Cell culture
Hepatocellular carcinoma-derived cells (HepG2) were purchased from ATCC (HB-8065). Cells were maintained in DMEM (gibco, 41966-029) containing 4.5 g/l glucose supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; HyCloneTM, SV30160.03) and 1% Penicilin/Streptamycin (gibco, 15140-122). Cells were cultured in a humidified atmosphere of 5% CO2, 95% air at 37°C. From those cells, HepG2 p53 knockout cell lines (HepG2 p53KO) were generated using p53 CRISPR/Cas9 KO plasmid (h) (sc416469, Santa Cruz Biotechnology). Hepatocellular carcinoma-derived cells (HepG2) were maintained in DMEM (gibco, 41966-029) (“growth medium”, GM) containing 4.5 g/l glucose supplemented with 1 % (v/v) heat inactivated fetal bovine serum (FBS; HyCloneTM, SV30160.03) and % Penicilin/Streptamycin (gibco, 15140-122). Cells were cultured in a humidified atmosphere of % CO2, 9 % air at 37°C. For starvation of cells, medium was removed, cells were washed with phosphate buffered saline (PBS; gibco, 10010-015), and treated in HBSS (gibco, 14175-053) (“starvation medium”, SM) containing 1 g/l glucose supplemented with 10 mM HEPES (gibco, 1560-080).
Compound treatments
For treatments with different small molecules, the following concentrations were used:
10 µM nutlin-3a (Biomol, Plymouth Meeting, PA, USA),
10 mM cycloheximide (CHX; Caymen Chem 14126).
10 µM MG132 (Calbiochem 474790)
20 nM FRAX597 (Selleckchem, S7271)
Cell lysis
Lysis buffers were supplemented with 1x protease inhibitor cocktail (PIC) (cOmplete Tablets EASYpack, Roche, 04693116001) and 1x PhosSTOP (Roche, 04906837001). Total protein lysates were obtained after washing the cells with PBS, and application of the respective buffers provided in each protocol segment.
Western Blot (WB)
Whole cell lysates: Cultured cells were scraped and collected in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1% SDS, 0.5% Na-deoxycholate, 1% NP-40, adjusted to pH 7.2 – 7.4) mixed with PhosStop and PIC. Samples were sonicated for 5 min (30 secs on/off) in a cooled water bath ultrasound sonicator (Bioruptor, Diagenode). Cell lysates were centrifuged (14500 rpm 15 min 4°C) and clear supernatants were used to measure protein concentrations with a bicinchoninic acid assay (BCA; Thermo Fisher Scientific). Immunoblotting was performed as described elsewhere (Prokesch et al. 2016), using 50 µg protein of each sample.
Subcellular fractionation: Cytoplasmic and nuclear fractions were separated using a method previously described (Baldwin, Rockland). Briefly, cells were scraped in hypotonic cytoplasmic lysis buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 1 mM, adjusted to pH 7.6) and incubated for 10 min on ice. After addition of IGEPAL 10% to a final concentration of 0.5%, cells were vortexed, incubated on ice for 1 min, vortexed and centrifuged for 4 min at 1500 rpm. The supernatant was collected as the cytoplasmic fraction. Crude nuclei cell pellet was washed once with cytoplasmic lysis buffer (without PIC/PhosSTOP) and resuspended in nuclear extraction buffer (usually one fifth of cytoplasmic buffer volume used; 20 mM Tris-HCl, 100 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.05% SDS and 25% (v/v) glycerol, adjusted to pH 8.0). The nuclear fraction was sonicated for 5 min (30 secs on/off) in a cooled water bath ultrasound sonicator (Bioruptor, Diagenode). After sonication, 100 U/ml benzonase (Chem Cruz, sc-202391) was added to the nuclear lysates and incubated on ice for 1 – 2 hours, vortexing the mixture periodically (15 min) to resuspend the pellet. Digested sample was centrifuged at 14500 rpm for 15 min and 4°C to precipitate any remaining cell debris. The supernatant was used as the nuclear fraction.
RNA isolation and reverse transcription
After cell lysis with RNA-Lysis-Puffer T (peqGOLD VWR, 12-TRK-88) RNA was isolated with PeqGOLD Total RNA Kit (C-Line) (peqGOLD VWR, 12-6634-02) according to the manufacturer’s manual. Sample purification and concentrations was measured with NanoDrop® ND-1000 (peqlab Biotechnologie GmbH). Reverse transcription was performed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Thermo Fischer Scientific, 4368814). RT Puffer (10x), 0,8 µl dNTP (100 mM), 2 µl RT Random Primer (10x), nuclease free water and MultiScribe Reverse Transcriptase (50 U/µl) were added to defined RNA concentrations. The thermo cycler program was set to 10 min at 25°C, 120 min at 37°C, 5 min at 85°C and subsequent cooling at 4°C. cDNA was diluted afterwards to a concentration of 1 - 20 ng/µl and stored at -80°C.
Real time quantitative PCR (qPCR)
qPCR was performed in 96-well plate or 384 well plates (BioRad, HSP3805) with a cDNA concentration between 1 - 20 ng/µl. A master mix of Blue SybrGreen qPCR (Biozym, 331416) and cDNA (2:1) were prepared and added to each well to pre-pipetted primer pairs (800 nM). After performing the qPCR in a C1000TM Thermal Cycler (BIO –RAD, CFX96TM Real-Time System) (Program: 1 cycle (10 min) at 95°C; 40 cycles: 15 sec at 95°C, 1 min at 60°C, 1 min at 72°C; 1 cycle: 30 sec at 95°C, 30 sec at 60°C, 30 sec at 95°C) data was analyzed with Bio-Rad CFX Manager 3.1. The list of primers is provided in Table S6.
Viability assay
For the viability assay, 2×105 cells/well were seeded in a 96-well plate (Thermo Fisher Scientific) in 200 µl of GM to reach full confluency. Cells were allowed to attach for 24 hours at 37°C in 5% CO2, after which the medium was discarded, and cells were washed with PBS and treated with either GM or SM for 24 hours. Cell viability was analyzed using an EZ4U assay (Biomedica Immunoassays) according to the manufacturer’s instructions. Briefly, at the end of treatment the media was replaced with fresh GM (200 µl/well) and 20 µl/well EZ4U working solution. After 2 hours of incubation at 37°C, the absorbance was measured at 492 nm with a reference-wavelength of 620 nm (Spark® 10M multimode microplate reader).
Molecular cloning
Inserts (p53 wt sequence or EGFP wt sequence) for V5-p53-miniTurbo-NES and V5-EGFP-miniTurbo-NES were amplified from Flag-p53 (Addgene #10838; Primer: F: 5’- ACAGCGCTAGCGAGGAGCCGCAGTCAGATC-3’ R: 5’-ACAGCGCTAGCGGATCCGTCTGAGTCAGGCCCTTCTGT-3’) or EGFP (Addgene #13031; Primer: F: 5’- ACAGCGCTAGCGTGAGCAAGGGCGAGGAG-3’ R: 5’-ACAGCGGCTAGCGGATCCCTTGTACAGCTCGTCCATGCCG AGAG-3’), cloned into the NheI site in V5-miniTurbo-NES (Addgene #107170) and validated by Sanger sequencing.
Proximity-biotinylation (BioID)
HepG2 p53KO cells were cultured in collagen-coated T75 flasks (approx. 1.5×108 cells) and transiently transfected (VIAfect transfection reagent, E4981, Promega) with either V5-p53-miniTurbo-NES or V5-EGFP-miniTurbo-NES. After 24 hours of transfection, intracellular biotinylation was induced by changing cell culture media to either GM or SM (medium composition described in subsection cell culture) both containing 250 µM biotin. Each group was analyzed in 3 biological replicates (GM_p53, SM_p53, GM_EGFP, SM_EGFP; 12 samples total). After 2 hours of biotin labeling, samples were harvested and processed as previously published (Roux, 2018). In brief, cells were lysed in lysis buffer (8 M urea, 50 mM Tris-HCl, pH 7.4) containing protease inhibitor and 1 mM dithiothreitol (DTT). After addition of 20% Triton X-100 to a final concentration of 1% and sonication for 10 min (30 secs on/off) in a cooled water bath ultrasound sonicator (Bioruptor, Diagenode), samples were subjected to affinity purification with streptavidin beads (Invitrogen, Cat.#65001) on a rotator o/n at 4°C. Afterwards, beads with captured biotinylated proteins were thoroughly washed (10 times) with washing buffer (50 mM Tris-HCl, pH 7.4, 8 M urea) and final washing steps were performed with 50 mM Tris-HCl pH 7.4 and 50 mM AMBIC containing 1 mM biotin to block remaining streptavidin on the beads. The beads including the captured proteins were reconstituted in 100 mM Tris-HCl pH 8.5, 2% sodium deoxycholate (SDC) and reduced/alkylated with 5 mM TCEP/30 mM chloroacetamide at 56°C for 10 min. Next, the proteins were quantified (using a BCA assay) and digested with 1:100 Lys-C and 1:50 trypsin overnight at 37°C with continuous shaking. Digestion was stopped by adding 1% trifluoroacetic acid (TFA) to a final pH of 2, in which SDC was completely precipitated. SDC and the beads were removed by centrifugation at 14000 rpm for 10 min and the supernatant, containing peptides was desalted on an Oasis HLB plate (Waters). Peptides were dried and dissolved in 2% formic acid before liquid chromatography-tandem mass spectrometry analysis.
Mass spectrometry run: One thousand ng of each sample was analyzed using an Ultimate3000 high-performance liquid chromatography system (Thermo Fisher Scientific) coupled to an EXPLORIS 480 mass spectrometer (Thermo Fisher Scientific). Buffer A consisted of water acidified with 0.1% formic acid, while Buffer B was 80% acetonitrile (ACN) and 20% water with 0.1% FA. The peptides were first trapped for 1 min at 30 µl/min with 100% Buffer A on a trap (0.3 × 5 mm with PepMap C18, 5 µm – 100 Å Thermo Fisher Scientific); after the trapping peptides were separated by a 50 cm analytical column packed with C18 beads (Poroshell 120 EC-C18, 2.7 µm, Agilent Technologies). The gradient was 9 – 40% B in 40 min at 400 nL/min. Buffer B was then raised to 55% in 5 min and increased to 99% for the cleaning step. Peptides were ionized using a spray voltage of 2 kV and a capillary heated at 275°C. The mass spectrometer was set to acquire full-scan MS spectra (350–1400 m/z) for a maximum injection time of 120 ms at a mass resolution of 60,000 and an automated gain control (AGC) target value of 300%. For a total cycle of 1 second the most intense precursor ions were selected for tandem MS (MS/MS). HCD fragmentation was performed in the HCD cell, with the readout in the Orbitrap mass analyzer at a resolution of 15,000 (isolation window of 1.4 Th) and an AGC target value of 200% with a maximum injection time of 25 ms and a normalized collision energy of 28%.
BioID data analysis: All raw files were analyzed by MaxQuant v1.6.17 software using the integrated Andromeda Search engine and searched against the Human UniProt Reference Proteome (October 2020 release with 75,088 protein sequences). MaxQuant was used with the standard parameters (“Label-Free Quantification”, “iBAQ” and “Match between runs” were selected with automatic values) with only the addition of Deamidation (N) as variable modification. Data analysis was performed with Perseus v1.6.14: proteins reported in the file “proteinGroups.txt” were filtered for reverse, potential contaminants and identified by site. For the quantitation, we used the iBAQ values calculated by MaxQuant and we kept only proteins found in at least 2 biological replicates in each group. At this point intensities were log10 transformed and missing values were imputed by Perseus with the automatic settings (width: 0.3, down shift: 1.8, mode: separately for each column) leading to 2,500 proteins left for statistical analysis with ANOVA testing (Benjamini-Hochberg FDR 0.05), z-score (mean per row) and hierarchical clustering (distance: Euclidian). Clusters were selected if enriched over EGFP background control under both nutrient conditions. Volcano bots were generated with Perseus 1.4.16 using the setting t-test, GM_p53 vs SM_p53, FDR0.05. KEGG overrepresentation analysis was performed in webgestalt.org [80] functional database ‘pathway – KEGG’ against ‘genome protein-coding’ reference list with 272 high confidence p53 interactors originating from the hierarchical clustering analysis (3 clusters enriched over EGFP background, Fig. 2C). In a second MaxQuant screen with the annotated proteins from the initial search only, biotinylated peptides were identified, by adding the modification Biotin(K)="C(10) H(14) N(2) O(2) S" (+226.077598) possible on any K or protein N-term as search criteria. The resulting biotinylated peptides were filtered for occurring only in the p53 samples (Table S2). Biotinylated peptides are added in the search output (PRIDE) with file name “Biotin (K)Sites.txt” together with the other submitted files. Unique peptides found only in p53 samples were analyzed in Perseus v1.6.14: peptides from “peptideGroups.txt” were first filtered for reverse, potential contaminant, identified by site and afterwards only for occurring in p53 samples (Table S2). For SAINTscore analysis, the peptide counts for each sample were retrieved from MaxQuant output file “proteinGroups.txt” and prepared as input file for analysis in the Contaminant Repository for Affinity Purification (CRAPome, lit). Analysis in CRAPome was conducted with experiment type: Proximity Dependent Biotinylation; Quantitation type: SPC without selecting CRAPome internal controls, instead only experimental controls as background (GM_p53, SM_p53, and CONTROL) EGFP control groups were merged to a single group, as we specifically look for proteins enriched over background control in both conditions (GM and SM), similar to the hierarchical clustering analysis. Interaction scoring was conducted with standard analysis options. For generating dot plots, the CRAPome output file was downloaded via the External Tools section and used as input file in ProHits-viz [48].
Fluorescence microscopy
Cells were seeded onto collagen-coated glass Chamber Slides and transfected with ViaFECT (Promega) according to the manufacturer’s protocol. After 18 hours, cells were washed two times with PBS and fixed with 1% paraformaldehyde (PFA; Thermo scientific) for 15 min at room temperature. PFA was removed and cells were washed two times with PBS. After washing, cells were blocked using Ultravision Hydrogen Peroxide Block (Thermo scientific, Rockford, USA) for 5 min at RT. Then, cells were incubated with primary antibodies (listed in Table 11) diluted in Antibody diluent (Dako, California, USA) for 45 min in a dark environment. Cells were then washed two times with PBS, followed by incubation with secondary antibody Anti-rabbit IgG (H+L) Alexa Fluor 555 (Cell Signaling) for 30 min in a dark environment. Secondary antibodies were diluted 1:1000 in Antibody diluent. After another two times PBS washing, nuclei were stained using DAPI 1:2000 (Thermo scientific) diluted in PBS for 5 min at RT. Finally, chambers were removed from the slides and the slides were washed twice with 1x PBS before mounting with ProLongTM Gold antifade reagent (Invitrogen) and applying a cover slip. Slides were stored in the dark until visualizing on the PALM Microbeam Laser Microdissection microscope (Zeiss, New York, USA) using 40x magnification. Images were analyzed using Zen software 2.3. Brightness and contrast were modified for enhanced visualization.
Expression and purification of recombinant proteins
Expression constructs for the fragments of p53 (Uniprot ID P04637) corresponding to amino acid 1 to 94 (p53TAD) and 94 to 312, as well as for PAK2 (Uniprot ID Q13177) corresponding to amino acid 1 to 212 were generated by synthesis of the corresponding optimized cDNA-constructs of p53 or PAK2 respectively and insertion of these cDNA into a pETM11-ZZ-His6 vector via NcoI/BamHI restriction digest.
For expression of recombinant unlabeled or 15N labelled ZZ-His6 proteins, the bacterial expression vectors were transformed into Escherichia Coli BL21-DE3 Star strain cells. Cells were either grown in lysogeny broth medium for unlabeled proteins or minimum medium supplemented with 6g 12C6H12O6 and 1 g 15NH4Cl at 37°C until they reached an OD600 of 0.8, when protein expression was induced by addition of 0.5 mM IPTG. After proteins were expressed at 20°C for 16 hours, cells expressing disordered fragments (p531–94 and PAK2 1–212) were harvested in denaturing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM Imidazol, 6 M Urea) and cells expressing folded fragments (p5394–312) were harvested in non-denaturing harvesting buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM Imidazol, 2 mM tris(2-carbocyethyl)phosphine)), followed by sonication and centrifugation at 6198 rcf for 45 min. Proteins were purified from the lysate using Ni-NTA agarose (Qiagen) and the ZZ-His6 tag was cleaved by 2% (w/w) His6-tagged TEV protease treatment for 16 hours at 4°C. After a desalting step (HiPrep 26/10, GE Healthcare) on an ÄKTA Pure system (GE Healthcare) into a low imidazole buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM Imidazol), the cleaved protein was separated from the uncleaved protein and the His6 tagged TEV protease by Ni-NTA affinity chromatography. Finally, the proteins were purified using size exclusion chromatography (Superdex 75 Increase, GE Healthcare).
Nuclear magnetic resonance (NMR) binding assay
For binding studies all proteins were equilibrated in the same buffer containing 20 mM Hepes pH 7.0, 50 mM NaCl, 2 mM TCEP. Samples for NMR measurements contained 100 µM 15N labelled p5394–312 in presence of 0 µM, 50 µM, 100 µM or 200 µM unlabeled PAK21–212 and 10% D2O. For titrations using 15N labelled PAK21–212 50 µM were used in presence 0 µM, 50 µM and 100 µM p5394–312 and 10% D2O. 1H15N HSQC spectra of the aforementioned samples were recorded at 25°C on a Bruker 600 Mhz Avance Neo NMR spectrometer equipped with a TXI room temperature probe.
Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis chromatin complexes
Wild-type (p53-proficient) HepG2 cells were grown to 100% confluency and treated with either SM or GM for 24 hours. After crosslinking with 1% FA (prepared in either SM or serum–free GM) for 8 min, the reaction was quenched with glycine at a final concentration of 0.1 M and cells washed within the flask twice with ice-cold PBS. Cells were immediately frozen at -80°C and stored in flasks until use. Cell lysates were prepared as previously described (Mohammed et al. 2016) and nuclei extracted for immune-precipitation. IP was conducted with either DO1X antibody for p53-specific pulldown or IgG2A isotype control. After thorough washing of the bead-bound proteins with AMBIC, proteins were digested on-bead with trypsin (1:50) o/n and for an additional 4 hours the next day. Derived peptides were desalted with C18 ultra micro spin columns and analyzed with LC-MS/MS.
Mass spectrometry run: One-tenth of each sample was measured by nano-HPLC (Dionex Ultimate 3000) equipped with an Aurora Series Emitter nanocolumn with CSI fitting (C18, 1.6 µm, 120 Å, 250 x 0.075 mm) (IonOpticks, Melbourne, Australia). Separation was carried out at 50°C at a flow rate of 300 nl/min using the following gradient. Solvent A is 0.1% formic acid in water and solvent B is acetonitrile containing 0.1% formic acid: 0-18 min: 2% B; 18-100 min: 2-25% B; 100-107 min: 25-35% B, 107-108 min: 35-95% B; 108-118 min: 95% B, 118-118 min: 95-2% B; 118-133 min: 2% B. The Bruker maXis II ETD mass spectrometer was operated with the captive source in positive mode with following settings: mass range: 150 - 2200 m/z, 4 Hz, precursor acquisition control top20 (CID), capillary 1600 V, dry gas flow 3 L/min with 150°C, nanoBooster 0.2 bar.
RIME data analysis: The MS/MS data were analyzed for protein identification and label-free quantification using MaxQuant 1.6.1.0 against the public database Swiss-Prot with taxonomy Homo sapiens and common contaminants (downloaded on 16.04.2019, 20482 sequences). Detailed search criteria were used as follows: trypsin, max. missed cleavage sites: 2; oxidation on Met as variable modification; search mode: MS/MS ion search with decoy database search included; precursor mass tolerance +/- 0.006 Da; product mass tolerance +/- 80 ppm; acceptance parameters for identification: 1% PSM FDR; 1% protein FDR and 1% site decoy fraction. In addition, a label free quantitation and iBAQ values including the match between runs feature of MaxQuant was performed (Cox et al. 2014) requiring a minimum of 2 ratio counts of quantified razor and unique peptides while omitting the normalization step. Data processing was performed using Perseus software version 1.6.6.0, contaminants and reverse proteins created during database search were removed. Intensities were log10 transformed in order to lower the effect of the outlier values, filtered for 3 valid values in either GM_p53 or SM_p53, or at least 4 valid values in total, allowed no values in the IgG control groups. Missing intensities were replaced with random values taken from the Gaussian distribution of values using default parameters (width: 0.3, down shift: 1.8, mode: separately for each column), in order to simulate a value for low abundant proteins. Two-sample t-tests and volcano blotting were used to identify altered proteins between GM and SM conditions. PCA analysis and multi-scatter plot were generated in Perseus. For SAINTscore analysis, the peptide counts for each sample were retrieved from MaxQuant output file “proteinGroups.txt” and prepared as input file for analysis in the Contaminant Repository for Affinity Purification (CRAPome, lit). Analysis in CRAPome was conducted with experiment type: Endogenous pull-down; Quantitation type: SPC without selecting CRAPome internal controls, instead only experimental controls as background (GM_p53, SM_p53, and IgG CONTROL) IgG control groups were merged to a single group, as we specifically look for proteins enriched over background control in both conditions (GM and SM). Interaction scoring was conducted with standard analysis options. For generating dot plots and comparison of average spectral counts, the CRAPome output file was downloaded via the External Tools section and used as input file in ProHits-viz [48].
Co-Immunoprecipitation (IP)
HA-p53 (Genscript, OHu20059C) and/or UGP2-FLAG (Genscript, OHu02074D) were transiently overexpressed in HepG2 p53KO cells with ViaFECT (Promega) according to the manufacturer’s protocol. After 24 hours of transfection, cell culture media was changed to either GM or SM and kept under those conditions for another 24 hours. Next, cells were harvested by washing with ice-cold PBS and lysed in Co-IP buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 5% glycerol; adjusted to pH 7.4) including PIC and PhosSTOP. After 30 min incubation on ice (passive lysis), the suspensions were sonicated for 5 min (30 secs on/off) in a cooled water bath ultrasound sonicator (Bioruptor, Diagenode) at 4°C. The lysates were centrifuged for 5 min at 14500 rpm and 4°C. Protein concentration was determined with a BCA protein assay kit. 500 µg of protein lysates was normalized to equal volumes with Co-IP buffer and used for precipitation. 30 µl Flag-beads slurry (Sigma, M8823) was used per mg protein, equilibrated in 500 µl Co-IP buffer for 3 times and incubated with the protein lysates over night at 4°C with reciprocal shaking (BioSan MultiBio RS-24). Next day, flow-through was collected and used for control blots. Beads were washed 3 times with Co-IP buffer and proteins eluted from the beads with elution buffer (100 mM glycine, pH 3) at room temperature for 5 min.
RNA-seq experiments and data analysis
Three SEQ libraries (TruSeq® Stranded mRNA Library Prep) each of vehicle control (pcDNA4/HisMax, Invitrogen) and FLAG-p53 (pcDNA3 flag-p53, Addgene) re-expressing cells (electroporation) were prepared from purified mRNAs of HepG2 p53KO cells under untreated (GM) or treated (SM) conditions for a total of 4 x 3 (12) libraries. Raw sequencing data was generated on an Illumina NextSeq 550 and mapped to the human genome (hg18) using STAR alignment (Dobin et al. 2013). Differentially expressed genes were found using DEseq2 package in R, data was transformed (VST), Wald-Test performed on individual genes, and Benjamini-Hochberg corrected for multiple testing. Genes with an adjusted P-value≤ 0.05 were used for further analysis.
p53 motif analysis and motif density
Analysis was conducted using HOMER (http://homer.ucsd.edu/homer/index.html) (Heinz et al. 2010). A motif file was generated based on p53 consensus sequence “GGACATGCCCGGGCATGTCY” (Human, hg18) derived from MotifMap (http://motifmap.ics.uci.edu/) (Daily et al. 2011) using HOMER’s seq2profile.pl, allowing 6 mismatches (Table Sx). Motif sites were identified via HOMER’s annotatePeaks.pl module in “TSS mode”, searching ± 10 kb around each gene’s TSS on either the sense or the antisense strand in the provided lists of genes of interest. A Gaussian density plot was generated in R (https://R-project.org/) (R Core Team, 2017) using the ggplot2 package (https://ggplot2.tidyverse.org) (Wickham H. 2016).