Ab7766

ChIP assay were performed as previously described [67 (link)]. Briefly, dissected ovaries were fixed in 1.8% formaldehyde at room temperature for 10 min. Chromatin was sonicated and used for immunoprecipitation with anti-trimethyl-Histone H3 Lys9 (ab8898; Abcam), or anti-dimethyl-Histone H3 Lys4 (ab7766; Abcam) antibodies. DNA precipitates were amplified by real-time quantitative PCR. PCR product levels were normalized to input and expressed relative to a positive control gene (the 1360-element for the immunoprecipitation with the anti-H3K9me3 antibody and Rpl15 for the immunoprecipitation with the anti-H3K4me2 antibody). The relative DNA levels were calculated using the following formula: E(target)^CtIP *E(ref)^CtInput / E(ref)^CtIP * E(target)^CtInput, where E is the efficiency of each primer pair and ref the positive control. Primers are listed in S3 Table.

Histone extraction was modified from the method previously described (68 (link)). Fresh mycelia were ground with liquid nitrogen and homogenized in 10 mL extraction buffer (10 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.25 M HCl, 5 mM DTT, and 1× protease inhibitor cocktail). The fragments were removed by filtration with one layer of Miracloth (Calbiochem). The supernatants were separated after centrifugation (12,000 × g, 10 min) and 20% trichloroacetic acid was added to it to precipitate soluble proteins. The pellets were collected after centrifugation (17,000 × g, 30 min) and washed twice with ice-cold acetone. Histones were resuspended in lysis buffer (7 M urea, 2 M thiourea, 2% carrier ampholytes, 4% CHAPS, and 1% DTT) and quantified by the Bradford method. The procedure of Western bolt was performed as previously described with a few modifications (66 (link)). An equal amount of histone (10 μg) was separated by 15% SDS-PAGE. Primary antibodies used in this study include anti-Histone H3 (Abcam; ab1791) and anti-H3K4me1 (Abcam; ab8895), anti-H3K4me2 (Abcam; ab7766), and anti-H3K4me3 (Abcam; ab8580).

Chromatin from cells was fragmented to a size range of 200–500 bases with a Sonicator. Solubilized chromatin was immune‐precipitated with antibody against H3K4me2 (Abcam, ab7766, 1:1,000), H3K4me3 (Abcam, ab8580, 1:1,000), H3K9me2 (Abcam, ab1220, 1:1,000), H3K9me3 (Abcam, ab8898, 1:1,000), H3K27me3 (Millipore, 17‐622, 1:2,000), and H3K36me3 (Abcam, ab9050, 1:1,000). Antibody–chromatin complexes were pulled down with protein A/G (Invitrogen), washed, and then eluted. After cross‐link reversal and proteinase K treatment, immune‐precipitated DNA was extracted with phenol–chloroform, ethanol precipitated, and treated with RNase. ChIP DNA was quantified using PicoGreen. For ChIP‐qPCR, primer sequences are listed in Dataset EV6. qPCR was performed on the CFX96 Real‐Time System (Bio‐Rad) with SsoAdvanced SYBR Green Supermix (Bio‐Rad).

To identify histone modifications, acid extraction of histone was performed as previously reported [48 (link)]. To detect other proteins, cells were extracted with lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1.5 mM PMSF and protease inhibitors cocktail). Equal amounts of protein were size fractionated on 6.0 to 15.0% SDS-PAGE gel. The antibodies used were anti-COX-2 (sc-19999, Santa Cruz), anti-c-Fos (ab7963, Abcam), anti-c-Jun (sc-44, Santa Cruz), anti-KMT3A (ab31358, Abcam), anti-KMT3B (17-10264, Merck Millipore), anti-KDM2A (ab31739, Abcam), anti-KDM2B (ab108276, Abcam), anti-KDM4A (ab70786, Abcam), anti-KDM4B (ab80473, Abcam), anti-NF-κB (sc-372G, Santa Cruz), anti-p300 (H-272, Santa Cruz), anti-CEBPβ (sc-150, Santa Cruz), anti-H3K4me1/2/3 (ab8895/ab7766/ab1012, Abcam), anti-H3K9me1/2/3 (ab9045/ab1220/ab8898, Abcam), anti-H3K27me1/2/3 (ab113671/ab24684/ab6002, Abcam), anti-H3K36me1/2/3 (ab9048/ab9049/ab9050, Abcam), anti-H3K79me1/2/3 (ab2886/ab3594/ab2621, Abcam), anti-H4K20me1/3 (ab9051/9053, Abcam), anti-histone H3 (ab131711, Abcam) and anti-β-actin (sc-1615, Santa Cruz).

Immunofluorescence was performed as in Ding et al. for H3K4me3 staining. For mono or di methyl staining, animals were fixed in 1% paraformaldehyde and permeabilized in cold 100% methanol before proceeding with the remainder of the protocol used in Ding, et al. 2015. Antibodies used were: Tri-Methyl-Histone H3 (Lys4) Rabbit mAb #9751 (Cell Signaling), Abcam Anti-Histone H3 (di methyl K4) antibody—ChIP Grade (ab7766) (Abcam) and Anti-Histone H3 (mono methyl K4) antibody—ChIP Grade (ab889) (Abcam).

LoVo, HCT-116, COLO-320, SW1116, and Caco2 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI-1640 (COLO-320) supplemented with 10% fetal bovine serum at 37°C. Normal colonic epithelial cells (NCM460) were cultured in F12 supplemented with 20% fetal bovine.
Antibodies against LSD2 (ab193080 for CHIP and WB, ab234863 for IHC), p53 (ab32389), H3K4me2 (ab7766), p21 (ab227443) and Ki-67(ab15580) were purchased from Abcam (Cambridge, MA, USA), while CL-caspase 9, CL-caspase 3, BAX, Bcl-2, Phospho-Rb (Ser807/811)(9308) and GAPDH antibodies were purchased from Cell Signaling (Danvers, MA, USA).