Histone ChIP was performed as previously described (Lee et al., 2006). Briefly, cell samples were cross-linked in 1% formaldehyde for 10 minutes, and the reaction was stopped by addition of glycine to 125 mM final concentration. Fixed cells were lysed in SDS lysis buffer, and the chromatin was fragmented by sonication (Covaris). Sheared chromatin was incubated with a final 10 μg/mL concentration of antibodies against either H3K4me3 (Abcam, ab8580, Lot:GR164706-1), H3K27ac (Abcam, ab4729, Lot:GR200563-1), H3K4me1 (Abcam; ab8895, Lot:GR114265-2) or normal rabbit IgG (Abcam, ab46540) at 4 °C for overnight. Antibodies were recovered by binding to protein A/G agarose (Millipore), and the eluted DNA fragments were used directly for qPCR or subjected to high-throughput sequencing (ChIP-Seq) using a HiSeq 2000 platform (Illumina). High-throughput reads were aligned to mouse genome assembly NCBI37/mm9 as previously described (Barradas et al., 2009). Reads that aligned to multiple loci in the mouse genome were discarded. The ChIP-Seq signal for each gene was quantified as total number of reads per million (RPM) in the region 2 kb upstream to 2 kb downstream of the transcription start site (TSS). The complete dataset is available at NCBI Gene Expression Omnibus (GSE85055). Primers used for ChIP-qPCR of mouse Cdkn2a promoter (Barradas et al., 2009) are included in Supplementary Table S1.

For the MLL3 ChIP-Seq, the following protocol was used. Cross-linking ChIP in mouse and human HCC cells was performed with 10–20×10⁷ cells per immunoprecipitation. Cells were collected, washed once with ice-cold PBS, and flash-frozen. Cells were resuspended in ice-cold PBS and cross-linked using 1% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 5 minutes at room temperature with gentle rotation. Unreacted PFA was quenched with glycine (final concentration 125mM) for 5 minutes at room temperature with gentle rotation. Cells were washed once with ice-cold PBS and pelleted by centrifugation (800g for 5 minutes). To obtain a soluble chromatin extract, cells were resuspended in 1mL of LB1 (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1X complete protease inhibitor cocktail) and incubated at 4°C for 10 minutes while rotating. Samples were centrifuged (1400g for 5 minutes), resuspended in 1mL of LB2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 1X complete protease inhibitor cocktail), and incubated at 4°C for 10 minutes while rotating. Finally, samples were centrifuged (1400g for 5 minutes) and resuspended in 1mL of LB3 (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-Lauroylsarcosine, 1X complete protease inhibitor cocktail). Samples were homogenized by passing 7-8 times through a 28-gauge needle and Triton X-100 was added to a final concentration of 1%. Chromatin extracts were sonicated for 14 minutes using a Covaris E220 focused ultrasonicator. Lysates were centrifuged at maximum speed for 10 minutes at 4°C and 5% of supernatant was saved as input DNA. Beads were prepared by incubating them in 0.5% BSA in PBS and antibodies overnight (100μL of Dynabeads Protein A or Protein G (Invitrogen) plus 20μL of antibody). Antibody used the anti-MLL3/4 (kindly provided by the Wysocka laboratory (Dorighi et al., 2017)). Antibody-Beads mixes were washed with 0.5% BSA in PBS and then added to the lysates overnight while rotating at 4°C. Beads were then washed six times with RIPA buffer (50mM HEPES pH 7.5, 500mM LiCl, 1mM EDTA, 0.7% sodium-deoxycholate, 1% NP-40) and once with TE-NaCl Buffer (10mM Tris-HCl pH 8.0, 50mM NaCl, 1mM EDTA). Chromatin was eluted from beads in Elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) by incubating at 65°C for 30 minutes while shaking, supernatant was removed by centrifugation, and crosslinking was reversed by further incubating chromatin overnight at 65°C. The eluted chromatin was then treated with RNaseA (10mg/mL) for 1 hour at 37°C and with Proteinase K (Roche) for 2 hours at 55°C. DNA was purified by using phenol-chloroform extraction followed with ethanol precipitation. The NEBNext Ultra II DNA Library Prep kit was used to prepare samples for sequencing on an Illumina NextSeq500 (75bp read length, single-end, or 37bp read length, paired-end).