Distinct modulation of IFNγ-induced transcription by BET bromodomain and catalytic P300/CBP inhibition in breast cancer

Cell lines and reagents

AT3 breast cancer cell lines were obtained from the institutional cell line bank at the Peter MacCallum Cancer Center (PMCC; Melbourne, Australia) and tested bi-monthly for the presence of mycoplasma. Cells were cultured in vitro for < 3 months before a fresh aliquot was thawed to minimize culture adaptations/genetic drift. AT3 cells were grown at 37 °C and 10% CO2 in Gibco Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 u/mL), and streptomycin (100 mg/mL). JQ1 was provided by Dr. James E. Bradner (Boston MA, USA). Catalytic P300/CBP inhibitors A-241 and A-485 were provided by AbbVie (North Chicago, IL, USA). All small molecules were reconstituted at 10 mM in 100% dimethyl-sulfoxide (DMSO) and stored at – 20 °C. Recombinant murine and human IFNy was purchased from BioLegend (Catalog #575304 and # 570206, respectively) and diluted to 20 µg/mL in 0.5% BSA in PBS and stored in single-use aliquots at – 80 °C.

Immunoblot

AT3 cells were pre-treated with A-241 (250 nM) for 1 h prior to the addition of recombinant murine IFNy (1 ng/mL) for an additional 2 h. Cells were harvested by centrifugation and washed once in ice-cold PBS prior to whole cell lysis using Lamelli buffer (60 mM Tris HCl pH 6.8, 10% v/v glycerol, 2% v/v glycerol SDS) and incubated at 95 °C for 5–10 min until completely homogenized. Cell lysate protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, 23225) according to the manufacturer’s instructions. Prior to running SDS-PAGE, protein lysates were prepared with sample loading buffer (120 mM Tris HCl pH 6.8, 20% v/v glycerol, 4% w/v SDS, 71.5 mM β-mercaptoethanol, bromophenol blue). Protein lysates were separated on Mini-PROTEAN TGX 4–15% gels (Bio-Rad, 465-1086) prior to transfer at 0.25A onto Immobilon-P (IPVH00010) membranes in transfer buffer (25 mM Tris HCl, 192 mM Glycine, 5% v/v methanol) at 4 °C. Membranes were blocked with Tris-buffered saline (TBS) supplemented with 5% w/v skim milk powder. Next, membranes were incubated overnight using the following primary antibodies: anti-phospho[Y701]-STAT1 (#9167, Cell Signaling Technologies), anti-STAT1[total] (#610185, BD Biosciences), and anti-α-Tubulin (#05-829, Millipore Sigma). Membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h and washed at least three times in TBS supplemented with 0.1% v/v Tween20. Immunoreactive bands were revealed using ECL reagents (Amersham ECL or ECL Prime, GE Healthcare) by film exposure (Fujifilm Super RX, Fujifilm) using an Agfa CP1000 developer (Agfa). For both of the cropped immunoblots presented in Fig. 4B, the corresponding uncropped blots are also shown in Additional File 5: Fig. S5A, B.

TCGA correlation analysis

RNA-Seq by Expectation Maximization (RSEM) [31] scaled expression values for TCGA were downloaded from the GDAC Firehose website [32]. Entrez gene IDs were mapped to HGNC gene symbols using the biomaRt (v2.42) R package [33] and collapsed to unique values per gene symbol by selecting the most variable entrez ID among all samples for each gene symbol. Primary samples from the TCGA BRCA cohort were selected using the TCGAbiolinks (v2.14.0) R package [34] and were matched with progression-free interval end points from the TCGA Pan-Cancer Clinical Data Resource [35]. IFNg signature scores were calculated using the Singscore (v1.6) R package [36] from a set of genes found to be strongly interferon induced across multiple cell lines (Additional file 1: Fig. S1C). Samples were then stratified into 'High' (top 90th percentile) and 'Low' (bottom 10th percentile) signature score groups and log-rank p values were calculated using the Survival (v2.38) R package [37].

RNA-sequencing

5e6 AT3 cells were plated in technical triplicate and each pre-treated with A-241 (250 nM), A-485 (1 µM), or DMSO vehicle for 1 h prior to the addition of recombinant murine IFNy (1 ng/mL), or vehicle control, for an additional 2 h (3 h total incubation with small molecules). Following indicated treatments, cells were collected by centrifugation and washed once with ice-cold PBS prior to resuspension in TRIzol™ (ThermoFisher Scientific, 15596026). RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research, R2052) according to the manufacturers instructions and eluted in 50 µL nuclease-free H2O. Sequencing libraries were prepared by the Molecular Genomics Core Facility (Peter MacCallum Cancer Center) with 500 ng input RNA using the QuantSeq 3’-mRNA Seq Library Prep Kit for Illumina (Lexogen, Vienna, Austria). Libraries were then pooled and sequenced on the Illumina NextSeq 500 to obtain 75 b.p. single-end reads. Sequencing files were demultiplexed using Bcl2fastq (v2.17.1.14) to generate individual FASTQ files on which QC was performed using FASTQC (v0.11.5). Sequencing reads were trimmed using cutadapt (v1.7) and aligned to the mouse reference genome (GRCm38/Mm10) using HISAT2 (v2.1.0). Read counting across genomic features was performed using featureCounts and the following settings: -p -T 20 -O -F GTF -t exon. Differential gene expression analysis was performed on the resultant counts matrix in Rstudio (v3.5.1) using the Limma/Voom workflow [38, 39]. Gene set enrichment analyses were performed using Gene Set Enrichment Analysis (GSEA) software (v3.0; https://www.gsea-msigdb.org/gsea/index.jsp) using pre-ranked (ranked by t-statistic) and enrichment plots were re-plotted from GSEA output using replotGSEA function in Rstudio. All additional figure generation for RNA-sequencing datasets was performed in in Rstudio (v3.5.1).

ChIP-sequencing

Cells were pre-treated with JQ1 (1 µM), A-241 (250 nM), or A-485 (1 µM) for 1 h prior to the addition of recombinant murine IFNy, or vehicle control, for an additional 2 h (3 h total incubation with small molecules). Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) was performed with reference exogenous genome (ChIP-Rx) using a modified protocol [27]. MM1.S cells (25e6/IP) were cultured in the presence or absence of A-485 or DMSO vehicle for indicated timepoints. At harvest, cells were washed once in ice-cold PBS prior to cross-linking. For cross-linking, 1/10th volume of fresh formaldehyde solution (11% formaldehyde, 0.05 mM EGTA, 1 mM EDTA, 100 mM NaCl, 50 mM Hepes–KOH pH 7.5) was added and incubated for 20 min at room temperature with rotation. Cross-linking was quenched by the addition of 1/20th volume of 2.5 M glycine and incubated for 5 min at room temperature with rotation. For isolation of nuclei, cell pellets were washed once in ice-cold PBS and then resuspended in ice-cold nuclear extraction buffer (0.5% NP-40, 2 mM EDTA, 10 mM NaCl, 20 mM Tris–HCl pH 8) and incubated for 5 min on ice. Following three sequential incubations in nuclear extraction buffer, cell nuclei were pelleted and resuspended in sonication buffer (0.3% SDS, 1% NP-40, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl pH 7.5) at a concentration equivalent to 50e6 cells per mL. Samples were sonicated in 12 × 24 mm Covaris tubes using the Covaris S2 instrument for 18 min using the following settings: 20% Duty Cycle, 1000 cycles/burst, and 10 Intensity. Prior to immunoprecipitation, sheared chromatin was diluted 1:1 in ChIP dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl pH 8) and quantified using Qubit dsDNA HS assay kit. For ChIP-Rx, sheared Drosophila chromatin from S2 cells was spiked into immunoprecipitations at 1:40 ratio of Drosophila/human and processed as a single sample until ChIP-Rx normalization following DNA sequencing. Immunoprecipitations were performed overnight (12–16 h, 4 °C, with rotation) using Protein A and Protein G Dyna beads (Invitrogen) and the following antibodies: H3K27Ac (Abcam, ab4729) and IRF1 (Santa Cruz Biotechnology Inc., sc-497). Samples were washed once with ChIP dilution buffer, wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris–HCl pH 8), wash buffer 2 (0.5% deoxycholate, 0.5% NP-40, 2 mM EDTA, 250 mM LiCl, 20 mM Tris–HCl pH 8), and TE buffer (1 mM EDTA, 10 mM Tris–HCl pH 7.5) prior to incubation in reverse cross-linking buffer (200 mM NaCl, 100 mM NaHCO3, 1% SDS, 300 μg/mL Proteinase-K) for 4 h at 55 °C with shaking. Finally, the supernatant was reverse-cross-linked overnight (12–16 h) at 65 °C prior to ChIP DNA isolation using Zymogen ChIP DNA Clean and Concentrator Kit (Zymo Research, D5205). For ChIP-Rx, libraries were generated using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645) and sequenced on an Illumina NextSeq 550 with 75 b.p. single-end reads. Library QC and quantification were performed using D1000 high-sensitivity screen tape with 4200 TapeStation Instrument (Agilent Technologies), and the size is selected between 200 and 500 bp using a Pippin Prep system (Sage Science).

ATAC-sequencing

Cells were pre-treated with JQ1 (1 µM) or A-241 (250 nM) for 1 h prior to the addition of recombinant murine IFNy (1 ng/mL), or vehicle control, for an additional 2 h (3 h total incubation with small molecules). Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) was performed using an improved protocol to reduce mitochondria from the transposition reaction [40]]. Briefly, 5e5 MM1.S cells were cultured in duplicate with JQ1, A-485, or DMSO vehicle as described above. Cells were washed once in ice-cold PBS and lysed in ATAC lysis buffer (0.1% Tween-20, 0.1% NP-40, 3 mM MgCl2, 10 mM NaCl, 10 mM Tris HCl pH 7.4). Tagmentation was then performed with Tn5 transposase and 2 × TD Buffer (Nextera DNA Library Prep Kit, Illumina) for 30 min at 37 °C (in a thermocycler). Tagmented DNA was immediately purified using a MinElute column (Qiagen, #28004) and then amplified for 12 cycles using 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) and Illumina-compatible/barcoded primers. The amplified libraries were purified using MinElute columns (Qiagen) and sequenced on an Illumina NextSeq 500 with 75 b.p. single-end reads. Library QC and quantification were performed using D1000 high-sensitivity screen tape with 4200 TapeStation Instrument (Agilent Technologies), and the size was selected between 200 and 500 bp using a Pippin Prep system (Sage Science).

ATAC-seq and ChIP-seq analysis

Sequencing files were demultiplexed using Bcl2fastq (v2.17.1.14) to generate Fastq files on which QC was performed using FASTQC (v0.11.5). Sequencing reads were then aligned to custom reference genome consisting the mouse genome (Mm10) and the Drosophila melanogaster genome (Dm3) using Bowtie2 (v2.3.3). The resulting SAM files were converted to BAM files using Samtools (v1.4.1) using the view command, which were subsequently sorted, indexed, and potential PCR duplicates removed using the rmdup function. BAM files were converted into BigWig files using the bamCoverage function (Deeptools, v3.0.0) using the following settings (—normalizeUsing CPM—smoothLength 150—binSize 50—e 200 scaleFactor 1). For experiments with external normalization, the reads mapping to either Mm10 or Dm3 genomes were quantified using FeatureCounts (Subread package, v1.5.0) and the percentage of mapped Dm3 reads as a total of total mapped Dm3 + Hg19 reads was calculated. A scale factor was then calculated as the ratio of Dm3 reads in the control treatment condition and the treatment sample, which was then manually applied as the scaleFactor in the bamCoverage function. BigWig files were imported into Integrative Genomics Viewer (IGV, v2.7.0) for visualization of specific loci. Using Deeptools (v3.0.0), heatmaps were generated by computing read average read density (from BigWig files) across defined genomic intervals using computeMatrix, which we subsequently plot using the plotHeatmap command. Average profile plots were created using matrices generated by computeMatrix using a custom script in Rstudio. Annotation of putative super-enhancer regions from H3K27ac ChIP-seq data was performed using Ranking Ordering of Super-Enhancer (ROSE) using a 12.5 k.b. stitching distance and a 2.5 k.b. TSS exclusion to reduce promoter bias. Peak calling was performed with MACS2 with default parameters. Annotation of ATAC-Seq/ChIP-Seq peaks to proximal genes was performed using annotatePeaks.pl (Homer, v4.8). Rstudio (v1.1.46) and R (v3.5.1) were used for all additional analyses and figure preparation using the following R packages: ggplots2, rcolorbrewer.

Super-enhancer and Coltron analysis

MACS2 (v2.2.1) was used for identification of (1) ATAC-seq peaks on BAM files from vehicle-treated and IFNγ-treated cells using the following parameters:—call-summits—nomodel—extsize 300, and (2) H3K27ac peaks on BAM files from vehicle-treated and IFNγ-treated cells using the following parameters:—cutoff-analysis. ATAC-seq and H3K27ac ChIP-seq peaks (.narrowPeak) mapping to ENCODE’s recommended Blacklisted regions [41] for the Mm10 genome were then excluded using bedtools intersect. Rose2 (v1.0.5) was then used to identify super-enhancers using the following parameters: -g mm10 -s 12,500 -t 2000. Coltron (https://pypi.org/project/coltron/) was used to perform core regulatory circuit analysis using the following parameters: -g MM10 -l 300. Rose2 and Coltron steps were repeated for both vehicle-treated and IFNγ-treated samples. Rstudio (v1.1.46) was used for figure preparation.

Data availability

RNA-sequencing data of B16-F10 cells stimulated with IFNγ or vehicle control were downloaded from NIH’s Gene Expression Omnibus (GEO) under the accession number GSE134264. RNA-sequencing of MC38 cells stimulated with IFNγ or control was downloaded from GEO (GSE112252). RNA-sequencing of AT3 cells stimulated with IFNγ ± JQ1, or vehicle control, from our previous study was downloaded from GEO (GSE94057). ChIP-sequencing for RNA polymerase II, BRD4, IRF1, and H3K27ac in AT3 cells stimulated with IFNγ ± JQ1, or vehicle control, from our previous study was downloaded from GEO (GSE94130). Next-generation sequencing data generated in this study have been deposited in the NIH’s Gene Expression Omnibus (GEO) under the accession number GSE201883.

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