Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR–Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.
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Any data generated or analyzed during this study, associated protocols, materials within the manuscript and public databases (GSE163162, GSE145789) are included in the article and related Supplementary information, or are available from the corresponding author. Source data are provided with this paper.
Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu. Rev. Biochem. 83, 129–157 (2014).
Bishop, J. R., Schuksz, M. & Esko, J. D. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037 (2007).
Esko, J. D. & Selleck, S. B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435–471 (2002).
Presto, J. et al. Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation. Proc. Natl Acad. Sci. USA 105, 4751–4756 (2008).
Kreuger, J. & Kjellen, L. Heparan sulfate biosynthesis: regulation and variability. J. Histochem. Cytochem. 60, 898–907 (2012).
Weiss, R. J. et al. ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis. Proc. Natl Acad. Sci. USA 117, 9311–9317 (2020).
Naba, A. et al. The extracellular matrix: tools and insights for the "omics" era. Matrix Biol. 49, 10–24 (2016).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Elson-Schwab, L. et al. Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway. J. Biol. Chem. 282, 13585–13591 (2007).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Spahn, P. N. et al. PinAPL-Py: a comprehensive web-application for the analysis of CRISPR/Cas9 screens. Sci. Rep. 7, 15854 (2017).
Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).
Shishido, Y., Sharma, K. D., Higashiyama, S., Klagsbrun, M. & Mekada, E. Heparin-like molecules on the cell surface potentiate binding of diphtheria toxin to the diphtheria toxin receptor membrane-anchored heparin-binding epidermal growth factor-like growth factor. J. Biol. Chem. 270, 29578–29585 (1995).
Su, X., Lin, Z. & Lin, H. The biosynthesis and biological function of diphthamide. Crit. Rev. Biochem. Mol. Biol. 48, 515–521 (2013).
Szklarczyk, D. et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 47, D607–D613 (2019).
Yan, M., Yang, X., Wang, H. & Shao, Q. The critical role of histone lysine demethylase KDM2B in cancer. Am. J. Transl. Res. 10, 2222–2233 (2018).
Vacik, T., Ladinovic, D. & Raska, I. KDM2A/B lysine demethylases and their alternative isoforms in development and disease. Nucleus 9, 431–441 (2018).
Farcas, A. M. et al. KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. eLife 1, e00205 (2012).
He, J. et al. Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat. Cell Biol. 15, 373–384 (2013).
Frescas, D., Guardavaccaro, D., Bassermann, F., Koyama-Nasu, R. & Pagano, M. JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genes. Nature 450, 309–313 (2007).
Zhang, X., Ong, C., Su, G., Liu, J. & Xu, D. Characterization and engineering of S100A12-heparan sulfate interactions. Glycobiology 30, 463–473 (2020).
Banito, A. et al. The SS18-SSX oncoprotein hijacks KDM2B-PRC1.1 to drive synovial sarcoma. Cancer Cell 34, 346–348 (2018).
Ashikari-Hada, S. et al. Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library. J. Biol. Chem. 279, 12346–12354 (2004).
Kreuger, J., Salmivirta, M., Sturiale, L., Giménez-Gallego, G. & Lindahl, U. Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2. J. Biol. Chem. 276, 30744–30752 (2001).
Sugaya, N., Habuchi, H., Nagai, N., Ashikari-Hada, S. & Kimata, K. 6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture. J. Biol. Chem. 283, 10366–10376 (2008).
Thacker, B. E., Xu, D., Lawrence, R. & Esko, J. D. Heparan sulfate 3-O-sulfation: a rare modification in search of a function. Matrix Biol. 35, 60–72 (2013).
Lamanna, W. C., Frese, M. A., Balleininger, M. & Dierks, T. Sulf loss influences N-, 2-O-, and 6-O-sulfation of multiple heparan sulfate proteoglycans and modulates fibroblast growth factor signaling. J. Biol. Chem. 283, 27724–27735 (2008).
Habuchi, H. et al. The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine. J. Biol. Chem. 275, 2859–2868 (2000).
Shworak, N. W. et al. Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate D-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 272, 28008–28019 (1997).
Tian, S. et al. Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation. PLoS Biol. 16, e2006951 (2018).
Labeau, A. et al. A genome-wide CRISPR-Cas9 screen identifies the dolichol-phosphate mannose synthase complex as a host dependency factor for dengue virus infection. J. Virol. 94, e01751–19 (2020).
Bassaganyas, L. et al. New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells. J. Cell Biol. 218, 3861–3879 (2019).
Klose, R. J., Kallin, E. M. & Zhang, Y. JmjC-domain-containing proteins and histone demethylation. Nat. Rev. Genet. 7, 715–727 (2006).
Nagamine, S. et al. Organ-specific sulfation patterns of heparan sulfate generated by extracellular sulfatases Sulf1 and Sulf2 in mice. J. Biol. Chem. 287, 9579–9590 (2012).
Lamanna, W. C. et al. Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity. Biochem. J. 400, 63–73 (2006).
Miller, R. L. et al. Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides. Nat. Commun. 11, 1481 (2020).
Wu, J. et al. Sequencing heparan sulfate using HILIC LC-NETD-MS/MS. Anal. Chem. 91, 11738–11746 (2019).
Wei, J. et al. Characterization and quantification of highly sulfated glycosaminoglycan isomers by gated-trapped ion mobility spectrometry negative electron transfer dissociation MS/MS. Anal. Chem. 91, 2994–3001 (2019).
Wang, Y. et al. KDM2B overexpression correlates with poor prognosis and regulates glioma cell growth. Onco Targets Ther. 11, 201–209 (2018).
Zheng, Q. et al. Histone demethylase KDM2B promotes triple negative breast cancer proliferation by suppressing p15INK4B, p16INK4A, and p57KIP2 transcription. Acta Biochim. Biophys. Sin. (Shanghai) 50, 897–904 (2018).
Tzatsos, A. et al. Lysine-specific demethylase 2B (KDM2B)-let-7-enhancer of zester homolog 2 (EZH2) pathway regulates cell cycle progression and senescence in primary cells. J. Biol. Chem. 286, 33061–33069 (2011).
Galbiati, A. et al. Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells. Oncotarget 8, 37091–37103 (2017).
Kuang, Y. et al. Histone demethylase KDM2B upregulates histone methyltransferase EZH2 expression and contributes to the progression of ovarian cancer in vitro and in vivo. Onco Targets Ther. 10, 3131–3144 (2017).
Zhao, E. et al. Inhibition of cell proliferation and induction of autophagy by KDM2B/FBXL10 knockdown in gastric cancer cells. Cell Signal. 36, 222–229 (2017).
Kottakis, F. et al. FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. Mol. Cell 43, 285–298 (2011).
Andricovich, J., Kai, Y., Peng, W., Foudi, A. & Tzatsos, A. Histone demethylase KDM2B regulates lineage commitment in normal and malignant hematopoiesis. J. Clin. Invest. 126, 905–920 (2016).
Ye, B. et al. LncKdm2b controls self-renewal of embryonic stem cells via activating expression of transcription factor Zbtb3. EMBO J. 37, e97174 (2018).
Bracken, A. P., Dietrich, N., Pasini, D., Hansen, K. H. & Helin, K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 20, 1123–1136 (2006).
Lawrence, R., Lu, H., Rosenberg, R. D., Esko, J. D. & Zhang, L. Disaccharide structure code for the easy representation of constituent oligosaccharides from glycosaminoglycans. Nat. Methods 5, 291–292 (2008).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).
Lawrence, R. et al. Evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling. J. Biol. Chem. 283, 33674–33684 (2008).
Kreuger, J., Lindahl, U. & Jemth, P. Nitrocellulose filter binding to assess binding of glycosaminoglycans to proteins. Methods Enzymol. 363, 327–339 (2003).
We thank C. van der Kooi, University of Kentucky, for providing the b1b2 domain of NRP1, and D. Xu, University of Buffalo, for providing the biotinylated S100A12 protein. We also thank the GlycoAnalytics Core Facility at University of California, San Diego for help with analytical experiments. We thank C. Kuo for processing of RNA sequencing data. RNA sequencing and CRISPR amplicon sequencing were conducted at the IGM Genomics Center, University of California, San Diego, La Jolla, CA (MCC grant no. P30CA023100). This work was supported by grant nos. R21 CA199292 (to J.D.E. and N.E.L.), GM33063 (to J.D.E.), GM119850 (to N.E.L.), NSF CHE 200424 (to K.G.) and T32 GM008326 (fellowship support for B.M.T.); and DFG research fellowship no. 420160411 from the German Research Foundation (to S.R.) and no. K12HL141956 (fellowship support for R.J.W.).
The University of California San Diego and J.D.E. have a 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 conflict-of-interest policies.
Peer review information Nature Chemical Biology thanks Ulf Lindahl, Linda Troeberg and the other, anonymous, reviewer for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Left: Frequency distribution of gene counts after treatment with either diphtheria toxin (DTX) or PBS (Plasmid = GeCKO plasmid library). Right: Lorenz curves showing the distribution of sequencing reads over the gene library. Numbers represent Gini coefficients. b, Scatterplot of sgRNA counts (log10, normalized) in samples after DTX treatment versus PBS treatment. The sgRNA fraction with significant fold-change is shown in green. sgRNA fraction representing non-targeting controls shown in orange. HBEGF-targeting sgRNAs shown in red, and DPH genes are shown in purple and blue. c, Table of the top 10 ranked genes showing enrichment after DTX treatment. The enrichment level of the six gene-targeting sgRNAs is indicated by color.
a, Sanger sequencing of two A375 KDM2B knockout clones (C5 and C13) and (b) Sanger sequencing of one HeLa knockout clone (C9) after targeting with the indicated sgRNAs. Intron sequence denoted in lower case.
a, Protein binding in A375 KDM2B knockout clone C13 (t-tests; n = 3). b, Fold change in KDM2B mRNA expression in A375 KDM2BC5 cells, and in A375 KDM2BC5 cells expressing a KDM2B cDNA from a lentiviral construct (hKDM2B) (n = 2). c, FGF1 binding in HeLa KDM2BC9 cells, and in HeLa KDM2BC9 cells expressing KDM2B cDNA (hKDM2B) (t-test on log10 fluorescence data, n = 5). d, Fold change in KDM2B mRNA expression in A375 KDM2BC5 cells expressing a KDM2B cDNA with a point mutation (H242A) in the demethlase domain (n = 2).
a, Fold change in SULF1 mRNA expression in A375 KDM2BC5 cells, and in A375 KDM2BC5 cells expressing a siRNA against SULF1 (n = 2). b, top: Western blot of SULF1 protein levels in conditioned media (CM) collected from A375 wild-type and KDM2BC5 cells (CM 10x equals 10-fold concentrated solution) bottom: Total protein (coomassie-stained gel), red frame indicates position of SULF1. c, Gene track for H3K4me3, KDM2B, and H3K36me2 ChIP-Seq at the MNX1 locus, a known target of KDM2B. d, Transcriptome-wide expression data displaying differentially expressed genes in KDM2BC5 cells compared to wild-type. HS biosynthetic genes and extracellular matrix genes are highlighted. e, Gene set enrichment analysis of significantly downregulated (log2 ≤ -0.5, p ≤ 0.05; FDR-corrected) and upregulated (log2 ≥ 0.5, p ≤ 0.05; FDR-corrected) genes in A375 wild-type and KDM2BC5 RNA-Seq datasets (n = 3). f, MMP-9 and TIMP-3 levels in the supernatant from cultured A375 wild-type and KDM2BC5 cells (t-test, n = 3). g, Histograms showing FGF1 binding in A375 wild-type, KDM2BC5 cells, and in KDM2BC5 cells upon treatment with an siRNA targeting SULF1. h, Fold change in HS6ST2 mRNA expression in A375 KDM2BC5 cells, and in A375 KDM2BC5 cells expressing a HS6ST2 cDNA from a lentiviral construct (hHS6ST2) (n = 2). i, Fold change in HS3ST3A1 mRNA expression in A375 KDM2BC5 cells, and in A375 KDM2BC5 cells expressing a HS3ST3A1 cDNA from a lentiviral construct (hHS3ST3A1) (n = 2). Source data
a, Sanger sequencing of a A375 KDM2B SULF1 double knock-out clone (KDM2BC5 SULF1#1) after targeting with the indicated sgRNA. Both sequence changes result in frameshift mutations. b, Clonogenic assay under normal growth conditions. After 14 days, colony growth was quantified by methylene blue staining and absorption readings at 650 nm (t-test, n = 3). Scale bar = 5 mm.
Synthesis scheme for GNeo-biotin (1). Supplementary figure explaining gating strategy and Tables 1–3.
sgRNA enrichment (GNeo-SAP screen). sgRNA enrichment in the GNeo-SAP resistance screen. Counts: normalized read counts in sample treated with GNeo-SAP (average of two replicate experiments). Control mean: normalized read count in sample treated with PBS (average of two replicate experiments). Control stdev: normalized read count standard deviation in PBS-treated sample across two replicate experiments. Fold change: counts divided by control mean. P value: P value of negative binomial test. Significant: statistically significance (true/false), based on FDR = 0.1.
sgRNA enrichment (GNeo-Cy5 screen). sgRNA enrichment in the GNeo-Cy5 binding screen. Counts: normalized read counts in GNeo-Cy5-negative sample (average of two replicate experiments). Control mean: normalized read count in GNeo-Cy5-positive sample (average of two replicate experiments). Control stdev: normalized read count standard deviation in GNeo-Cy5-positive sample across two replicate experiments. Fold change: counts divided by control mean. P value: P value of negative binomial test. Significant: statistically significance (true/false), based on FDR = 0.1.
Gene enrichment scores (GNeo-SAP screen, permissive setting). Ranking of genes based on accumulated sgRNA enrichment (Methods) in the GNeo-SAP resistance screen. SigmaFC: gene-ranking score. P value: estimated P value of obtained gene-ranking score. Significant: statistically significance (true/false), based on FDR = 0.1. #sgRNAs: number of sgRNAs targeting a gene in the library. #signif. sgRNAs: number of gene-targeting sgRNAs reaching statistically significant enrichment.
Gene enrichment scores (GNeo-Cy5 screen, permissive setting). Ranking of genes based on accumulated sgRNA enrichment (Methods) in the GNeo-Cy5 FACS screen. SigmaFC: gene-ranking score. P value: estimated P value of obtained gene-ranking score. Significant: statistically significance (true/false), based on FDR = 0.1. #sgRNAs: number of sgRNAs targeting a gene in the library. #signif. sgRNAs: number of gene-targeting sgRNAs reaching statistically significant enrichment.
Gene candidates shared among both the GNeo-SaAP and GNeo-Cy5 screen (permissive setting). List of genes yielding significant enrichment under both the GNeo-SAP resistance and GNeo-Cy5 FACS screen. SigmaFC (FACS): gene-ranking score in FACS screen. #sgRNAs (FACS): number of gene-targeting sgRNAs reaching statistically significant enrichment in the FACS screen. SigmaFC (Saporin): gene-ranking score in resistance screen. #sgRNAs (Saporin): number of gene-targeting sgRNAs reaching statistically significant enrichment in the resistance screen.
Gene enrichment scores (GNeo-SAP screen, restrictive setting). Ranking of genes based on accumulated sgRNA enrichment (Methods) in the GNeo-SAP resistance screen. SigmaFC: gene-ranking score. P value: estimated P value of obtained gene-ranking score. Significant: statistically significance (true/false) at the 1% level (Sidak correction). #sgRNAs: number of sgRNAs targeting a gene in the library. #signif. sgRNAs: number of gene-targeting sgRNAs reaching statistically significant enrichment.
Gene enrichment scores (GNeo-Cy5 screen, restrictive setting). Ranking of genes based on accumulated sgRNA enrichment (Methods) in the GNeo-Cy5 FACS screen. SigmaFC: gene-ranking score. P value: estimated P value of obtained gene-ranking score. Significant: statistically significance (true/false at the 1% level (Sidak correction). #sgRNAs: number of sgRNAs targeting a gene in the library. #signif. sgRNAs: number of gene-targeting sgRNAs reaching statistically significant enrichment.
Transcriptomic analysis of KDM2B knockout cells. Raw RNA-seq reads from KDM2BC5 cells and Cas9-expressing control cells (three replicates each) and differential expression analysis. BaseMean: mean counts, normalized for sequencing depth. lfcSE: standard error of log2 fold change. Stat: Wald statistic (log2 fold change divided by lfcSE). P value: P value from negative binomial test. Padj: adjusted P value (FDR correction).
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Weiss, R.J., Spahn, P.N., Chiang, A.W.T. et al. Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate. Nat Chem Biol (2021). https://doi.org/10.1038/s41589-021-00776-9