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Cholesterol Accessibility at the Ciliary Membrane Controls Hedgehog

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Cholesterol Accessibility at the Ciliary Membrane Controls Hedgehog 1 Cholesterol accessibility at the ciliary membrane controls 2 Hedgehog signaling 3 4 Maia Kinnebrew1#, Ellen J. Iverson1#, Bhaven B. Patel1, Ganesh V. Pusapati1, Jennifer H. Kong1, 5 Kristen A. Johnson3, Giovanni Luchetti1,5, Kaitlyn M. Eckert6, Jeffrey G McDonald3,6, Douglas F. 6 Covey4, Christian Siebold2, Arun Radhakrishnan3* and Rajat Rohatgi1* 7 8 1Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, 9 California, United States of America 10 2Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, 11 Oxford, UK 12 3Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, 13 TX 75390 14 4Department of Developmental Biology and Taylor Family Institute for Innovative Psychiatric 15 Research, Washington University School of Medicine, St. Louis, Missouri, United States of 16 America 17 5Current address: Department of Physiological Chemistry, Genentech, South San Francisco, CA 18 6Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 19 75390 20 21 #MK and EJI contributed equally to this study. 22 *Correspondence to [email protected] or [email protected] 23 Abstract 24 Previously we proposed that transmission of the Hedgehog signal across the plasma membrane 25 by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is 26 cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can 27 cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct 28 pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by 29 a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. 30 Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol 31 in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol 32 accessibility in the membrane of the primary cilium by inactivating the transporter-like protein 33 Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across 34 the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can 35 be modified by extracellular ligands to control the activity of cilia-localized signaling proteins. 36 37 38 39 Introduction 40 A long-standing mystery in Hedgehog (HH) signaling is how Patched 1 (PTCH1), the receptor 41 for HH ligands, inhibits Smoothened (SMO), a G-protein-coupled receptor (GPCR) family 42 protein that transduces the HH signal across the membrane (Kong et al., 2019). We and others 43 demonstrated that cholesterol directly binds and activates SMO and proposed that PTCH1 44 regulates SMO by restricting its access to cholesterol (Byrne et al., 2016; Huang et al., 2016; 45 Luchetti et al., 2016). Biochemical studies show that PTCH1 can bind and efflux sterols from 46 cells (Bidet et al., 2011), and structural studies highlight the homology of PTCH1 to the 47 cholesterol transporter Niemann-Pick C1 protein (NPC1) (Gong et al., 2018; Kong et al., 2019; 48 Kowatsch et al., 2019; Qian et al., 2019; C. Qi et al., 2019; Qi et al., 2018a, 2018b; Zhang et al., 49 2018). However, the resolution of the PTCH1 cryo-EM structures is not high enough to 50 distinguish cholesterol from other sterol lipids as PTCH1 substrates, and PTCH1 transport 51 activity has not yet been demonstrated in a purified system or at endogenous expression levels 52 in cells. 53 A challenge to this model is presented by the fact that cholesterol constitutes up to 50% 54 of the lipid molecules in the plasma membrane (Colbeau et al., 1971; Das et al., 2013; Lange et 55 al., 1989; Touster et al., 1970): how can such an abundant lipid be kept away from SMO to 56 prevent inappropriate pathway activation? Indeed, other less abundant lipids can bind and 57 regulate SMO activity, including oxysterols, phosphoinositides, endocannabinoids and 58 arachidonic acid derivatives (Arensdorf et al., 2017; Jiang et al., 2016; Khaliullina et al., 2015; 59 Nachtergaele et al., 2012). Side-chain oxysterols, synthesized through the enzymatic or non- 60 enzymatic oxidation of cholesterol, are appealing alternatives to cholesterol because of their 61 lower abundance, higher hydrophilicity and structural similarity to cholesterol (Corcoran and 62 Scott, 2006; Dwyer et al., 2007). To find the endogenous lipidic activator of SMO, we took an 63 unbiased genetic approach to identify lipid-related genes whose loss influences the strength of 64 HH signaling. 65 Results 66 A focused CRISPR screen targeting lipid-related genes 67 Using our previously described strategy (Pusapati et al., 2018b) to identify positive and negative 68 regulators of the HH pathway, we conducted focused loss-of-function CRISPR screens using a 69 custom library targeting 1,244 lipid-related genes compiled by the LIPID MAPS consortium 70 (Supplementary File 1 provides a list of all genes and guide RNAs in the library). This CRISPR 71 library targeted all annotated genes encoding enzymes involved in the synthesis or metabolism 72 of lipids as well as proteins that bind or transport lipids. We used a previously characterized 73 NIH/3T3 cell line (NIH/3T3-CG) that expresses Cas9 and GFP driven by a HH-responsive 74 fluorescent reporter (GLI-GFP) (Pusapati et al., 2018b). To ensure that HH signaling in the 75 plasma membranes of these cells would be sensitive to perturbations of endogenous lipid 76 metabolic pathways, we minimized sterol uptake from the media by growing the entire 77 population of mutagenized cells in lipoprotein-depleted media for one week prior to the screen 78 and then further treating with U18666A, a drug that a drug that traps any residual lipoprotein- 79 derived cholesterol in lysosomes (Figure 1A) (Lu et al., 2015). In the screen for positive 80 regulators, we treated cells with a high, saturating concentration of the ligand Sonic Hedgehog 81 (HiSHH) and used Fluorescence Activated Cell Sorting (FACS) to collect poor responders, 82 those with the lowest 10% of GLI-GFP fluorescence (Figure 1A,1B; full screen results in 83 Supplementary File 2). These candidate positive regulators are hereafter referred to as 84 “HiSHH-Bot10%”. In the screen for negative regulators, we treated cells with a low, sub- 85 saturating concentration of SHH (LoSHH) that activated the reporter to <10% of maximal 86 strength and selected super-responders, cells with the top 5% of GLI-GFP fluorescence (Figure 87 1A,1C; full screen results in Supplementary File 3). These candidate negative regulators are 88 hereafter referred to as “LoSHH-Top5%”. 89 The screens correctly identified all four positive controls included in the library: Smo and 90 Adrbk1 (or Grk2) as positive regulators and Ptch1 and Sufu as negative regulators (Figure 91 1B,1C). In addition, genes previously known to influence HH signaling (Gnas) and protein 92 trafficking at primary cilia (Inpp5e) were amongst the most significant hits (Chávez et al., 2015; 93 Garcia-Gonzalo et al., 2015; Regard et al., 2013). In addition to Inpp5e, other genes involved in 94 phosphoinositide metabolism (Mtmr3 and Plcb3) were also significant hits. Pla2g3, which 95 encodes a secreted phospholipase, was identified as a negative regulator of HH signaling, an 96 effect that may be related to its known role as a suppressor of ciliogenesis (Figure 1D)(Gijs et 97 al., 2015; Kim et al., 2010). 98 To identify lipid species that influence HH signaling, we separately analyzed the 99 intersection of all genes expressed in NIH/3T3-CG cells based on RNAseq and annotated as 100 part of a lipid metabolic pathway in the Kyoto Encyclopedia of Genes and genomes (KEGG) 101 (Figure 1D; gene lists used for each pathway are shown in Supplementary File 4; RNAseq 102 data in Supplementary File 5). Statistically significant hits clustered in two major pathways: (1) 103 genes encoding enzymes in the cholesterol biosynthesis pathway were positive regulators of 104 HH signaling and (2) genes encoding enzymes in the sphingolipid biosynthesis pathway were 105 negative regulators (positive regulators are shown in blue and negative regulators in orange in 106 Figure 1D). We focused on these two pathways for the work described in the rest of this study. 107 108 Enzymes in the cholesterol biosynthesis pathway positively regulate Hedgehog signaling 109 Mutations in Dhcr7 and Sc5d, which encode enzymes that catalyze the terminal steps in 110 cholesterol biosynthesis, impair HH signaling in target cells and cause the congenital 111 malformation syndromes Smith-Lemli-Opitz and lathosterolosis, respectively (Blassberg et al., 112 2016; Cooper et al., 2003; Horvat et al., 2011; Porter and Herman, 2011). In addition to these 113 genes, many of the genes encoding enzymes in the pathway that converts squalene to 114 cholesterol were statistically significant hits with a FDR-corrected p-value threshold of 0.1 in the 115 HiSHH-Bot10% screen (Figure 2A). Of the post-squalene cholesterol biosynthesis genes that 116 did not meet this threshold, Nsdhl came close (FDR-corrected p-value = 0.25, Supplementary 117 File 2) and Tm7sf2 is redundant (Sharpe and Brown, 2013), leaving Cyp51 as the only gene 118 that was not identified. CRISPR-mediated loss-of-function mutations in Lss, required for an early 119 step in the pathway, and in Dhcr7 and Dhcr24, required for the terminal steps, impaired the 120 transcriptional induction of endogenous Gli1 (an immediate target gene used as a measure of 121 signaling strength) (Figure 2B, 2C; CRISPR-edited alleles shown in Figure 2--figure 122 supplement 1A). Quantitative mass spectrometry measurements and intact cell staining with a 123 cholesterol-binding probe both confirmed that the abundance of cholesterol was reduced in both 124 Dhcr7-/- and Dhcr24-/- cells (Figure 2--figure supplement 1B,1C). Conversely, the abundances 125 of substrates for Dhcr7 and Dhcr24, 7-dehydrocholesterol and desmosterol, respectively, were 126 elevated (Figure 2--figure supplement 1D,1E).
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