycoprotein D of herpes simplex virus, input genes.