Mammalian cell culture growth conditions
Cell lines were obtained from American Type Culture Collection and verified using cell line fingerprinting. HCC70 and HCC1937 cell lines were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS, 20 mM HEPES (Thermo Fisher Scientific) and 1 mM Sodium Pyruvate (Thermo Fisher Scientific). MDA-MB-468 and OVKATE cell lines were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS, 20 mM HEPES (Thermo Fisher Scientific) and 0.25% (v/v) insulin (Human) (Clifford Hallam Healthcare). Cells stably-transduced with SMARTChoice lentiviral vectors were grown in the presence of 1 μg/mL puromycin.
Imaging
Immunohistochemistry images were obtained using an inverted epifluorescence microscope (Carl Zeiss, ICM-405, Oberkochem, Germany). Images were captured by the Leica DFC280 digital camera system (Leica Microsystems, Wetzlar, Germany). The Leica DM 5500 Microscope with monochrome camera (DFC310Fx) or Leica DMI SP8 Confocal with 4 lasers (405 nm, 488 nm, 552 nm and 638 nm) and two PMT detectors were used to capture standard fluorescent and confocal images.
SMARTChoice inducible lentiviral system
ID4 and control lentiviral shRNA constructs (SMARTchoice) were purchased commercially (Dharmacon, GE, Lafayette, CO, USA). Successfully transfected cells were selected using puromycin resistance (constitutive under the humanEF1a promoter). For ID4 knockdown analysis, HCC70 cells with SMARTChoice shID4 #1 #178657 (VSH6380-220912204), SMARTChoice shID4 #2 #703009 (VSH6380-221436556), SMARTChoice shID4 #3 #703033 (VSH6380-221436580), SMARTChoice shNon-targeting (VSC6572) and mock-infected cells. Cells were treated with vehicle control or with 1 μg/mL doxycycline for 72 hours before harvesting protein and RNA directly from adherent cells. The SMARTChoice shID4 #2 #703009 (VSH6380-221436556) produced the highest level of ID4 knockdown and was used for further analysis.
Non-lethal DNA damage induction with ionising radiation
Cells were seeded at 2.5 x105 (HCC70) or 2.2 x105 (MDA-MB-468) cells/well in a 6-well plate in normal growth medium. One day post seeding, cells were exposed to 2-5 Gy of ionising radiation using an X-RAD 320 Series Biological Irradiator (Precision X-Ray, CT, USA). Cells were returned to normal tissue culture incubation conditions and harvested at designated time points.
Gene expression analysis
Total RNA was prepared for using the miRNeasy RNA extraction kit (Qiagen), according to the manufacturer’s instructions. cDNA was generated from 1000ng RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) using oligo-dT primers and following the manufacturer’s instructions. qPCR analysis was used to analyse mRNA expression levels using Taqman probes (Applied Biosystems/Life Technologies) as per manufactures specifications (Table 2) using an ABI PRISM 7900 HT machine. qPCR data was analysed using the ΔΔCt method (64).
Gene
|
Taqman assay
|
ID4
|
Hs02912975_g1
|
B2M
|
Hs99999907_m1
|
GAPDH
|
Hs02758991_s1
|
NEAT1
|
Hs01008264_s1
|
MALAT1
|
Hs00273907_s1
|
ELF3
|
HS00963881_M1
|
GBA
|
HS00986836_G1
|
ZFP36L1
|
Hs00245183_m1
|
FAIM
|
HS00216756_M1
|
Genes analysed and the corresponding Taqman assay used to analyse their expression level
Protein analysis
Cells were lysed, unless specified, using RIPA [0.88% (w/v) Sodium Chloride, 1% (v/v) Triton X-100, 0.5% (w/v) Sodium Deoxycholate, 0.1% (w/v) SDS, 0.61% (v/v) Tris (Hydroxymethyl)Aminomethane and protease and phosphatase inhibitors (Roche)] or Lysis Buffer 5 (10 mM Tris pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100 and protease and phosphatase inhibitors). If required, protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Western blotting analysis was conducted as previously described (12). MDC1 protein was analysed using 3-8% tris/acetate gels and PVDF nitrocellulose membrane for MDC1 analysis (BioRad). All other proteins were analysed using the LiCor Odyssey system (Millenium Science, Mulgrave, VIC, Australia). Protein expression was analysed using antibodies targeting ID4 (Biocheck anti-ID4 rabbit monoclonal BCH-9/82-12, 1:40,000), β-Actin (Sigma anti-Actin mouse monoclonal A5441, 1:5,000) and MDC1 (Sigma anti-MDC1 mouse monoclonal M2444, 1:1000).
Co-immunoprecipitation
Co-immunoprecipitations (IP) were conducted using 10 μL per IP Pierce Protein A/G magnetic beads (Thermo Fisher Scientific) with 2 μg of antibody: IgG rabbit polyclonal (Santa Cruz sc-2027), ID4 (1:1 mix of rabbit polyclonal antibodies: Santa Cruz L-20: sc-491 and Santa Cruz H-70: sc-13047), MDC1 (rabbit polyclonal antibody Merck Millipore #ABC155). Beads and antibody were incubated at 4°C on a rotating platform for a minimum of 4 h. Beads were then washed three times in lysis buffer before cell lysate was added to the tube. Lysates were incubated with beads overnight at 4°C on a rotating platform. Beads were washed three times in lysis buffer and resuspended in 2 x NuPage sample reducing buffer (Life Technologies) and 2 x NuPage sample running buffer (Life Technologies) and heated to 85°C for 10 min. Beads were separated on a magnetic rack and supernatant was analysed by western blotting as described above.
Rapid Immunoprecipitation and Mass Spectrometry of Endogenous Proteins (RIME)
Cells were fixed using paraformaldehyde (PFA) (ProSciTech, Townsville, QLD, Australia) and prepared for RIME (38) and ChIP-seq (37, 65) as previously described. Cross-linking was performed for 7 min for RIME experiments and 10 min for ChIP-seq, ChIP-exo and ChIP-qPCR experiments. Samples were sonicated using a Bioruptor Standard (Diagenode, Denville, NJ, USA) for 30-35 cycles of 30 sec ON/30 sec OFF (sonication equipment kindly provided by Prof. Merlin Crossley, UNSW). IP was conducted on 60 (ChIP-seq/ ChIP-exo) to 120 (RIME) million cells using 100 μL beads/20 μg antibody. Correct DNA fragment size of 100-500bp was determined using 2% agarose gel electrophoresis.
Patient-derived xenograft tumour models were cross-linked at 4°C for 20 min in a solution of 1% Formaldehyde (ProSciTech), 50 mM Hepes–KOH, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors (H. Mohammed, personal communication). Samples (0.5 mg of starting tumour weight) were dissociated using a Polytron PT 1200E tissue homogeniser (VWR) and sonicated using the Branson Digital Sonifer probe sonicator (Branson Ultrasonics, Danbury, CT, USA) with a microtip attachment for 3-4 cycles of 10 x [0.1 sec ON, 0.9 sec OFF].
Mass spectrometry analysis was conducted at the Australian Proteomic Analysis Facility (APAF) at Macquarie University (NSW, Australia (66). Briefly, samples were denatured in 100 mM triethylammonium bicarbonate and 1% w/v sodium deoxycholate, disulfide bonds were reduced in 10 mM dithiotreitol, alkylated in 20 mM iodo acetamide, and proteins digested on the dynabeads using trypsin. After C18 reversed phase (RP) StageTip sample clean up, peptides were submitted to nano liquid chromatography coupled mass spectrometry (MS) (nanoLC-MS/MS) characterisation. MS was performed using a TripleToF 6600 (SCIEX, MA, USA) coupled to a nanoLC Ultra 2D HPLC with cHipLC system (SCIEX). Peptides were separated using a 15 cm chip column (ChromXP C18, 3 μm, 120 Å) (SCIEX). The mass spectrometer was operated in positive ion mode using a data dependent acquisition method (DDA) and data independent acquisition mode (DIA or SWATH) both using a 60 min acetonitrile gradient from 5 – 35%. DDA was performed of the top 20 most intense precursors with charge stages from 2+ – 4+ with a dynamic exclusion of 30 s. SWATH-MS was acquired using 100 variable precursor windows based on the precursor density distribution in data dependent mode. MS data files were processed using ProteinPilot v.5.0 (SCIEX) to generate mascot generic files. Processed files were searched against the reviewed human SwissProt reference database using the Mascot (Matrix Science, MA, USA) search engine version 2.4.0. Searches were conducted with tryptic specificity, carbamidomethylation of cysteine residues as static modification and the oxidation of methionine residues as a dynamic modification. Using a reversed decoy database, false discovery rate was set to less than 1% and above the Mascot specific peptide identity threshold. For SWATH-MS processing, ProteinPilot search outputs from DDA runs were used to generate a spectral library for targeted information extraction from SWATH-MS data files using PeakView v2.1 with SWATH MicroApp v2.0 (SCIEX). Protein areas, summed chromatographic area under the curve of peptides with extraction FDR ≤ 1%, were calculated and used to compare protein abundances between bait and control IPs.
Chromatin immunoprecipitation-quantitative real-time PCR analysis
Chromatin immunoprecipitation (ChIP) was conducted as described previously (65); however, following overnight IP, the samples were processed using a previously described protocol (67).
DNA was purified then quantified using quantitative real-time PCR analysis. Control regions analysed and primers used are listed in Table S4.
Relative enrichment of each region/primer set was calculated by taking an average of each duplicate reaction. The input Ct value was subtracted from the sample Ct value and the Ct converted using the respective PE for each primer set. The relative ChIP enrichment is then calculated by dividing the gene region of interest by the specific control region that is negative for both ID4 and H3K4Me3 binding (IFF01/NOP2 #1 primer). The formula for this normalisation is below:
ΦCt = Ctregion of interest – Ctinput region
ChIP enrichment = PE[-ΦCt(region of interest)] - PE[-ΦCt(IFF01)]
A sample was considered to be enriched if the fold-change over IgG control for each region was >2.
Chromatin immunoprecipitation-sequencing
Chromatin Immunoprecipitation and sequencing (ChIP-seq) was conducted as previously described (65). Samples were prepared and sequenced at Cancer Research United Kingdom (CRUK), Cambridge, UK. Antibody conditions for ChIP are the same as those used for RIME, with the addition of antibodies targeting H3K4Me3 (Active Motif #39159) and γH2AX (Ser139) (1:1 mix of Cell Signalling #2577 and Merck Millipore clone JBW301). Samples were sequenced at CRUK using an Illumina HiSeq 2500 single-end 50 bp sequencing. Quality control was conducted using FastQC (68) and sequencing adapters trimmed using cutadapt (69). Reads were aligned using Bowtie for Illumina v0.12.7 (70) followed by Sam-to-Bam conversion tool (71) and alignment using Bwa v0.705a (71). Alignment statistics were generated using samtools flagstat [version 0.1.18 (r982:295)] (71). ChIP-seq peaks were called using the peak calling algorithm HOMER v4.0 and MACS v1.4.2 (72, 73).
Chromatin Immunoprecipitation-exonuclease sequencing (ChIP-exo) was conducted as previously described (37). Samples were prepared and sequenced at CRUK.
qPCR analysis of ChIP DNA
Publicly available H4K3Me3 ChIP-sequencing data and the ID4 ChIP-sequencing data generated in this project were visualised using UCSC Genome Browser (genome.ucsc.edu and (74)). Regions of positive and negative enrichment were selected and the 500-1,000 bp DNA sequence was imported into Primer3, a primer design interface, web version 4.0.0 (75). Primers were designed with a minimum primer amplicon length of 70 bp. Primers were confirmed to align with specific DNA segments by conducting an in silico PCR using UCSC Genome Browser (genome.ucsc.edu and (74)). Oligo primers were ordered from Integrated DNA Technologies (Singapore). Primers were tested to determine adequate primer efficiency (between 1.7-2.3). All assays were set up using an EPmotion 5070 robot (Eppendorf, AG, Germany) and run on an ABI PRISM 7900 HT machine (Life Technologies, Scoresby, VIC, Australia). Briefly, reactions were performed in triplicate in a 384-well plate. Each reaction consisted of 1 μL 5 μM Forward primer, 1 μL 5 μM Reverse primer, 5 μL SYBR Green PCR Mastermix (Thermo Fisher) and 3 μL DNA. A standard curve was created using unsonicated, purified DNA extracted from the HCC70 cell line in 10-fold dilutions (1, 0.1, 0.01, 0.001, 0.0001).
PCR cycling was as follows: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. A dissociation step was conducted at 95°C for 15 sec and 60°C for 15 sec. Data was analysed and a standard curve created using SDS 2.3 software (Applied Biosystems). The slope was used to calculate the PE using the qPCR Primer Efficiency Calculator (Thermo Fisher Scientific, available at thermofisher.com).
Patient-derived xenograft and histology
All experiments involving mice were performed in accordance with the regulations of the Garvan Institute Animal Ethics Committee. NOD.CB17-Prkdcscid/Arc mice were sourced from the Australian BioResources Ltd. (Moss Vale, NSW, Australia). Assoc. Prof Alana Welm (Oklahoma Medical Research Foundation) kindly donated the patient-derived xenografts (PDX) models used in this study. Models were maintained as described elsewhere (47). Tumour chunks were transplanted into the 4th mammary gland of 5-week old recipient NOD.CB17-Prkdcscid/Arc mice. Tumours were harvested at ethical endpoint, defined as having a tumour approximately 1 mm3 in size or deterioration of the body condition score. At harvest, a cross-section sample of the tumour was fixed in 10% neutral buffered formalin (Australian Biostain, Traralgon, VIC, Australia) overnight before transfer to 70% ethanol for storage at 4°C before histopathological analysis. The formalin fixed paraffin embedded (FFPE) blocks were cut in 4 μm-thick sections and stained for ID4 (Biocheck BCH-9/82-12, 1:1,000 for 60 min following antigen retrieval using pressure cooker 1699 for 1 min, Envision Rabbit secondary for 30 min). Protein expression was scored by a pathologist using the H-score method (76).
Fluorescent In Situ Hybridisation
Tissue sections were analysed using Fluorescent In Situ Hybridisation (FISH) to examine the genomic region encoding ID4 (6p22.3). ID4 FISH Probe (Orange 552 nm-576 nm, Empire Genomics, NY, USA) was compared to the control probe CEP6 (Chromosome 6, Green 5-Fluorescein dUTP). This CEP6 probe marks a control region on the same chromosome as ID4 and is used to normalise ID4 copy number. Breast pathologist Dr Sandra O’Toole oversaw the FISH quantification for all samples.
Immunofluorescence and Proximity ligation assays
Immunofluorescence: Cells were seeded on glass coverslips (Coverglass, 13 mm, VITLAB, Germany). At harvest, media was removed, cells were washed twice with PBS without salts and fixed in 4% paraformaldehyde (PFA) (ProSciTech) for 10 min. Cells were again washed twice with PBS without salts (Thermo Fisher Scientific) before permeabilising for 15 min with 1% Triton-X (Sigma-Aldrich) in PBS and then blocking with 5% BSA in PBS without salts for 1 h at room temperature. Cells were washed twice with PBS without salts and antibodies were applied overnight at 4°C: ID4 (Biocheck BCH-9/82-12, 1:1,000), MDC1 (Sigma-Aldrich M2444, 1:1,000), BRCA1 (Merck Millipore (Ab-1), MS110, 1:250), γH2AX (Ser139) (Merck Millipore clone JBW301 05-636, 1:300), FLAG (Sigma-Aldrich M2, 1:500) and V5 (Santa Cruz sc-58052, 1:500). Cells were washed twice with PBS without salts then secondary antibodies were applied for 1 h at room temperature. Cells were washed twice with PBS without salts, with the second wash containing DAPI (1:500 dilution) and phalloidin (1:1,000 dilution) (CytoPainter Phalloidin-iFluor 633 Reagent Abcam ab176758). Cells were then mounted on slides using 4 μL of Prolong Diamond (Thermo Fisher Scientific).
Duolink Proximity ligation assay analysis (PLA): PLA was conducted using Duolink PLA technology with Orange mouse/ rabbit probes (Sigma-Aldrich, DUO92102) according to the manufacturers instructions. Images were captured using SP8 6000 confocal imaging with 0.4um Z-stacks. Maximum projects were made for each image (100-200 cells) and quantified using FIJI by ImageJ (77) as described previously (78). Quantification was conducted on a minimum of 50 cells. Data us represented as number of interactions (dots) per cell.
Quantification of DNA damage foci: Image quantification was conducted using FIJI v2.0.0 image processing software (Fiji is just ImageJ, available at Fiji.sc, (77)) as previously described (78). Four to five images were taken of each sample. The DAPI channel was supervised to enable accurate gating of cell nuclei for application to other channels. Size selection (pixel size 2,000 to 15,000) and circularity (0.30-1.00) cut-offs were used. Cells on the edge of the image were excluded from the analysis. The number of DNA damage foci per cell nucleus was calculated for approximately 100-200 cells. The information for individual samples was then collated and analysed using the Pandas package in Python 3.5.
Clinical Cohorts
Basal-like breast cancer: Samples were stratified into groups as follows: 42 BLBC (negative for ERα, PR, HER2 and positive for CK5/6, CK14 or EGFR), 14 triple negative non-BLBC (negative for ERα, PR, HER2, CK5/6, CK14 and EGFR) and 26 HER2-Enriched (negative for ERα and PR, positive for HER2). BRCA1-mutation status in this cohort us unknown, however it is expected to occur in approximately 6.5% of BLBC patients (79). Samples were obtained under the Garvan Institute ethical approval number HREC 08/145.
Kathleen Cuningham Foundation Consortium for research into familial breast cancer (KConfab): BRCA1-mutant BLBC was sourced from KConfab. A total of 97 BRCA1-mutant BLBC cases were obtained under the Garvan Institute ethical approval number HREC 08/145.
Ovarian Cancer: A total of 97 HGSOC cases were obtained under the Human Research Ethics Committee of the Sydney South East Area Hospital Service Northern Section (00/115) (51).