H3k4me1

Antibodies recognizing β-Actin (Abclonal, AC026, RRID: AB_2768234), H3 (abcam, ab1791, RRID: AB_302613), H3K27ac (abcam, ab4729, RRID: AB_2118291), H3K4me1 (Cell Signaling Technology, #5326, RRID: AB_10695148), p53 (Santa Cruz, sc-126, RRID: AB_628082), p21 (Cell Signaling Technology, #2947, RRID: AB_823586), MDM2 (abcam, ab3110, RRID: AB_303518), PUMA (Cell Signaling Technology, #4976, RRID: AB_2064551), MLL3(Merck ABE1851) and TNS3 (Abclonal A7991, RRID: AB_2772674) were purchased from indicated vendors. PCR primers were custom synthesized by TSINGKE and siRNAs by GenePharma. Nutlin-3A (S8059) was purchased from Selleck.
MDA-MB-231 and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) at 37 °C with 5% CO₂, and U2OS and 769-P cells in RPMI 1640 (Gibco). Both mediums were supplemented with 10% FBS (Biological Industries) and 1% penicillin-streptomycin solution (HyClone). All cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences.

POU2F3 (#36135), H3K27ac (#8173), H3K4me1 (#5326), H3K4me3 (#9751), H3K27me3 (#9733), Histone H3 (#4499), Cleaved-PARP (#5625), Cleaved Caspase 3 (#9664), SUZ12 (#3737), EZH2 (#5246), BRG1 (#49360), BRM (#11966), ARID1A (#12354), BAF57 (#11956), BAF155 (#11956), BAF60A (#35070), BAF47 (#91735), and PTEN (#9188) antibodies were purchased from Cell Signaling. Tubulin antibody (E7) was purchased from the Developmental Studies Hybridoma Bank. HSP90 (sc-7947) antibody was purchased from Santa Cruz. BRG1 (ab110641) and EZH2 (ab191250) antibodies were purchased from Abcam, and were used for ChIP-seq. The POU2AF2 (C11orf53) antibody was produced in rabbits at Pocono Rabbit Farm And Laboratory Inc. by using full-length POU2AF2 recombinant protein as antigen. For all of the western blot experiments, the antibody dilution is 1: 2k. For immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP), 5 μg of antibody per reaction was used. The small molecule inhibitors GSK126 (S7061) and JQ1 (S7110) were purchased from Selleck Chemicals. The small molecule inhibitor BRM014 (HY-119374) was purchased from Med Chem Express.

The following antibodies were used for chromatin immunoprecipitation: (i) H3K4me1: Cell Signaling Technologies, cat #5326BF, lot #2 (quantity: 3 µg/concentration: 0.015 µg/µl); (ii) H3K4me3: Cell Signaling Technologies, cat #9751BF, lot#6 (quantity: 5 µg/concentration: 0.025 µg/µl); (iii) H3K9me3: Abcam, cat #Ab8898, lot #GR93671-1 (quantity: 3 µg/concentration: 0.015 µg/µl); (iv) H3K27me3: Cell Signaling Technologies, cat #9733S, lot #6 (quantity: 10 µg/concentration: 0.05 µg/µl); (v) H3K27ac: Diagenode, cat #pAB-196-050, lot #A1723-0041D (quantity: 6 µg/concentration: 0.03 µg/µl); (vi) H3K36me3: Active motif, cat #MABI0333, lot #12003 (quantity: 2 µg/concentration: 0.01 µg/µl). Libraries were prepared using the automated protocol for the Kapa HTP Library Preparation Kit (Illumina), and sequencing was performed using the Illumina HiSeq 2000, as per the manufacturer’s instructions, to achieve at least 30 and 60 million reads for narrow (H3K27ac and H3K4me3) and broad (H3K27me3, H3K36me3, H3K4me1, and H3K9me3) marks, respectively (Supplementary Fig. 1a).

The ChIP assay procedure was modified from the manufacturer’s instructions (EZ-ChIP, millipore). Briefly, isolated BMDMs or mouse Raw264.7 cells (about 1 × 10⁷ cells) were fixed with 1% methanol-free formaldehyde (Thermo Fisher Scientific) at room temperature for 10 min, followed by quenching with 125 mM glycine. All subsequent steps were implemented as the standard ChIP protocols^{42 (link)}. And libraries were sequenced with an Hiseq 2500. The FASTQ data were mapped to the mouse genome (mm10) using Bowtie, and significant enrichments were identified by MACS2.0 using Broad Peak mode with p value ≤1 × 10⁻⁵, FDR ≤ 0.01 as a cutoff to call peaks from the aligned results. Reads-per-million-normalized wiggle files were displayed in the UCSC genome browser. Antibodies used in ChIP are ZMYND8 (Bethyl, catalog no. A302-090A, 2 μg/sample), H3K4me1 (Cell Signaling Technology, catalog no. 9751, 2 μg/sample), LSD1 (Abcam, catalog no. ab129195, 2 μg/sample), H3K4me2 (Abcam, catalog no. ab32356, 2 ug/sample), p65 (Santa Cruz, catalog no. sc-8008, 2 ug/sample). Shanghai DIATRE Biological Technology performed the ATAC assay. And libraries were sequenced with NovaSeq 6000.

Cells were lysed with RIPA buffer (25 mM Tris, pH7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitors (Roche, 4693132001) and phosphatase inhibitors (Roche, 4906845001) and sonicated with Bioruptor Plus at High setting. Cell lysates were resuspended in 4× sample buffer (200 mM Tris, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue and 20% 2-mercaptoethanol) and heated at 95 ℃ for 5 min. Antibodies used were OPA1 (67589), DRP1 (8570), Mitofusin-1 (14793), Mitofusin-2 (11925), H2AX (2595), γH2AX (2577), H3 (9715), H3K4me1 (5326), H3K4me3 (9751), IRF3 (11904), p-IRF3 (S396) (29047), TBK1 (3504), p-TBK1 (S172) (5483), p65 (8242) and p-p65 (S536) (3033) from Cell Signaling; H3K4me2 (39679), H3K9me1 (39887), H3K9me2 (39683), H3K9me3 (39765), H3K27me1 (61015), H3K27me2 (39919), H3K27me3 (39155), H3K36me1 (61351), H3K36me2 (39255), H3K36me3 (61101) from Active Motif; α tubulin (sc-23948) from Santa Cruz; Lamin A/C (GTX101127) from GeneTex. α tubulin was in 1:20000 dilution. For Lamin A/C and all histone-related antibodies, antibodies were used in 1:5000 dilution. Others were in 1:1000 dilution.

The ICC protocol previously developed by Huang et al. (ref) was followed, in which the cells were first fixed with 4% paraformaldehyde (PFA), and then double-washed, blocked with blocking buffer, and incubated in primary antibodies. Following the primary antibody incubation (H3K27ac, 1:1000, Cell Signaling; p300, 1:1000, Cell Signaling; iNOS, 1:750, Santa Cruz; H3K4me1, 1:1000, Cell Signaling; H3K4me3, 1:1000, Cell Signaling), the Alexa dye-conjugated secondary antibody was added to the cells following the repeated PBS wash step. After the secondary incubation period finished, the cells were washed with filtered PBS and then left in Millipore water for approximately 1 min prior to being mounted. Cell coverslips were mounted onto microscope slides via Fluoromount aqueous mounting medium (Sigma) and then dried at RT while protected from light. The images were taken with an inverted fluorescence microscope Keyence BZ-X800 (Itasca, IL). Z-stack images of mitochondria were captured by confocal imaging. Quantification of fluorescence intensity was performed by automated BZ-X800 analyzer. For Figure 2A, cell body size enlarged upon treatment, therefore, automated BZ-X800 analyzer was set up to quantify the total fluorescence intensity of H3K27ac per cell rather than intensity peaks.