Cell culture
E006AA-hT (CRL-3277) and LNCaP (CRL-1740) cell lines were purchased from the ATCC. E006AA-hT cells were maintained in DMEM supplemented with 10% FBS. LNCaP cells were maintained in RPMI 1640 supplemented with 10% FBS. Cells were assessed regularly for mycoplasma contamination.
siRNA knockdown
Human DUSP1 siRNA SMARTpool (#L-003484–02-005) and non-targeting control pool (#D-001810–10-05) was purchased from Dharmacon. PC3 cells (2.5 × 105 cells per well) were seeded on 6 well plates. PC3 cells were transfected with 25 and 50 nM siRNAs using Lipofectamine RNAiMAX reagent (Life Technologies) according to the manufacturer’s instructions. Transfections were performed on two consecutive days. RNA and protein were collected 72 h after the first transfection. Whole cell lysates were harvested in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.1% SDS, 10 mM EDTA, 10 μg/mL aprotonin and leuptin, 1 mM PMSF, and 1 mM Na3VO4) and protein concentration was quantified using a Bradford Assay.
shRNA knockdown
DUSP1 and scramble shRNA were cloned into a pTRIPZ backbone. pTRIPZ plasmid and viral packaging plasmids were transfected into HEK 293 T cells with Lipofectamine Reagent (Thermo Fisher 18324012) (2 μg pMD2G, 3 μg psPAX2, and 5 μg pTRIPZ) and virus was collected and filtered after 48 h. Viral supernatant (4 mL) was supplemented with fresh media (2 mL) and Polybrene (8 μg/mL) and incubated with E006AA-hT cells for 4 h. After 48 h, cells were treated with puromycin (1 μg/mL). Once selected, shRNA expression was induced with 1 μg/mL doxycycline for 48 h.
sgRNA creation and design
sgRNAs were designed using the MIT guide RNA design tool (CRISPR.MIT.edu). sgRNAs with at least one mismatch to older LINE-1 (L1PA2-L1PA7) sequences were prioritized. Once sequences were selected, sgRNAs constructs were cloned by into the pEJS614_pTetR-P2A-BFPnls/sgNS by replacing sgNS sequence with LINE-1 targeting guide sequence. Guide sequences can be found in Supplementary Table 2.
DUSP1 overexpression
DUSP1 sequence was amplified from a DUSP1 GenScript plasmid (OHu10841D) and cloned into a pCW57-MCS1-2A-MCS2 backbone (Addgene #71782). Overexpression plasmid and viral packaging plasmids were transfected into HEK 293 T cells with Lipofectamine Reagent (Thermo Fisher 18324012) (2 μg VSV-G, 3 μg gag-pol, and 5 μg Overexpression plasmid) and virus was collected and filtered after 48 h. Viral supernatant (4 mL) was supplemented with fresh media (2 mL) and Polybrene (8 μg/mL) and incubated with E006AA-hT or LNCaP cells for 4 h. After 48 h, cells were treated with puromycin (1 μg/mL). Once selected, DUSP1 expression was induced with 1 μg/mL doxycycline for 48 h.
BCI treatment
E006AA-hT cells were seeded in a 6-well plate and incubated overnight at 37 °C. Cells were then treated with DMSO or BCI (Axon Medchem #2178) for 3 h at 37 °C. RNA was collected with the Qiagen RNeasy Plus Mini Kit (74134) as described below and assessed by qPCR.
RNA isolation and qPCR
RNA was isolated from cells using the Qiagen RNeasy Plus Mini Kit (74134) and contaminating DNA was digested using a Turbo DNA-free DNase digestion (Thermo Fisher Scientific AM1907) according to manufacturer’s protocol. cDNA was made using the Verso cDNA kit (Thermo Scientific- AB1453A). qPCR was conducted using SYBR Green Master Mix (Life Technologies 4344463) and relative mRNA levels were calculated using ΔΔCT. RPL19 was used as an internal control for normalization. Primer sequences can be found in Supplementary Table 2. qPCR LINE-1 primers have been previously published [63].
Chromatin immunoprecipitation
E006AA-hT and LNCaP cells (~ 20 × 106) stably expressing dSpyCas9-mCherry-APEX2 and gRNA were grown to 80% confluency. Cells were treated with 250 nM Sheild1 (Clontech) and 2 μg/mL doxycycline for 21 h to induce dCas9 expression. Cells were crosslinked with formaldehyde (1% formaldehyde in PBS) at room temperature for 10 min, and quenched with 1 mL of 2.5 M glycine, gently shaking for 5 min. Cells were washed with PBS and pelleted at 425xg for 5 min at 4 °C and resuspended in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCL, 0.5% NP-40, Halt protease inhibitor (Thermo Fisher-87786)). Suspension was re-pelleted and flash frozen in liquid nitrogen. Pellets were resuspended in 1 mL Farnham lysis buffer with Halt protease inhibitor, passed through a 25 gauge syringe 15 times, and spun at 425xg for 5 min at 4 °C. Pellets were resuspended in RIPA buffer (1 × PBS, 1%NP-40, 0.5% Na-deoxycholate, 0.1% SDS, Halt protease inhibitor) and passed through a 25 gauge syringe 20 times. Lysates were sonicated in a Diagenode Bioruptor for 30 min, 30 s on, 30 s off at 2 °C and spun at 20800xg for 10 min. Sheared DNA was incubated with 4 μg mCherry antibody (Thermo PA5-34,974) overnight at 4 °C and incubated with 50μL protein A/G beads for 3 h at 4 °C. Beads were washed 5 × with LiCl wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% Na-deoxycholate) for 3 min, and once with TE buffer (10 mM tris–HCl pH7.5, 0.1 mM Na2EDTA) for 1 min. Beads were resuspended in Proteinase K/SDS solution (0.5% SDS, 0.2 mg/mL Proteinase K, 1X TE) and incubated at 55 °C for 3 h and 65 °C overnight to reverse crosslinks. Samples were placed on magnetic strip to collect supernatant. 600μL of PB and 4μL of RNaseA (17500u/mL) were added to the samples, and samples were purified using the Qiagen PCR purification kit (28104). Sample was eluted twice with 35μL of 10 mM Tris pH 8. Illumina libraries were generated using the NEB Next DNA Library Prep Ultra II kit (E7645S) according to manufacturer’s protocol. Libraries were sequenced on an Illumina NextSeq 500. Reads were demultiplexed with Illumina bcl2fastq v2.20 requiring a perfect match to indexing BC sequences.
Cell sorting
LNCaP and E006AA-hT cells were treated with doxycycline (2 μg/mL) and Sheild1 (250 nM) for 21 h prior to sorting. Cells were sorted using the SONY SY3200 parallel sorter (SONY Biotechnology, San Jose, CA), using a 100 µm orifice nozzle and system pressure of approximately 25 psi. Double positive cells for mCherry and BFP were purified as previously described [54].
C-BERST assay
Biotinylation: Seven 15 cm plates of E006AA-hT or LNCaP cells (~ 6 × 107) expressing a gRNA and dCas9-APEX2 were treated with 2 μg/mL doxycycline and 250 nM Sheild 1 for 21 h. Cells were incubated for 30 min with biotin-phenol (500 μM) at 37 °C, and 1 mM H2O2 was then added to cells for 1 min at room temperature. To stop the biotinylation reaction, quencher solution (5 mM trolox, 10 mM sodium ascorbate, and 10 mM sodium azide) was added and cells were placed on ice. Three additional washes with quencher solution were performed, followed by two washes with PBS.
Nuclear Isolation: Cells were scraped from plates and centrifuged at 300 × g for 5 min at 4 °C. Pellet was resuspended in 7.5 nuclei isolation buffer (10 mM PIPES pH 7.4, 0.1% NP-40, 10 mM KCl, 2 mM MgCl2, 1 mM DTT and Halt protease inhibitor). Cells were incubated on ice for 10 min and ruptured using a Dounce homogenizer (~ 20x). Cells were further incubated on ice for 20 min and homogenization was repeated. Lysate was gently added to a sucrose cushion that contained 20 mL of 30% sucrose and 3.5 mL 10% sucrose (10 mM PIPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 30% or 10% sucrose, and 1 mM DTT). Sucrose cushion and cell lysate was spun at 1000 × g for 15 min. Supernatant was removed and nuclei (pellet) was resuspended in 800μL PBS. Suspension was spun at 1500 × g for 5 min at 4 °C. 500μL RIPA lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.125% SDS, 0.125% sodium deoxycholate, 1% Triton X-100) was added and samples were incubated at 4 °C. Lysates were sonicated in a Diagenode Bioruptor for 15 min (30 s on, 30 s off), and centrifuged for 10 min at 15,800 × g, 4 °C. Protein concentrations were measured using a Bradford Assay and samples were normalized.
Immunoprecipitation: MyOne Streptavidin T1 Dynabeads (Thermo Fisher 65,601) (400μL) were added to each sample and incubated at 4 °C overnight. Beads were washed with RIPA (twice), 1 M KCl, 0.1 M Na2CO3, 2 M urea in 10 mM Tris–HCl pH 8.0, and again with RIPA (twice). After washes, beads were processed for mass spectroscopy (see On-beads digestion of streptavidin-bound proteins). Protocol was based off of the previously described C-BERST technique [54].
Western blot/streptavidin blot
Cell lysates were boiled at 98 °C for 5 min in SDS loading buffer. Samples were resolved by SDS-PAGE on polyacrylamide gels, and transferred to PVDF using the BioRad Trans-Blot Turbo Transfer System. Blots were blocked in 5% BSA in TBS and probed with streptavidin-HRP (Thermo Fisher SA10001), ORF1p (Millipore MABC1152), DUSP1 (Cell Signaling 48625), or HSP90 (BD Biosciences 610419). Western blots were developed using BioRad Clarity Western ECL Substrate (1705060S) and visualized on an iBrightCL1000 Imager. Protein bands were quantified using ImageJ [64].
On-beads digestion of streptavidin-bound proteins
Streptavidin beads were washed twice with 1 mL 50 mM NH4HCO3 to exchange the buffer. Washed beads were then resuspended in 50 μl 50 mM NH4HCO3 containing 20 ng/μl trypsin/Lys-C (Promega) followed by overnight incubation at 37 °C with vigorous mixing in a thermoshaker (Eppendorf). After incubation beads were pelleted and supernatants were transferred to new tubes. Samples were acidified by adding 5 μl 20% heptafluorobutyric acid, incubated at room temperature for 5 min and clarified by 5-min centrifugation at 16000 g. Peptides from clarified samples were desalted using C18 spin tips (Thermo Scientific) according to manufacturer’s instructions. Desalted peptides were dried under vacuum and redissolved in 0.1% formic acid prior to LC–MS analysis. Peptide concentration was measured at 205 nm on Nanodrop One (Thermo Scientific).
LC–MS analysis
Peptides were analyzed by LC–MS on Orbitrap Fusion Lumos mass spectrometer coupled with Dionex Ultimate 3000 UHPLC. During each run, 0.5–2 μg of peptides from individual samples were injected and resolved on 50-cm long EASY-Spray column (Thermo Scientific) by 90-min long linear gradient of 4–40% acetonitrile in 0.1% formic acids at flowrate 0.25 μl/min. The method for data-dependent acquisition was based on published protocol [65] with exception that each cycle was set to last for 2 s instead of 3 s.
Peptides identification and label-free quantitation was done in the Proteome Discoverer 2.1. The protein database for Sequest HT search engine included human proteome downloaded from UniProt (www.uniprot.org) and amino acid sequence of streptavidin from Streptomyces avidinii. Parameters were set to search for peptides of at least 5 amino acids long, containing at most 2 missed trypsin cleavages. Dynamic modifications were set to include: phosphorylation of serine, threonine or tyrosine, acetylation of protein N-terminus, mono- and dimethylation of lysine and arginine. MS1-based label-free quantitation was done using the “Precursor Ions Area Detector” module in Proteome Discoverer. Samples were first normalized by intensity of streptavidin detected. Samples with an area value of 0 were replaced with the lowest MS1 intensity detected. Next, H2O2 only (endogenous biotinylation) values were subtracted from the + H2O2 samples (gRNA NS, gRNA 4 and gRNA7). Mean was calculated from replicates and targeted guides (gRNA 4 and gRNA 7) were divided by gRNA NS values for each protein detected. Proteins with at least 1.5 × greater than gRNA NS control were considered enriched and included in further analysis.