Aim and design of the study
The aim was to study the difference of histone modifications in breast cancer tissues compared with normal tissues in the global scale. Cancer tissues and corresponding normal tissues were collected. Mass spec of histone modifications was performed and confirmed with western and patient tissue chips. Then, ChIP-seq study was performed to identify the different enhancer sites. Finally, an inhibitor of H3K27ac, which represent enhancer activity, was applied to test its effect on cancer.
Materials and reagents
Antibodies, H3K4me3 (clone MC315, Millipore 04-745), H3K27ac (Abcam Ab4729), H3K27me3 (clone C36B11, CST 9733), H4K8ac (Abcam Ab45166 for ChIP-Seq, IHC and western, Abclonal A7258 for western), H4K12ac (CST 13944), H3K27me1 (clone D3R8N, CST 84932), H3K23ac (CST 14932), H3K23me1 (Active motif, 39388), H3K9ac (clone C5B11, CST 9649), H3K9me2 (Abclonal A2359), H3K9me3 (Abcam ab176916), H3K4me1 (CST 5326), H4K20me1 (Abcam ab9051), H3K36me3 (Abcam ab9050), H4K20me3 (Abclonal A2372), H3 (Abcam Ab1791), p300 (Abcam ab14984), and β-actin (Abclonal AC026), were purchased from the indicated merchants.
Mice feeding and tissue collection
FVB/N-Tg(MMTV-PyVT)634Mul/J transgenic mice [34] and wild-type FVB mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (http://www.nbri-nju.com/). All mice were housed in the SPF grade room of Animal Center of College of Life Sciences, Wuhan University. All the animal operations were following the laboratory animal guidelines of Wuhan University and were approved by the Animal Experimentations Ethics Committee of Wuhan University (Protocol NO. 14110B).
Heterozygous female MMTV-PyVT (Mtv/-) mouse was backcrossed onto wild-type male FVB mouse and we selected male MMTV-PyVT (Mtv/-) mice for mating, female MMTV-PyVT (Mtv/-) and female wild-type FVB mice for subsequent experiments. Mammary tumors were gotten from female MMTV-PyVT in 12 weeks old; meanwhile, normal mammary glands from female wild-type FVB of 12 weeks old were taken out. Tumors grown in thoracic mammary gland were selected as the tumor samples, 1 mouse for 1 biological replicate. Abdominal and inguinal mammary glands were selected as the normal tissue samples, 3 mice for 1 biological replicate because of the low amount of tissue in normal mammary gland.
Tissue microarray and IHC
The tissue microarray slide was purchased from Xi’an Alenabio Co., Ltd. (Cat. NO. BR1503e and BR1505b). The BR1505b slide contained 75 cases (150 cores) of breast invasive ductal carcinoma samples. The BR1503e slide contained 3 cases (6 cores) of adjacent normal breast tissue, 3 cases (6 cores) of breast fibroadenoma samples, 2 cases (4 cores) of breast cystosarcoma phyllodes samples, 7 cases (14 cores) of breast intraductal carcinoma samples, and 60 cases (120 cores) of breast invasive ductal carcinoma samples.
The slides were deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval. For immunohistochemistry (IHC) analysis, the sections were incubated with 3% H2O2 for 15 min at room temperature to quench endogenous peroxidase activity. After incubating in normal goat serum for 1 h, sections were treated with primary antibody at 4 °C overnight. IHC analysis of tumor samples was performed using primary antibodies against H4K8ac (dilution 1:200; Abcam ab45166) and H3K27ac (dilution 1:200; Abcam ab4729). The sections were then washed three times in PBS and treated for 30 min with biotinylated goat-anti-rabbit IgG secondary antibodies. After washing three times in PBS, sections were incubated with streptavidin-conjugated HRP. After washing three times in PBS for 5 min each, specific detection was developed with 303-diaminobenzidine (DAB-2031). Images were taken by Leica Aperio VERSA 8 digital pathology scanner.
For information about other experimental methods, please refer to the supplemental materials.
Molecular subtypes of breast cancer
Molecular subtypes of breast cancer were determined as follows: Luminal A—breast cancer is hormone-receptor positive (estrogen-receptor and/or progesterone-receptor positive), HER-2 negative, and has low levels of the protein Ki-67. Luminal B—breast cancer is hormone-receptor positive (estrogen-receptor and/or progesterone-receptor positive) and either HER-2 positive or HER-2 negative with high levels of Ki-67. HER-2-type breast cancer is hormone-receptor negative (estrogen-receptor and progesterone-receptor negative) and HER-2 positive. Basal-like breast cancer is hormone-receptor negative (estrogen-receptor and progesterone-receptor negative) and HER-2 negative.
IHC image analysis
The digital slides were taken by Leica Aperio VERSA 8 digital pathology scanner and then analyzed by Aperio colocalization image analysis algorithm. First, we use ImageScope to view the digital slides and select the appropriate area to calibrate the color and threshold of hematoxylin and DAB. Then, we create an algorithm macro and register it on the eSlide Manager. The eSlide Manager provides a convenient tool for batch analysis of slides. We used the selected algorithm macro to analyze the digital slides. The percent of DAB represented the protein concentration.
Histone extraction for LC-MS/MS analysis
Mouse breast tissues were minced and homogenized on ice in 1:10 (wt/v) NIB buffer within 10 mM sodium butyrate, 1 × protease inhibitor cocktail, 1 × phosphatase inhibitor cocktail, and 0.2% NP-40. After homogenization for 10 min, the nuclei were pelleted by centrifugation for 5 min at 1000g. The nuclei samples were washed gently three times in 1:10 (v/v) NIB buffer without 0.2% NP-40. Histones were then extracted from the nuclei in 1:5 (v/v) 0.2 M H2SO4 for 3 h and precipitated by 33% TCA on ice overnight. Histones were washed using ice-cold acetone supplemented with 0.1% HCl and then ice-cold acetone without 0.1% HCl, respectively. Histones were dried in a vacuum centrifuge. The concentration of extracted histone was measured by the bio-rad protein assay. The purity and quality of histones were affirmed by 15% SDS-PAGE analysis and Coomassie staining.
Proteomics studies of histone modifications
Buffer preparation
The buffer was prepared with the following: HPLC-grade acetonitrile (ACN) (Millipore), propionic anhydride (Sigma), sequencing-grade modified trypsin (Promega), HPLC buffer A:0.1% formic acid (Sigma), and HPLC buffer B: 0.1% formic acid in ACN.
Chemical derivatization and digestion of histones
Twenty micrograms of histone proteins were redissolved in 30 μl 50 mM NH4HCO3. The lysine residues and N-terminus of histone were chemically derivatizated twice by propionic anhydride with 1:3 (v/v) acetonitrile at 37 °C and completely dried. Histone proteins were then re-suspended in 30 μl 50 mM NH4HCO3 and digested by 1:10 (wt/wt) trypsin at 37 °C overnight. Subsequently, the histone peptides were derivatizated twice with propionic anhydride to label the peptide N-terminus generated from the trypsin digestion and dried completely via a vacuum centrifuge.
Stage tip desalting
Stage tips were prepared in the house using double-layered reversed-phase material C18 in 200 μl of tip. Stage tips were washed using 70% ACN/0.1% TFA and balanced using 0.1% TFA. Histone peptides were redissolved in 0.1% TFA and loaded in stage tips twice. The stage tips within histone peptides were washed twice by 0.1% TFA for removing extra salt. Histone peptides were eluted from stage tips using 50% ACN/0.1% TFA and 70% ACN/0.1% TFA and dried in a vacuum centrifuge.
LC-MS/MS of histone peptides
Histone peptides were resuspended with HPLC buffer A. Peptides were loaded on to 100 μm i.d. × 2-cm Reprosil-Pur C18-AQ (5 μm; Thermo Scientific, CA) trap column and separated by 75 μm i.d. × 25-cm Reprosil-Pur C18-AQ (2 μm; Thermo Scientific, CA) analytical column. The HPLC gradient was as follows: from 5 to 35% buffer B in 50 min, from 35 to 95% buffer B for 5 min, and 95% buffer B for 5 min at a flow rate of 250 nL/min. NanoLC was coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). For data-independent acquisition (DIA), two full-scan MS spectra (m/z 300–1100) at a resolution of 60,000 were acquired in the orbitrap within a DIA duty cycle, and 16 MS/MS were performed at a resolution of 60,000 with an isolation window of 50 Da. Normalized collision energy (NCE) was set to 27.
Data analysis
The raw DIA MS data were processed using Epiprofile, which allowed quantitative analysis of histone peptides with multiple PTMs. Based on previous knowledge of histone peptide masses and elution profiles, the retention times and peak areas of histone peptides were extracted under the curve in mass spectra. A histone peptide with multiple PTMs was quantified in relative abundance, which was the percentage of the area of this histone peptide with particular PTM in summed total area of the histone peptide in all forms. The relative abundance of a single histone mark was obtained by summing the relative abundances in all the modified peptide forms. All quantitative results were expressed as the mean ± SD (standard deviation). For normal and tumor comparison, data were analyzed by homoscedastic t test based on biological replicates. Differences were considered statistically significant when the p values were less than 0.05.
Epigenomic studies of histone modifications of mouse tissues
Chromatin immunoprecipitation (ChIP) assay of mouse tissues
Sixty milligrams of each tissue was cut into 1-mm3 pieces in PBS with protease inhibitor. Tissue pieces were cross-linked for 10 min at room temperature with 1% formaldehyde and then quenched with 0.125 M of glycine for 5 min. Cross-linked tissues were triturated by trituration equipment for 30 s and then centrifuged at 12000 rpm, 4 °C for 5 min. Supernatant with massive oil was discarded and the precipitates were lysed with 1 mL lysis buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 5 mM EDTA) and incubated for 5 min with gentle rotation. After centrifugation at 12000 rpm, 4 °C for 2 min, lysates were washed once by digestion buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2, 0.2% Triton X-100). Washed lysates were incubated in 630-μL digestion buffer with 1-μL MNase (NEB, M0247S) at 37 °C for 20 min and then quenched with 8 μL 0.5 M EDTA. Whole lysates were sonicated and the supernatants were taken out after centrifugation. Thirty microliters of supernatants were taken for checking the efficiency of MNase digestion. Immunoprecipitation was further performed with 150-μL sheared chromatin, 2-μg antibody, 50-μL Protein G sepharose beads and 800-μL dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) overnight at 4 °C. Next day, immuno-complexes were washed once with Wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer II (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer III (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Na-deoxycholate, 1% NP-40), and twice with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The immune complexes were eluted twice with 100-μL elution buffer (1% SDS, 0 .1M NaHCO3, 20 mg/mL Proteinase K) at room temperature. The elution was incubated at 65 °C for 6 h and then purified with DNA purification kit (TIANGEN DP214-03).
ChIP-seq library construction
ChIP-seq libraries were constructed by VATHS Universal DNA Library Prep Kit for Illumina (Vazyme ND604). Briefly, 50 μL of purified ChIP DNA (8–10 ng) was end-repaired for dA tailing, followed by adaptor ligation. Each adaptor was marked with a barcode of 6 bp which can be recognized after mixing different samples together. Adaptor-ligated ChIP DNA was purified by AMPure XP beads (1:1) and then amplified by PCR of 11–13 cycles with the primer matching with adaptor universal part. Amplified ChIP DNA was purified again using AMPure XP beads (1:1) in 35-μL EB elution buffer. For multiplexing, libraries with different barcode were mixed together with equal molar quantities by considering appropriate sequencing depth (30–40 million reads per library). Libraries were sequenced by Illumina Hi-seq X Ten platform with pair-end reads of 150 bp.
RNA-seq library construction
For both normal mammary gland and tumor samples, a 20-mg tissue was used for RNA extraction. Total RNA was extracted by using EASYspin RNA Mini Kit (Aidlab RN07). Briefly, tissue was triturated by trituration equipment for 20 s in lysis buffer provided by kit and then centrifuged at 12000 rpm, 4 °C for 5 min. Liquid between precipitates on the bottom and oil on the top was taken out and pipetted 10 times using 1 mL syringe. The entire volume of liquid was added into adsorption column provided by kit and RNA would retain in column while other components include DNA and protein would be washed out by several buffers. Total RNA eluted in 50-μL RNase-free water. RNA-seq libraries were constructed by NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490) and NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB E6111). Briefly, mRNA was extracted by poly-A selected with magnetic beads with poly-T and transformed into cDNA by first and second strand synthesis. Newly synthesized cDNA was purified by AMPure XP beads (1:1) and eluted in 50-μL nucleotide-free water. Subsequent procedures were the same as ChIP-seq library construction described previously except the sequencing depth of 20 million reads per library. RNA-seq libraries were sequenced by Illumina Hi-seq X Ten platform with pair-end reads of 150 bp.
ChIP-seq data processing
All ChIP-seq raw fastq data were cleaned by removing the adaptor sequence. Cutadapt (version 1.16,
http://cutadapt.readthedocs.io/en/stable/guide.html
) was used for this step with the parameters -u 10 -u -15 -U 10 -U -15 -m 30. Cleaned reads were aligned to the mouse reference genome (mm10) using BWA (version 0.7.15,
http://bio-bwa.sourceforge.net
) with the default settings. Peaks calling was finished by MACS2 (version 2.1.1,
https://github.com/taoliu/MACS
) with the parameters --nomodel --keep-dup all -p 1E-10 --broad --broad-cutoff 1E-10 --extsize 147. Replicates of ChIP-seq data were pooled for downstream analysis.
RNA-seq data processing
All RNA-seq raw fastq data were removed of adaptor sequence as the same way of ChIP-seq data processing. Cleaned reads were mapped to the mouse reference genome (mm10) using TopHat (version 2.1.1,
http://ccb.jhu.edu/software/tophat/index.shtml
) with the default settings. The gene expression level was calculated by Cufflinks (version 2.2.1,
http://cole-trapnell-lab.github.io/cufflinks
) and normalized by fragments per kilobase of bin per million mapped reads (FPKM).
Comparison between different histone markers and between replicates
For the comparison in genome-wide, the entire genome was divided into massive 2 kb windows and the enrichment of modification in each bin would be considered to calculate correlation. For the comparison in enhancers, signal of modification in each enhancer was normalized by reads of bin per million mapped reads (RPM) and such enrichment was used for calculating correlation.
Identification of typical and super-enhancers
Enhancers were identified by the algorithm developed by Richard A. Young, 2013. Briefly, significant distal H3K27ac peaks (peak boundary 1.5 kb or peak center 3 kb away from gene TSS) were identified, and the peaks whose distance was shorter than 12.5 kb were merged together as distal enhancers. The distal enhancers were ranked by the total signal of H3K27ac, and a plot was drawn to show the increased H3K27ac signal. Then, a tangent line with slope 1 was found of the curve and the intersection point was determined as the infection point. Enhancers above this point were defined as super-enhancers; meanwhile, enhancers below this point were defined as typical enhancers.
Identification of H3K4me3-enriched typical and super-enhancers
Firstly, we identified significant distal H3K4me3 peaks using the same way of defining significant distal H3K27ac peaks described previously, and nearby peaks of a distance shorter than 12.5 kb were merged together. Similar to the strategy to identify super-enhancers, H3K27ac in the regions were ranked and a plot was drawn. A tangent line with slop 1 was drawn and the intersection point was found. The enhancers above the intersection point were defined as H3K4me3 super-enhancer.