H3k4me2

Small piece of frozen liver was cut and lysed with cold RIPA lysis buffer (ZhongHuiHeCai, Xi'an, China) with protease and phosphatase inhibitors on the ice. The BCA protein assay kits were used to determine the protein concentration of lysis supernatant. Then protein samples were denatured and performed for SDS-polyacrylamide gel electrophoresis separation to detect proteins expression. Detailed steps were reported to our previous study (Liu et al., 2018 (link)). The primary antibodies were used after 1:1,000 dilutions and their detailed information were as follows: β-actin (CWBIO, CW0096, Taizhou, China), SREBP (Wanleibio, wl01314, Shenyang, China), H3K27ac (Abcam, ab4729, MA), H3K4me2 (Abcam, ab32356, MA), and H3 (ABclonal, A2348, Wuhan, China). The secondary antibodies were used after 1:3,000 dilutions and goat anti-rabbit (No.122107) or mouse (No.117228) IgG HRP conjugated secondary antibody was bought from Jackson ImmunoResearch. The Image J software (National Institutes of Health, MD) was used to quantify blotting bands with β-actin or H3 as an internal control.

The ChIP assays were performed using a ChIP isolation kit (Millipore, Billerica, MA). We treated 1 × 10⁵ to 5 × 10⁵ murine naïve T cells with 1% formaldehyde to cross-link histones to DNA. The fixed cells were sonicated to yield chromatin fragments of 200–500 base pairs (bp). The antibodies used in the ChIP assays included: H3Ac (Millipore), H4Ac (Upstate Biotechnology, Lake Placid, NY), H3K9Ac (Active Motif, Carlsbad, CA), H3K4Me3 (Millipore), H3K4Me2 (Abcam), H3K4Me (Millipore), H3K9Me2 (Millipore), H3K9Me3 (Abcam) H3K27me2 (Abcam) H3K27me3 (Millipore), p300 (Abcam), PCAF (Abcam), SUV39H1 (Millipore), CBP (Abcam), ESET/SetDB1(Millipore), Ezh2 (Cell Signaling Technology, Beverly, MA), G9a/EHMT2 (C6H3) (Abcam), HP1α (Millipore), HP1β (Active Motif), and HP1γ (Millipore). DNA was recovered by a Chelex 10% slurry and sample boiling method,¹⁰ (link) or using the IP-STAR (Diagenode, Denville, NJ) direct chip protocol followed by the IPPURE DNA purification. 1% Pre-enriched chromatin (input) served as the percentage input for sample quantification, confirmed by fold over IgG changes. The UV-exposed 4% agarose gels were imaged, and the digitized images were analyzed using the software VisionWorks (UVP). Protein signals were indexed by measuring their relative mean grey value per area.

Western blots were carried out using standard protocols. Briefly, HCT116 and RPE cells were grown, treated and harvested as previously mentioned. Nuclear protein lysates were separated by SDS-PAGE and transferred to GVS nitrocellulose 0.22 μm membranes. Blots were probed with primary antibodies, followed by peroxidase-conjugated secondary antibodies (GE Healthcare Life). Signal for all immunoblots was acquired using the ImageQuant LAS 4000 biomolecular imager (GE Healthcare LS) with an average exposure of 30 s. Antibodies used are as follows: H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766); H3K4me3 (abcam, cat. # ab8580); total H3 (abcam, cat #1791).

Detailed protocols for ChIP are given in the Supplementary Materials. For histone modification, 3 × 10⁶ cells per IP and primary antibody (4 μg/ml) were used; for LSD1, GFI1, and PML ChIP, 40 × 10⁶ cells per IP were used (10 μg/ml). The obtained DNA was then quantified by PicoGreen and processed for ChIP-seq library preparation (as described for the Illumina protocol) or used for qPCR. For libraries preparation, 2 ng of immunoprecipitated DNA was used. Antibodies used for ChIP are as follows: H3K4me1 (Abcam, no. 8895), H3K4me2 (Abcam, no. 32356), H3K4me3 (Active Motif, no. 39159), H3K27ac (Abcam, no. 4729), LSD1 (Abcam, no. 17721), GFI1 (Abcam, no. 21061), PML (Santa Cruz Biotechnology, sc-5621), and immunoglobulin G rabbit (Jackson ImmunoResearch, lot no. 134230).