Data availability
All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Cell lines and reagents
HeLa, HEK293T, RCC4, HK-2, HepG2, cells were maintained in DMEM (Sigma Aldrich D6429) and supplemented with 10% foetal calf serum (FCS) and 100 units/ml penicillin with 100 µg/ml streptomycin, in a 5% CO2 incubator at 37 °C. 786-O cells were cultured in RPMI-1640 (Sigma R8758). HeLa HMGCR-Clover cells were generated as previously described12. HeLa mCherry CL1 cells have previously been described31. Cells were confirmed mycoplasma negative (Lonza, MycoAltert), and authenticated by short tandem repeat profiling (Eurofins Genomics). Cells were also cultured with lipid depleted serum as described for lipid deplete or sterol deplete conditions. Full details of reagents and antibodies used are shown in Supplementary Table 1.
Plasmids
All plasmids are detailed in the Supplementary Table 2. CRISPR sgRNAs were cloned into a lentiviral sgRNA expression vector pKLV-U6gRNA(BbsI)-PGKpuro2ABFP4640 or LentiCRISPRv241 as previously described42. Cas9 expressing cells were generated using LentiCas9 with puromycin, hygromycin or blasticidin as previously described43. sgRNA sequences are detailed in Supplementary Table 3.
SREBP2 constructs were generated from an I.M.A.G.E cDNA clone (Source Bioscience IRATp970B0781D 6169568) and cloned into the pHRSIN-pSFFV-HA backbone with puromycin resistance using NEBuilder HiFi (NEB). The primers are detailed in Supplementary Table 4.
Lentiviral production and transduction
Lentivirus was produced by transfection of HEK293T cells (Trans-IT 293 reagent, Mirus) or Fugene (Promega) at 70–80% confluency in six-well plates, with the appropriate pHRSIN vector and the packaging vectors pCMVR8.91 (gag/pol) and pMD.G (VSVG), as previously described42. Viral supernatants were harvested at 48 hr, filtered (0.45 μm filter), and stored at −80 °C. For transduction, cells were seeded on 24-well plates in 500 µl media, and 500 µl viral supernatant added. Plates were centrifuged at 1800 rpm at 37 °C for 1 hr. Antibiotic selection was applied from 48 hr.
CRISPR–Cas9 targeted deletions
Gene-specific CRISPR sgRNA sequences were taken from the TKO library, designed using E-CRISP (http://www.e-crisp.org/E-CRISP/) or VBC score (https://www.vbc-score.org/), with 5′CACC and 3′CAAA overhangs, respectively. SgRNAs were ligated into the LentiCRISPRv2 or pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector and lentivirus produced as described. Transduced cells were selected with puromycin, and were generally cultured for 9–10 days before subsequent experiments to allow sufficient times for depletion of the target protein. KO clones were isolated from the sgRNA-targeted populations by serial dilution or FACS. SgRNAs used are shown in Supplementary Table 3.
CRISPR/Cas9-mediated gene knock-in
The Hela HMGCR-Clover knock-in cells were generated as previously described12. HeLa SREBP2-Clover cells were generated using a knock-in donor template, in a similar method to HMGCR-Clover. SREBP2 homology arms were synthesised as gBlocks (IDT) and cloned into pDonor-Clover-LoxP using Gibson assembly (NEBuilder HiFi DNA), with ~1 kb flanking homology arms An EcoRV site was inserted upstream of KpnI in pDonor-Clover-LoxP to allow homology arms to be inserted without altering the Clover-LoxP cassette (Supplementary Table 4). The vector was cut using EcoRI and PvuI for the 5’ homology arm, and PacI and EcoRV for the 3’ homology arm. The final vector, pDonor-Clover-LoxP-SREBP2, was sequence verified and transfected into HeLa cells alongside Cas9 and sgRNA targeting the 3’ SREBP2 untranslated region (pSpCas9(BB)-T2A-Puro vector) (Supplementary Table 4). Cells were treated with puromycin for 14 days and expanded. The presence of SREBP2-Clover was verified by flow cytometry and immunoblot. Cells were then transfected with pHRSIN- pSFFV Cre pGK Hygro to remove the puromycin resistance and single cell clones isolated. HeLa SREBP2-Clover KI was confirmed by PCR amplification using primers SPfor and SPrev 3’-GAGTGGGAAGGAACAGGACAATTA-5’ (Supplementary Table 4). The presence of the 1596 bp fragment confirmed incorporation of Clover to the C-terminus in the knock-in cells (KI), and was not observed in the parental cells that only encoded WT SREBP2 (1269 bp).
CRISPR-Cas9 forward genetic screens
HeLa HMGCR-Clover screen: Hela HMGCR-Clover cells were first transduced with Cas9-Hygro and maintained under selection for at least 14 days. For the screen, 108 cells were transduced with the CRISPR knockout Bassik library44. Cells were transduced at an MOI of ~30% with the transduction efficiency determined by measurement of mCherry by flow cytometry 48 hr post transduction. Transduced cells were enriched by puromycin selection for at least seven days. To maintain an even representation of guides throughout the screen the unsorted library was routinely maintained in excess of 100 x 106 cells ensuring approximately 500X representation of each guide. After eight days, cells were seeded at 30% confluency and allowed to settle before being sterol depleted (DMEM +10% LPDS+10 μM mevastatin+penicillin/streptomycin) overnight. The next day, the cells were placed in hypoxia for 18 hr. Cells were prepared for FACS sorting, and CloverHIGH cells were collected. These cells were cultured for a further six days before being subjected to sterol depletion and hypoxia and FACS as detailed above. DNA was extracted from the control library (transduced cells which had not undergone FACS) and sorted cells using the Puregene® Core kit A (Qiagen) according to manufacturer’s instructions. The sgRNA locus was amplified using two rounds of PCR, and the amplicons sequenced by Illumina MiniSeq and HiSeq as previously described45. Reads were extracted, the first 19 bp trimmed using Cutadapt46 and then aligned against the Bassik sgRNA library using HISAT247. Read counts for each sgRNA were compared between conditions, and false discovery rates for each gene were calculated using MAGeCK48 (Supplementary Data 1). Amplification, indexing and sequencing primers are detailed in Supplementary Table 5.
HeLa SREBP2-Clover screen: The Toronto human knockout pooled library (TKOv3) was a gift from Jason Moffat (Addgene #125517)49,50. 7.2x107 HeLa SREBP2-Clover cells expressing Cas9 were transduced with pooled TKOv3 sgRNA virus to maintain greater than 200-fold coverage at multiplicity of infection 0.3. After 27 hr cells were selected with puromycin 1 μg/ml, expanded and were pooled prior to any selection event to maintain representation throughout the screen. On day 9, 2.4x108 cells were harvested, washed in PBS supplemented with HEPES 10 mM, resuspended in sort media (PBS with 10 mM HEPES and 2% FBS) and filtered. FACS was undertaken using an Influx cell sorter (BD) selected the top and bottom 1% of fluorescent cells into collection media (50% DMEM, 50% FBS, 2% Penicillin-Streptomycin). Gating was set with reference to non-transduced controls treated with bortezomib or 1% oxgyen. Selected cells were divided for expansion and for immediate DNA extraction (Puregene Core Kit A, Qiagen 51304, 158388). Simultaneously, 2x107 phenotypically non-selected transduced cells were harvested for library DNA extraction and 4x107 cells continued for the later library. Selected cells were further expanded before repeat sorting and DNA extraction on day 17. A two-stage PCR was completed to amplify inserts using the following primers, where NNNNNNN represents a barcode for multiplexing (Supplementary Table 5) Following PCR, DNA was purified (AMPure XP, Agencourt A63880), quantified via Bioanalyzer (Agilent DNA 1000, 5067-1504) and sequencing performed by NovaSeq (custom primer, Supplementary Table 5). Reads were extracted and analysed with reference to the TKOv3 library as previously described (Supplementary Data 2).
Sterol depletion
Cells were seeded at 60% confluency. The following day, cells were washed twice with PBS and before being incubated in sterol depletion media (DMEM, 10% LPDS, 10 μM mevastatin, penicillin/streptomycin). For hypoxic experiments, cells were sterol depleted for 24 hr in 21% oxygen, prior to being incubated in 1% oxygen for a further 18 hr still under sterol depletion. Where indicated, sterol depletion media was also added at the same time as the hypoxic incubation was started.
Hypoxic incubation
All hypoxic experiments were performed in either a Whitley H35 Hypoxystation (Don Whitley Scientific) or a SCI-tive Dual Hypoxia workstation (Baker Ruskinn) maintained at 1%-5% oxygen, 94% N2, 5% CO2 at 37°C. During harvesting cells were kept upon ice to minimise re-oxygenation of samples
Immunoblotting
Cells were lysed in an SDS lysis buffer (1% SDS, 50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol and 5 µl ml−1 Benzonase (Sigma)) unless stated otherwise. Samples were incubated on ice for 10 min before heating at 90 °C for 5 min, 70 °C for 15 min (HMGCR or SREBP2). In specified cases cells were lysed using an appropriate volume of either RIPA buffer (50 mM TRIS pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% Sodium deoxycholate, protease inhibitors), or Digitonin buffer (1% digitonin, protease inhibitors). Cells were lysed on ice for at least 15 min before being subjected to centrifugation at 16.9 xg for 10 min and the supernatants collected. 6X SDS sample buffer (Laemmli) was added to the supernatant, and the samples typically heated at 90°C for 5 minutes prior to analysis. Proteins were separated by SDS–PAGE, transferred to PVDF (polyvinylidenedifluoride) membranes, probed with appropriate primary and secondary antibodies and developed using enhanced chemiluminescent or Supersignal West Pico Plus Chemiluminescent substrate (Thermo Scientific).
Quantitative PCR
Total RNA was extracted using the RNeasy Plus minikit (Qiagen) following the manufacturer’s instructions and then reversed transcribed using Protoscript II Reverse Transcriptase (NEB). Template cDNA (20 ng) was amplified using the ABI 7900HT Real-Time PCR system (Applied Biotechnology or Quantstudio 7, Thermo Scientific) reactions Transcript levels of genes were normalized to a reference index of a housekeeping gene (β-actin). The primers sequences are shown in Supplementary Table 6.
Flow cytometry
Cells were harvested and washed twice with PBS by centrifugation before either being subjected to live cell flow cytometry (for Clover) or being fixed in 3.6% PFA in PBS. Cells were run on an LSRFortessa™ (BD Biosciences). Resulting data was analysed using the FlowJo software. For cell surface staining, cells were trypsinised, washed in PBS and incubated with 100 μl of the primary antibody made up in PBS for 30 minutes at 4°C. The primary antibody was removed by a PBS wash by centrifugation (500 xg, 5 minutes) before incubation with the appropriate Alexa Fluor conjugated secondary antibody for 30 minutes at 4°C in the dark. Two final PBS washes were performed before cells were fixed in PBS with 3.7% PFA before analysis on a BD LSRFortessa™ (BD Biosciences).
siRNA-mediated depletion
HeLa cells were transfected with ON-TARGETplus siRNA (Dharmacon) for MARCHF6, TRC8 or NADK (Dharmacon). The MISSION siRNA Universal Negative Control (Sigma Aldrich) was used alongside, and siRNA were transfected uses Lipofectamine RNAi MAX (Thermo Fisher) according to manufacturer’s instructions. Cells were harvested after 48 hr for further analysis by flow cytometry, qPCR or immunoblot.
Transient Transfection assays
Cells were passaged 24 hours prior to transient transfection to ensure they were in the exponential growth phase when transfected. Transient transfections in HeLa cells were carried out using TransIT-HeLaMonster® (Mirus) following the manufacturer's instructions. Briefly, the transfection mix (200 μl OptiMEM, 1 ug DNA, 5 μl TransIT and 1 μl Monster) was incubated for 30 min at room temperature before being added dropwise to cells at 70% confluency in a 3 cm dish. 24 hr post transfection, the cells were split 1:10 and the appropriate antibiotic selection added for up to 48 hr to enrich for successful transfection.
Cycloheximide chase assays
HeLa SREBP2-Clover cells were seeded as previously described and sterol depleted for 24 hor. Cells were incubated in 21 % or 1 % oxygen with 10 µg/ml of cycloheximide and/or 5 µM bortezomib (Velcade) for 0, 2, 4, and 6 hr and analysed by flow cytometry. Alternatively, cells were sterol depleted for 24 hr, and then sterols were re-introduced to trigger HMGCR degradation (2 μg/ml 25-hydroxycholesterol and 20 μg/ml cholesterol). Cells were harvested at the indicated time points before analysis by immunoblot.
SREBP2 Immunoprecipitation and ubiquitination assays
HeLa cells were seeded in 10 cm plates, and when they were at 70 % confluency they were incubated in 1 % oxygen with or without 5 µM bortezomib (Velcade) for 6 hr. Cells were lysed in 500 µl of RIPA (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with cOmplete Protease Inhibitor Cocktail and Denarase for 30 min at 4 °C. Lysates were centrifuged at 14,000 r.p.m. for 10 min, supernatants collected and then diluted with 500 µl of 1% Triton for preclearing with Protein G magnetic beads (Thermo Scientific) for 2 hr at 4 °C. Supernatants were then incubated with primary antibody overnight (rotation at 4 °C). Protein G magnetic beads were then added for 2 hr, and samples were then washed three times with 1% Triton and once with PBS. Bound proteins were eluted in 1× SDS loading buffer, separated by SDS–PAGE and immunoblotted.
Statin and lipid depletion sensitivity assays
Lipid depletion. HK-2 and RCC4 cells were plated on six-well plates at a density of 5 × 104 for HK-2 or 1 × 105 for RCC4 cells and cultured in DMEM, with 10% FCS or 10% LPDS for 3 days before cells were counted using a hemocytometer.
Statin sensitivity. HK2 or HeLa cells were plated on 96 well plates at a density of 5 × 103 cells per well for HeLa and 1 × 104 for HK-2. After 24 hr, cells were treated with 0-40 µM of Simvastatin and/or 1 % oxygen for 48 hr. Cells were washed with PBS, stained with 100 µl 2.5 µM Hoesht in PBS, and visualised using CLARIOstar Plate Reader at 355-20/455-30 nm.
NADPH/NADP+ analysis
1x106 HeLa cells were cultured in RPMI with de-lipidated serum reconstituted, with or without lipid mixture (1%), and/or hypoxia (0.5% Oxygen) for 48 hr before harvesting the cells. The NADP/NADPH Assay Kit (Abcam) was used to measure NADPH and NADP+ levels. Cells were washed 3 times with ice cold PBS and scraped cells with 800 mL extraction buffer, prepared as directed by the manufacturer’s protocol, and analysed in a plate reader at OD450 nm.
Liquid-chromatography metabolic measurements
[C13]glucose labelling and isotope tracing. HeLa cells were seeded on 15cm plates and allowed to recover. At 40% confluency the cells were cultured in DMEM or DMEM containing lipid depleted FCS, or DMEM containing 5µM mevastatin at 21% or 1% oxygen for 24hr. Cells were then supplemented with media containing [13C]glucose and labelled for a further 24 hr. Cells were washed in PBS and then harvested using trypsin. The cells were then washed twice in PBS and pellets were frozen at -80oprior to analysis by LC-MS.
LC-MS sample preparation. Washed cell pellets was added 1 mL of 2:1 chloroform:methanol followed by 400 µL of acetone and the samples were vortexed, sonicated and lysed after adding a 5 mm stainless steel ball bearing (Qiagen, Manchester, UK) using a VelociRuptor tissue lyser (SLS, Wilford, Nottingham, UK). After centrifugation at 20,000 g for 5 min the supernatant was transferred to a separate glass vial and dried down using a centrifugal evaporator (Savant, Thermofisher, Horsham, UK). Dried samples were reconstituted in 200 µL of 2:1:1 2-propanol: acetonitrile: water for analysis.
LC-MS analysis of cholesterol. Lipid analysis for cholesterol was carried out using a Waters Premier CSH C18 column (100 × 2.1 mm, 2.0 µm). Mobile phase A consisted of 6:4 acetonitrile: water with 10 mM ammonium formate and mobile phase B was 9:1 2-propanol: acetonitrile with 10 mM ammonium formate. For gradient elution of compounds mobile phase B started at 40% with an increase to 43% at 1.6 min, an increase to 50% at 1.7 min followed by a linear gradient to 54% B to 9.6 min, a further increase to 70% over 0.1 min followed by a linear gradient to 99% B over 4.7 min with re-equilibration for 1.5 min giving a total run time of 16 min. The flow rate was 0.5 ml/min and the injection volume was 5 µl. The needle wash used was 9:1 2-propanol: acetonitrile: water with 0.1% formic acid. Source parameters used for the mass spectrometer were a vaporizer temperature of 450°C and ion transfer tube temperature of 320°C, an ion spray voltage of 3.5 kV and a sheath gas of 55, auxiliary gas of 15 and a sweep gas of 3 arbitrary units with an S-lens RF (radio frequency) of 60%. For MS analysis a full scan of 120-1500 m/z was used in positive ion mode at a resolution of 70,000 ppm. All solvents and additives used for all extractions and mobile phases were LC-MS or Optima grade and obtained from Fisher Scientific or Merck.
LC-MS data processing. All data were acquired using Xcalibur (Version 4.1, ThermoFisher Scientific). Targeted processing was carried out using Xcalibur. In order to confirm identification of compounds retention times were validated against known external standard solutions. Where appropriate peak areas corresponding to cholesterol levels were normalized to unlabeled cholesterol to account for varying amounts of material (Supplementary Data 3).
Statistical analyses
Quantification and data analysis of experiments are expressed as mean ± standard deviation and P values were calculated using analysis of variance (ANOVA) or two-tailed Student’s t-test for pairwise comparisons, and were calculated using Graphpad Prism v.8. Qualitative experiments were repeated independently to confirm accuracy. Immunoblots were quantified using ImageJ software 51. The mutagenesis screens were analysed using MAGeCK v.0.5.548, as described earlier.