Cell culture

MCF7 (HTB-22), ZR-75-1 (CRL-1500), CAMA-1 (HTB-21), MDA-MB-453 (HTB-131), MDA-MB-231 (HTB-26), OVCAR-3 (HTB-161) and Kuramochi (JCRB0098) cells were obtained from the American Type Culture Collection. MCF7 ESR1^Y537S cells were provided by S. Chandarlaparty³⁵. MCF7, ZR-75-1, MDA-MB-231 and CAMA-1 cells were cultured in RPMI-1640 medium (11875-093, Gibco) supplemented with 10% fetal bovine serum (FBS; SH30054.03, GE HyClone; SFBS-AU, Bovogen Biologicals), 1× GlutaMAX (35050061, Gibco) and 1% HEPES (15630080, Gibco). OVCAR-3 and Kuramochi cells were cultured in RPMI-1640, supplemented with 10% FBS and 1× GlutaMAX. MDA-MB-453 cells were cultured in DMEM supplemented with 10% FBS. All cells were maintained at 37 °C and 5% CO₂ in a humidified incubator. Cells were maintained below 90% confluency and routinely tested to confirm lack of mycoplasma contamination via PCR testing. Cell lines were authenticated by STR fingerprinting.

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In vitro drug treatment studies

Abemaciclib methanesulfonate (HY-16297) and palbociclib isethionate (HY-A0065) were purchased from MedChemExpress. INX-315 was obtained from Incyclix Bio. Fulvestrant (S1191) and imlunestrant (E1301) were purchased from Selleck Chemicals. All drugs were diluted in dimethyl sulfoxide (DMSO) for in vitro studies. Unless otherwise noted, abemaciclib and palbociclib were used at 500 nM, INX-315 was used at 300 nM, fulvestrant was used at 5 nM and imlunestrant was used at 40 nM.

For cell cycle analysis by BrdU, MCF7, OVCAR-3 and Kuramochi cells were treated with DMSO or drug (abemaciclib, palbociclib or INX-315, respectively) for 2 days; ZR-75-1 cells were treated for 4 days (abemaciclib or palbociclib). For CUT&RUN, MCF7, OVCAR-3 and Kuramochi cells were treated for 2 days, and ZR-75-1 cells were treated for 4 days. For dose matrix drug combination assays, cells were treated with abemaciclib and fulvestrant in a dose matrix using the Tecan D300e Digital Dispenser, with a treatment length of 5 days.

For hormone-deprivation and stimulation experiments, cells were cultured in phenol-free RPMI-1640 (11835030, Gibco) supplemented with 1× GlutaMAX, 1% HEPES and 10% charcoal-stripped FBS. Oestradiol (E2758, Sigma) dissolved in ethanol was used at 1 nM.

Cell cycle analysis by flow cytometry

Thirty minutes before end point, 10 μM BrdU (B5002, Sigma) was applied to the cells. Cells were stained using the Near-IR Fixable Viability Stain (L34976, Invitrogen) for 20 min at room temperature. Cells were fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience). DNA was denatured using 2 N HCl + 0.5% (v/v) Triton X-100 for 30 min. The acid was neutralized using 0.1 mol l⁻¹ Na₂B₄O₇•10H₂O (pH 8.5) followed by 0.5% BSA in PBS. Cells were then stained with BrdU antibody (clone 3D4, 560209, BioLegend; 1:20) for 1 h. Before acquisition on the BD A3 Symphony, DNA was stained using FxCycle Violet Stain (F10347, Invitrogen). A minimum of 10,000 live cell events were recorded for each sample and data were analysed using FlowJo (v10.8.1).

Dose matrix drug combination assays

Cell counts

Cells were fixed by adding 16% paraformaldehyde (PFA; C004, ProSciTech) to the media to attain a concentration of 4% PFA and incubated at room temperature for 10–15 min. Washed cells were then incubated with the DAPI (D1306, Invitrogen)–Hoechst 33342 (H1399, Thermo Fisher) stain solution (0.19% Triton X-100, 2.5 μg ml⁻¹ DAPI and 5 μg ml⁻¹ Hoechst in 50 mM Tris pH 7.6). The stained nuclei were imaged using the Cellomics CX7 LED or LZR (Thermo Fisher), and the CX7 software was used for segmentation and nuclei counting. Nuclei counts were used for synergy analysis.

Cell cycle

EdU was added to the cells at 10 μM and incubated at 37 °C for 45 min. Of PFA, 16% was added to attain 3.7% final concentration to fix the cells. EdU was labelled using the Click-iT EdU Cell Proliferation Kit for Imaging (C10340, Thermo Fisher) as per the manufacturer’s protocol. Once the staining cocktail was removed, cells were washed once in PBS containing 3% BSA, and nuclei were stained with DAPI and Hoechst as above. The EdU signal and nuclei were imaged using the CX7 LZR. The per cell data were analysed using the CellProfiler (v3.0.0) software. The percentage of cells in S phase was used for synergy analysis.

Cell death

Media and PBS washes from each well were collected to capture all floating or dead cells. Adherent cells were trypsinized and combined with floating cells in the media. Cells were centrifuged at 400g for 4 min at 4 °C. All cells were washed with PBS twice. Cells were stained with 80 μl propidium iodide from the Annexin V/PI Kit (556547, BD) at 1:20 in 1× binding buffer on ice for 15 min as per the manufacturer’s protocol. Cells were then analysed on the LSR II flow cytometer using the BD FACSDiva software (v9.0). Percentages of live and dead cells were calculated using FlowJo (v10.5.3), and this information was used for synergy analyses.

Synergy analysis

Loewe synergy scoring in the SynergyFinder R package (v3.14.0)⁴² and ggplot2 (v3.2.5) were used to analyse and visualize the data.

Cell proliferation assays

Live cells constitutively expressing H2B–GFP were imaged using the Incucyte SX5 at multiple time points. Using the images, cells were segmented based on GFP expression to obtain cell counts.

In vivo experiments

The KCC-P-3837 PDX model has been previously described⁴³. NOD.Cg-Prkdc^scidIl2rg^tm1Wjl (NSG) mice were used for all in vivo experiments. Mice were housed under specific pathogen-free conditions at 19–21 °C and 40–60% relative humidity with ad libitum access to food and water. Animals were maintained on a 14-h light–10-h dark cycle (lights on at 07:00 and off at 20:00) with 30-min dawn and dusk transitions. Red lighting was used after hours during the dark phase. No formal statistical methods were used to predetermine sample sizes. Sample sizes were selected based on previous experiences on the PDX model used in this study. Before treatment initiation, aged-matched mice were randomized into groups of equal average tumour volume. Downstream analyses of mouse tissue, including immunohistochemistry and image analysis, were performed in a blinded manner. Individuals conducting the assay and/or analysing the data were unaware of treatment allocation until data gathering was complete.

Rb chromatin profiling and immunohistochemistry

Tumour pieces of 1–2 mm³ were implanted into the thoracic mammary fat pads of 7-week-old NGS female mice (obtained from the Peter MacCallum Cancer Centre Animal Facility) implanted with 0.3 mg oestradiol silicon pellets. When tumours reached an average of 250 mm³ (using the formula width² × length × 0.5), mice were randomized into two treatment groups: abemaciclib or vehicle control. Abemaciclib and vehicle were prepared as previously described⁴⁴. Mice were treated with 90 mg kg⁻¹ abemaciclib by daily oral gavage, five doses in total, and then euthanized by CO₂ asphyxiation or cervical dislocation 2–4 h after the last dose. This study was performed in compliance with federal laws and institutional guidelines as approved by the Animal Ethics Experimentation Committee of the Peter MacCallum Cancer Centre. Humane end points included any diameter of any single tumour measuring at greater than or equal to 15 mm, weight loss of more than or equal to 20%, any tumour ulceration or infection, loss of ability to ambulate, laboured respiration, impaired movement or ruffled fur. No animals exceeded the approved humane end point criteria during the course of this study.

RNA-seq and tumour growth

The KCC-P-3837 has been previously described⁴³. At surgery, 4-mm³ sections of tumour tissue were implanted into the left and right fourth inguinal mammary glands of 6–8-week-old female NSG mice (Australian BioResources). Tumour growth was assessed by calliper measurement and mice were randomized to treatment arms when tumours reached 200–250 mm³. Mice were treated with palbociclib (100 mg kg⁻¹ in water 5 days per week by oral gavage), fulvestrant (5 mg per mouse in 100 μl peanut oil once weekly by subcutaneous injections), the combination of palbociclib and fulvestrant, or vehicles only. One cohort of mice was treated for 56 days to produce tumour growth kinetics, whereas a second cohort was treated for 19 days and then euthanized by CO₂ exposure and cervical dislocation. Tumours were resected, processed and snap frozen immediately following euthanasia. Experiments, procedures and end points were approved by the Garvan Institute of Medical Research Animal Ethics Committee. In brief, mice were monitored regularly for tumour burden and clinical signs of distress and were euthanized upon reaching predefined humane end points, including tumour ulceration, necrosis, infection, evidence of local invasiveness, impaired mobility, inability to access food or water, development of ascites, 20% or more body weight loss, or other indicators of compromised welfare. Animals were euthanized when tumour volume reached or exceeded 1,500 mm³. As euthanasia was initiated following tumour measurement, the final recorded tumour volume occasionally exceeded this threshold at the terminal assessment.

CRISPR–Cas9 gene editing

Alt-R S.p. Cas9 nuclease-purified Cas9 protein (1081059, Integrated DNA Technologies (IDT)) and single guide RNAs (sgRNAs; custom ordered from IDT) were complexed at a 1:5 molar ratio to form ribonucleoproteins (RNPs). For each nucleofection reaction, 36.6 pmol of Cas9 and 150 pmol sgRNA were used. The RNP complexes were incubated at room temperature for 10 min. For each reaction, cells were suspended in SF Cell Line Nucleofector solution (V4XC-2032, Lonza) containing Cas9 protein and gRNA complexes. The Lonza 4D-Nucleofector X Unit and the MDA-MB-453 program were used for nucleofection.

sgRNA sequences were as follows. For RB1, sgRB1-2: 5′-AAA CAA TCA AAG GAC CGA GA; for KRT18 enhancer, gRNA-1A: 5′-CAG GGC TCT AGA GTT CAC AG; gRNA-1B: 5′-AGC TTG GCA GCA GAG GAG GG; gRNA-2A: 5′-GTG GTG GCG GTA AGA GTC TG; gRNA-2B: 5′-GTG GGG AGG AGT TTT CAC AG; for CCND1 enhancer #1, set 1A: 5′-ACACAAGACACGCTGCACGG; set 1B: 5′-AGGAAGCTTGCTGAACACCG; set 2A: 5′-GAGGTTACCCCTCATAATGG; set 2B: 5′-GGGGTCAAGAGGAAGCTTAG; for CCND1 enhancer #2, set 1A: 5′-GAGCTGACTCGATTTGCCCG; set 1B: 5′-GCAGCACTCTGTACCCAGAA; set 2A: 5′-TGTTTCTGAGATCACAGGCG; set 2B: 5′-TCAGTGGTTTGCAGAGACGT; and for non-targeting control sgRNA, sgControl: 5′-CAT TTC TCA GTG CTA TAG AG.

Generation of isogenic RB1-knockout cell lines

Viable bulk populations of edited cells defined by negative staining for propidium iodide at 1 μg ml⁻¹ were single cell sorted by the BDFACS Aria Fusion 3 or 5 Flow Cytometer into each well of a 96-well plate. PCR amplification from genomic DNA was performed using the following primers: forward 5′-CTG TCC CTT GAA TGT TTG GTA G, reverse 5′-TCT GGA GAG GAA GAT TAA GAG GAC; and PCR amplicons were sequenced by Sanger sequencing. Cell lysates were analysed for RB1 knockout by western blotting. Verified RB1-knockout clones were expanded and cryopreserved for further studies.

Generation of nuclear GFP-expressing cells

Cell lines expressing H2B–GFP were generated using the lentiviral plasmid LV-GFP (Addgene #25999). In brief, lentivirus was generated using HEK 293T cells transfected with FuGENE HD (E2311, Promega) and pCMV-VSVG (Addgene #8454), pRSV-REV (Addgene #12253), pMDLg/pRRE (Addgene #12251) and LV-GFP. Concentrated lentivirus was used to transduce cell lines. Ten days post-transduction, GFP-positive cells were sorted on BD FACSAria Fusion 3 or 5 Flow Cytometers to capture cells within 50–75% brightness. At least 400,000 cells were collected post-sorting, and cell line identity was revalidated by STR profiling.

siRNA knockdown assays

Attached cells were transfected with siRNA by complexation with DarmaFECT 3 Transfection Reagent (T-2003, Horizon Discoveries) in Opti-MEM (31985062, Gibco). After 24 h, the media were replaced with fresh culture medium with drug treatment or DMSO control. Knockdown efficiency was measured by gene expression by quantitative PCR (qPCR) with reverse transcription or 3′ RNA-seq.

siRNA used were purchased from Horizon Discoveries and are as follows: for CCND1 knockdown, siRNA targeting CCND1-1 (siCCND1-1; J-003210-17) and siCCND1-2 (J-003210-18); for KDM5A knockdown, siKDM5A-1 (J-003297-22) and siKDM5A-2 (J-003297-23); and for non-targeting control siRNA, siControl (DHA-D-001810-01-05).

Chromatin profiling

Cell line sample preparation

Cells were trypsinized with TrypLE (12604021, Gibco), centrifuged at 400g and resuspended in fresh complete medium. Cells were counted manually with a haemocytometer, and an average of two counts per sample were used.

CUT&RUN assay, library preparation and sequencing

The CUT&RUN Assay Kit (86652, CST) was used. All buffers were prepared as per the CST CUT&RUN Assay Kit protocol. For each CUT&RUN reaction, 500,000 cells were used. Cells were washed, bound to Con A beads and incubated with primary antibody overnight at 4 °C as per the manufacturer’s instructions. After one wash with digitonin buffer, pA/G-MNase was added to cells for binding at 4 °C for 1 h. Cells were washed twice, and calcium chloride was added to activate the MNase for chromatin digestion at 4 °C for 30–60 min. A modification to the manufacturer’s protocol, 0.1% SDS, proteinase K and 300 mM NaCl were added to the STOP buffer. The modified STOP buffer was added to cells to stop the digestion reaction and release all DNA material. Lysed samples in STOP buffer were incubated at 55 °C for 1 h for cell lines or overnight for tumour tissue, and then centrifuged at room temperature at 16,000g for 2 min. Using a magnetic concentrator to collect Con A beads, the supernatant was collected for DNA purification with the DNA Purification Buffers and Spin Columns kit, as per the manufacturer’s protocol (14209, CST). Size selection was performed by adding AMPure XP beads (A63881, Beckman Coulter) to the purified CUT&RUN DNA at 0.5× to remove large DNA fragments. The remainder of DNA fragments were collected and purified by adding an extra 1.3× (to attain a total of 1.8×) AMPure beads. DNA-bound beads were washed with 80% ethanol twice, and DNA fragments were eluted in 10 mM Tris pH 8.0. Concentration and fragment sizes of the DNA were measured using the TapeStation D1000 High Sensitivity kit (5067-5584 and 5067-5585, Agilent) on the Agilent TapeStation 4150.

Primary antibodies used included Rb (clone 4H1; 61121, CST; 1 μg per reaction for cell lines and 1.5 μg for tumour tissue), ER (8644, CST; 0.8 μg), H3K4me3 (9751, CST; 1 μg), KDM5A (ab194286, Abcam; 1 μg), mouse IgG2a (61656, CST; 1 μg per reaction for cell lines and 1.5 μg for tumour tissue) and rabbit IgG (66362, CST; 1 μg).

For library construction, the concentration of DNA was determined from the TapeStation concentration reading between 100 and 700 bp. The NEBNext Ultra II DNA Library Prep Kit (E7645S, NEB) and Unique Dual Indices (E6440S, NEB) were used. We followed the CST DNA Library Prep Kit for Illumina Protocol that was optimized for CUT&RUN, which included modifications to adaptor dilutions, AMPure XP bead ratios, determining the number of PCR amplification cycles, and the PCR anneal and extension step, which had been shortened to 13 s compared with the standard protocol. Amplified DNA was cleaned up using AMPure XP beads and eluted in 10 mM Tris pH 8.0. CUT&RUN libraries were quantified using the TapeStation D1000 (5067-5582 and 5067-5583, Agilent) on the TapeStation, then pooled and sequenced on an Illumina NextSeq 2000 instrument, paired end, obtaining 5–10 million clusters per sample.

Tissue sample preparation

Tumours were extracted from euthanized mice and rinsed in PBS. The tissue was first cut longitudinally, then sliced into thin sheets using a microtome blade. The resulting pieces were approximately 4 mm × 4 mm × 1 mm, one piece per tumour. Each piece was used for one single DynaTag reaction.

DynaTag assay, library preparation and sequencing

Cleavage under DynaTag, a modified CUT&Tag method adapted from Hunold et al., was used²¹. In brief, tissue slices were washed in DynaTag buffer (25 mM HEPES pH 7.5 (15630-080, Gibco), 110 mM KCl (AM9640G, Invitrogen), 10 mM NaCl (AM9760G, Invitrogen), 1 mM MgCl₂ (AM9530G, Invitrogen), 1× spermidine (27287S, CST), 1× protease inhibitor cocktail (7012L, CST)), bound to Con A beads, and incubated overnight at 4 °C with primary antibody (Rb (61121, CST) or mouse IgG2a) in antibody buffer (DynaTag buffer + 1% w/v BSA (A8577, Sigma; 10 ml) and digitonin (16359L, CST)). Anti-mouse secondary antibody (0.5 μg; ab46540, Abcam) and pre-loaded pA–Tn5 complex (1:100 dilution; C0107001, Diagenode) binding were performed at room temperature for 1 h each. Tagmentation was initiated by incubating samples at 37 °C for 1 h in antibody buffer supplemented with 10 mM MgCl₂. Upon tagmentation, samples were resuspended in denaturation buffer (10 mM Tris-HCl (15568-025, Invitrogen), 50 mM NaCl (AM9760G, Invitrogen), 0.2% SDS (15553-035, Invitrogen), 0.15 mg ml⁻¹ proteinase K (10012S, CST) and 16.67 mM EDTA (15575020, Invitrogen)) and incubated at 58 °C for 3 h. Denaturation was stopped by purification of the tagmented DNA fragments using the MinElute PCR Cleanup Kit (28004, Qiagen), as per the manufacturer’s protocol. The library was prepared using Illumina DNA/RNA UD Indexes Set A, Tagmentation (20091654, Illumina) and NEBNext 2× PCR Mix (M0541L, NEB). Amplified DNA was cleaned up using AMPure XP beads and eluted in EB buffer. Libraries were quantified using the TapeStation D1000 on the TapeStation, then pooled and sequenced on an Illumina NextSeq 2000 instrument, paired end, obtaining 5–10 million clusters per sample.

RNA extraction

RNA was extracted using the NucleoSpin RNA plus kit (740984, Macherey-Nagel) per the manufacturer’s protocol.

Reverse transcription quantitative PCR

RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher) as per the manufacturer’s protocol. SYBR Select Master Mix (4472919, Applied Biosystems) was used for the qPCR, which was run on an Applied Biosystems StepOne Plus instrument. The ^ΔΔCt method was used to analyse the data. Primer sequences used for qPCR were as follows: KRT18 forward: 5′-TCG CAA ATA CTG TGG ACA ATG C, reverse: 5′-GCA GTC GTG TGA TAT TGG TGT; MKI67 forward: 5′-AGA AGA AGT GGT GCT TCG GAA, reverse: 5′-AGT TTG CGT GGC CTG TAC TAA; TK1 forward: 5′-GCC AAA GAC ACT CGC TAC AG, reverse: 5′-CCC CTC GTC GAT GCC TAT G; CCND1 forward: 5′-GCT GCG AAG TGG AAA CCA TC, reverse: 5′-CCT CCT TCT GCA CAC ATT TGA A; and HSP90AB1 (housekeeping) forward: 5′-AGA AAT TGC CCA ACT CAT GTC C; reverse: 5′-ATC AAC TCC CGA AGG AAA ATC TC.

RNA library preparation and sequencing

Libraries were prepared using the Quanteq Lexoen 3′ mRNA FW kit by the Peter MacCallum Cancer Centre Molecular Genomics Core. Single-end 100-bp reads were sequenced on a NextSeq 2000 instrument (Illumina), with a goal of 3–5 million reads per sample.

Western blotting

Protein lysate preparation

For the detection of ER, protein lysates were prepared using 1× cell lysis buffer (CST) as previously described²³. For the detection of Rb and KDM5A expression, lysates were prepared by lysing equal numbers of cells in 4× Laemmli sample buffer (1610747, Bio-Rad) diluted to 1× with water and with 5% β-mercaptoethanol added and heated to 95 °C for 13 min.

Western blotting

Western blotting was performed as previously described²³. Primary antibodies used include Rb (554136, BD; 1:1,000), phospho-Rb S780 (8180, CST; 1:1,000), phospho-Rb S807/811 (8516, CST; 1:1,000), ER (8644, CST; 1:1,000), KDM5A (ab194286, Abcam; 1:5,000), histone H3 (9715, CST; 1:1,000), cyclin D1 (55506, CST; 1:1,000), KRT18 (4548, CST; 1:1,000), TFF1 (15571, CST; 1:1,000), cyclin A2 (ab32386, Abcam; 1:1,000), actin (A3854, Sigma-Aldrich; 1:10,000) and vinculin (V9131, Sigma-Aldrich; 1:5,000). Secondary antibodies used include StarBright Blue 520 goat anti-mouse IgG (12005867, Bio-Rad) and StarBright Blue 700 goat anti-rabbit IgG (12004162, Bio-Rad), both at 1:2,500. Western blot images were acquired on ChemiDoc MP Imaging System (Bio-Rad) and visualized using Image Lab Software (v6.1).

Immunohistochemistry

Tumour tissue extracted from euthanized mice were immediately fixed in 10% neutral-buffered formalin overnight and stored in 70% ethanol. Fixed tissue pieces were dehydrated with ethanol and xylene and embedded in paraffin. The embedded tissues were sectioned at 10 μm thickness and dewaxed, rehydrated and subject to heat-mediated antigen retrieval in citrate buffer pH 6.0. After washing, samples were incubated in 3% hydrogen peroxide for 5 min, washed again and incubated for 1 h at room temperature in primary antibodies to Ki-67 (ab16667, Abcam), phospho-Rb Ser807/811 (8516, CST) and total-Rb (554136, BD Biosciences). Secondary antibodies were ImmPRESS anti-rabbit (MP-7451-NB, Novus) or ImmPRESS anti-mouse (MP-7452-NB, Novus). Signal development was performed using the Dako Liquid DAB⁺ Substrate Chromogen System (K3468, Agilent Technologies) before counterstaining with haematoxylin. Whole-slide images were acquired using an Olympus SLIDEVIEW VS200 microscope. The number of positive tumour cells was calculated using QuPath⁴⁵.

Nuclear co-immunoprecipitation

Nuclear co-immunoprecipitation (co-IP) was performed using the Active Motif Nuclear Complex Co-IP kit (54001, Active Motif) as per the manufacturer’s protocol. In brief, nuclei were extracted with the hypotonic buffer. Nuclear lysates were prepared using the digestion cocktail. Lysates were cleared using mouse IgG2a (61656, CST) and rabbit IgG (ab37415, Abcam) bound to protein A/G Dynabeads (10002D and 10004D, Thermo Fisher), incubated at 4 °C for 1–3 h. Cleared lysates were incubated with primary antibody in 1× low buffer overnight at 4 °C, and protein A/G Dynabeads were added to the lysate for another 1 h at 4 °C. The beads were washed for a total of six times using the 1× low buffer. Proteins were eluted using 4× Laemmli sample buffer (1610747, Bio-Rad) diluted to 2× with water and with 10% (v/v) β-mercaptoethanol added, and heated to 95 °C for 5 min. Primary antibodies used for co-IP include Rb (9039, CST), ER (8644, CST), mouse IgG2a (61656, CST) and rabbit IgG (ab37415, Abcam).

Mass spectrometry

Peptide preparation

Samples eluted from nuclear co-IP were reduced with 5 mM TCEP (646547-10X1ML, Merck), alkylated with 20 mM methyl methanethiosulfonate (23011, Thermo Fisher) and acidified using phosphoric acid (695017-100ML, Sigma-Aldrich). Samples were then loaded onto S-Trap Micro Spin Columns, following the manufacturer’s instruction (Protifi) and digested with trypsin (90057, Thermo Fisher) overnight at 37 °C. Peptides were sequentially eluted with 50 mM Tris-HCl (pH 8.5), 0.2% formic acid (695076-100ML, Sigma-Aldrich) and 50% acetonitrile (1000291000, Merck). Eluates were snap frozen in liquid nitrogen and freeze dried overnight. Immediately before mass spectrometry, samples where resuspended in 30–40 μl of resuspension buffer (2% acetonitrile + 0.05% trifluoroacetic acid in mass spectrometry grade water) and briefly sonicated in a water bath. Samples were centrifuged at maximum speed for 10 min, and the top 15 μl of each supernatant was collected for analysis.

Data-independent acquisition

Liquid chromatography–tandem mass spectrometry analyses were performed on an Orbitrap Ascend mass spectrometer (Thermo Fisher Scientific) as previously described⁴⁶. The liquid chromatography system (Ultimate 3000) was equipped with an Acclaim PepMap nano-trap column (Dionex C18, 100 Å, 75 µm × 2 cm) and an Acclaim PepMap RSLC analytical column (Dionex C18, 100 Å, 75 µm × 50 cm). Tryptic peptides were loaded onto the enrichment column at an isocratic flow rate of 5 μl min⁻¹ with 2% (v/v) acetonitrile containing 0.05% (v/v) trifluoroacetic acid for 6 min, after which the enrichment column was switched in-line with the analytical column. The eluents were 0.1% (v/v) formic acid in water (solvent A) and 100% (v/v) acetonitrile with 0.1% formic acid (solvent B), both supplemented with 5% DMSO. The gradient was run at 300 nl min⁻¹ as follows: (1) 0–6 min, 3% B; (2) 6–7 min, 3–4% B; (3) 7–82 min, 4–25% B; (4) 82–86 min, 25–40% B; (5) 86–87 min, 40–80% B; (6) 87–90 min, 80–3% B; and (7) 90–90.1 min, 80–3% B, followed by equilibration at 3% B for 10 min before the next injection.

For data-independent acquisition (DIA) experiments, full mass spectrometry scans were acquired at a resolution of 120,000 (at m/z 200), with a scan range of 350–1,400 m/z in profile mode. The full mass spectrometry AGC target was set to 250% with an injection time of 50 ms. The AGC target for fragment spectra was 2,000%. Fragmentation was performed across 50 windows of 13.7 Da width with a 1-Da overlap. MS2 resolution was set to 30,000 with a maximum injection time of 55 ms. Normalized collision energy was set to 30%, and data were acquired in centroid mode with positive-ion polarity.

Bioinformatic analysis

RNA-seq

Fastq files were trimmed for adapters and low-quality reads using BBDuk (v38.90) via the bbduk.sh script⁴⁷ with the following parameters: ktrim = r k = 23 mink = 11 hdist = 1 tpe tbo qtrim = r trimq = 5 minlen = 20. Trimmed reads were aligned to the hg38 reference genome (GRCh38.p15; https://www.ncbi.nlm.nih.gov/assembly/GCA_000001405.15) using STAR (v2.7.5b)⁴⁸. For PDX samples, mouse reads were filtered out using XenofilteR (v1.6)⁴⁹. Read counts were generated using featureCounts from the Subread package (v2.0.1)⁵⁰, with annotations from GENCODE (gencode.v35.annotation.gtf⁵¹). Normalization was performed using the median-of-ratios size-factor method implemented in DESeq2. Differential gene expression analysis was performed using DESeq2 (v1.46.0)⁵². Genes with an adjusted P < 0.05 were considered differentially expressed.

Gene expression heatmaps were generated using ComplexHeatmap (v2.22.0)⁵³. Gene-wise z-scores were calculated from DESeq2 size-factor-normalized read counts and used for heatmap visualization. Genes were clustered by hierarchical clustering using Euclidean distance and complete linkage.

Microarray data

Raw microarray data for palbociclib-treated patients and untreated control patients were accessed from our previous publication²⁷ (http://microarrays.curie.fr/publications/U981-GustaveRoussy/pop/). All participants provided written informed consent before study participation. Ethical approval and trial conduct have been previously described²⁷. The dataset was filtered to include only ER-positive and human epidermal growth factor receptor 2 (HER2)-negative patients, resulting in 18 control patients and 44 patients treated with palbociclib. Raw CEL files were imported into R using the read.celfiles function from the oligo R package. Data normalization was performed using the robust multichip average algorithm implemented in the oligo package with the parameter target = ‘core’. Probeset annotations were mapped to gene symbols using the hugene21sttranscriptcluster.db annotation package via the annotateEset function from the affycoretools R package. Quality control filtering was applied to remove lowly expressed genes using a median intensity threshold of 1.5, determined through visual inspection of transcript median distributions in histogram plots⁵⁴. Differential expression analysis was conducted using the limma R package⁵⁵ to identify genes significantly altered between control and palbociclib-treated groups.

GSEA of transcriptomics datasets

GSEA was conducted using clusterProfiler (v4.14.6)⁵⁶. Hallmark gene sets were sourced from the Molecular Signatures Database (MSigDB)^25,26 via msigdbr (v24.1.0). For cell line and PDX RNA-seq datasets, genes were ranked by shrunken log₂ fold changes calculated using the adaptive shrinkage ‘ashr’ method. For the POP clinical transcriptomics dataset, genes were ranked by the Wald statistic. Enrichment scores were computed using the running-sum statistic implemented in GSEA. Single-sample GSEA was performed using GSVA (v2.0.7) with α = 0.25.

CUT&RUN and DynaTag

Adapters and low-quality reads were removed from Fastq files using BBDuk (v38.90)⁴⁷ with the following parameters: ktrim = r k = 23 mink = 11 hdist = 1 tpe tbo qtrim = r trimq = 5 minlen = 20. Trimmed reads were aligned to hg38 (GRCh38.p15; https://www.ncbi.nlm.nih.gov/assembly/GCA_000001405.15) using Bowtie2 (v2.3.4.1) with the following parameters: –dovetail–local–very-sensitive–no-mixed–no-discordant–phred33 -I 10 -X 700. For PDX DynaTag samples, trimmed reads were aligned to a hybrid genome consisting of hg38 (GCA) and mm10 (UCSC). The mm10 genome FASTA and annotation files were obtained from UCSC (https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/). Reads aligning to ChIP blacklisted regions or mitochondrial DNA were removed using Picard (v3.0.0)⁵⁷, samtools (v1.9)⁵⁸ and bedtools (v2.27.1)⁵⁹. Duplicate reads were retained for downstream analysis.

Peaks were called using MACS2 (v2.2.7.1)⁶⁰ callpeak with the matched IgG controls specified using the–control parameter and the options–format BAMPE -gsize hs -q 0.05. Broad H3K4me3 peaks were called using MACS2 callpeak with the options–format BAMPE -g hs –broad–broad-cut-off 0.1 -q 0.05, using matched IgG controls as input.

Differential analysis was performed using DiffBind (v3.16.0)⁶¹, with relative log expression (RLE) background normalization using the following parameters: method = DBA_DESEQ2, normalize = DBA_NORM_RLE, library = DBA_LIBSIZE_BACKGROUND, background = TRUE. Differential binding was defined using a false discovery rate-adjusted P value threshold of 0.1 for CUT&RUN datasets and 0.2 for DynaTag datasets.

Peaks were annotated to genomic features using ChIPseeker (v1.42.0)⁶². Promoters were defined as regions spanning −2,000 bp to +500 bp relative to the transcription start site (TSS). Peaks overlapping promoter regions were classified as promoter peaks, and all remaining peaks were classified as non-promoter peaks.

BigWig files were generated using the bamCoverage function from deepTools with the following parameters: -binSize 20, -smoothLength 60, -scaleFactor scalefactor, -extendReads, where scale factors were derived from DiffBind RLE background normalization. BigWig files for negative input, H3K4me3 and H3K4me1 samples were normalized using bins per million mapped reads. BigWig tracks were visualized using the Integrative Genomics Viewer⁶³ or deepTools.

Motif enrichment analysis was carried out using HOMER (v4.11)⁶⁴ with the parameters; -size 200 -len 6,10,15,20 -p 14. Enrichment ratios represent motif frequency in the input regions relative to HOMER-generated random genomic background.

GSEA of genomic regions was conducted using ChIP-Enrich (v2.30.0)²⁴, assigning each peak region to the gene whose TSS was nearest to the peak midpoint. Gene set over-representation analysis (ORA) was performed using clusterProfiler (v4.14.6)⁵⁶ on genes located within 100 kb of the genomic regions of interest and significantly upregulated by treatment (log₂ fold change > 0, adjusted P < 0.05). MSigDB hallmark gene sets were used in both methods. Odds ratios represent the observed versus expected frequency of genes associated with the genomic regions of interest within each gene set.

To assess the transcriptional regulatory function of Rb-bound non-promoter regions, Rb CUT&RUN and RNA-seq data were integrated using BETA (v1.0.7)²². Genes were ranked according to regulatory potential scores calculated by BETA based on the proximity and abundance of Rb-bound regions relative to gene promoters. The regulatory potential distributions of upregulated and downregulated genes were compared using the one-tailed Kolmogorov–Smirnov test implemented in BETA.

Chromatin interaction and transcriptional hub analysis

H3K27ac HiChIP loop data from our previous study (GSE157381)²³ were used for these analyses. Processed loop calls, binned into 5-kb genomic regions, were obtained from the published dataset. To identify chromatin interactions associated with chromatin-bound Rb, each 5-kb region was annotated for overlap with Rb CUT&RUN peaks using a minimum threshold of 1 bp. Rb-bound regions were classified as promoter or non-promoter based on the genomic annotation of the overlapping Rb peak. Regions overlapping both promoter and non-promoter Rb peaks were classified as promoter regions.

For HiChIP ORA, gene set ORA using MSigDB hallmark gene sets was performed on protein-coding genes whose promoters were directly connected to non-promoter Rb regions through H3K27ac HiChIP interactions. To assess co-occupancy with ER, all 5-kb genomic windows were annotated for overlap with ER CUT&RUN peaks using a minimum overlap threshold of 1 bp.

Transcriptional hubs were identified from the H3K27ac HiChIP interaction network by constructing an igraph object with 5-kb regions as nodes and HiChIP interactions as edges. Network communities were detected using the Louvain clustering algorithm implemented in the igraph (v2.1.4)⁶⁵, with default parameters. For hubs ORA, protein-coding genes located within transcriptional hubs containing at least one non-promoter Rb peak were subjected to gene set ORA using MSigDB hallmark gene sets. Hub visualizations were generated using ggraph (v2.2.2).

To identify hubs associated with KDM5A, ER and Rb, hubs were required to contain at least one non-promoter Rb up peak overlapping a KDM5A consensus peak (more than 1 bp) and at least one ER peak (promoter or non-promoter) within the hub. Protein-coding genes with promoters located within these hubs were subjected to gene set ORA. For promoter H3K4me3 width analysis, this gene set was further restricted to genes with promoter-associated H3K4me3 broad peaks present in all samples and overlapping the TSS.

Mass spectrometry

DIA data were analysed using the direct DIA workflow with default settings in Spectronaut (v19.9) against the reviewed UniProt Homo sapiens database (downloaded October 2024). Trypsin digestion was specified with up to two missed cleavages. Methyl methanethiosulfonate alkylation of cysteine was set as a fixed modification, whereas N-terminal acetylation and methionine oxidation were specified as variable modifications. Protein and peptide-spectrum match (PSM) identifications were filtered at a 1% false discovery rate. Precursor filtering was based on Q value, quantification was performed at the MS2 level and cross-run normalization was set to automatic.

For differential binding analysis, raw protein group quantity data from Spectronaut were processed as follows. Proteins with two or three NaN values in both immunoprecipitation conditions were excluded. Remaining NaN values were imputed using one of two methods: (1) if one replicate was missing, the average of the other two replicates within the same condition was used; (2) if only one replicate in the treatment arm had a value, the two NaN values were imputed as zero. log₂-transformed quantity values were then analysed for differential binding using the limma package (v3.62.2)⁵⁵ with empirical Bayes moderation, and results were visualized with ggplot2.

Other statistical analyses

For datasets that passed the normality test, a two-tailed unpaired Student’s t-test or one-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test was used. A two-tailed Wilcoxon rank-sum test was applied to non-normally distributed or ranked data.

Information on replication relevant to each figure is included in the legends. No statistics were used to predetermine sample sizes.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.