H2aub

Triplicate pellets of 1 × 10⁶ cells were collected for all conditions, flash frozen and stored at −80 °C before use. Mouse feeder removal microbeads (Milteny Biotech, 130-095-531) were used according to the manufacturer’s protocol to reduce the amount of MEFs in the naïve cell samples. Samples were prepared for ChIP–seq following the MINUTE-ChIP protocol (10.17504/protocols.io.8nkhvcw)^{42 (link)}. Briefly, native cell pellets were lysed, MNase digested to mono- to tri-nucleosome fragments and ligated with double-stranded DNA adaptors (containing T7 promoter, 8 bp sample barcode and a 6 bp unique molecular identifier (UMI)) in a one-pot reaction. Barcoded samples were then pooled and aliquoted into individual ChIP reactions with Protein A/G magnetic beads (Bio-Rad, 161- 4013/23) coupled with the desired antibodies (5 μg each of H3K27me3 (Millipore, 07-449), H3K4me3 (Millipore, 04-745) and H2AUb (Cell Signaling, 8240 S)). Upon incubation for 4 h at 4 °C with rotation and washing steps, ChIP DNA was isolated and set up in sequential reactions of in vitro transcription, RNA 3′ adapter ligation, reverse transcription and PCR amplification to generate final libraries for each ChIP (Extended Data Fig. 1a). After quality assessment and concentration estimation, libraries were diluted to 4 nM, combined and sequenced on the Illumina NextSeq500 platform with paired-end settings.

Co-IP was performed with Protein G-coupled Dynabeads^® (Thermo Fisher, Waltham, MA, USA) according to manufacturer’s instructions. Briefly, 30 μl beads were incubated with 3 μg anti-Flag antibody (F1804; Sigma-Aldrich) for 10 min in PBS-T (0.2% Tween-20) at room temperature. Antibody-coupled beads were incubated overnight with 200 µg protein lysate in PBS-T at 4 °C. After washing beads in PBS-T, protein complexes were eluted in SDS loading buffer for 5 min at 70 °C. Subsequently SDS-PAGE with pre-cast 4–20% gradient gels (Bio-Rad, Feldkirchen, Germany) and Western Blotting were performed as previously described [33 (link)]. Proteins were detected with primary antibodies against Flag (F1804; Sigma-Aldrich), MYSM1 (orb137033, Biorbyt; St Louis, MO, USA), H2Aub (8240; Cell Signaling, Leiden, The Netherlands), H2AX (Abcam; ab2893, Cambridge, UK), Actin (sc-1615HRP; Santa Cruz, Heidelberg, Germany), and other specific antibodies as indicated followed by Peroxidase-coupled specifies-specific secondary antibodies (711-035-152, 715-035-150; Jackson ImmunoResearch, West Grove, PA, USA).

Protein lysates were extracted from cells and blotted as described in Schulte et al. [13 (link)]. The following antibodies were used: KDM5C/JARID1C (1:1000, ab34718, Abcam); β-ACTIN (1:10.000, A5441; Sigma-Aldrich); E-cadherin (1:1000, #3195, Cell Signaling, Cambridge, UK); N-cadherin (1:1000, #14215, Cell Signaling); SNAI2/Slug (1:1000, #9585, Cell Signaling); α-TUBULIN (1:1000, #2144, Cell Signaling); SMAD1 (1:1000, #6944, Cell Signaling); SMAD4 (1:1000, #38454, Cell Signaling); H2Aub, (1:1000, #8240, Cell Signaling); H3K4me2, (1:1000, #035050, Diagenode); H3K4me3 (1:1000, ab1012, Abcam); STAT6 D3H4 (1:1000, #5397, Cell Signaling); Phospho-SMAD1 Ser463/465/Smad5 Ser463/465/Smad9 Ser463/465 D5B10 (1:1000, #13820, Cell Signaling).

Seedlings (2 g, 10 days old, grown on MS plates) were harvested, ground to a powder in liquid nitrogen, and then homogenized in two volumes of lysis buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl₂, 250 mM sucrose, 1 mM PMSF, and 5 mM β-mercaptoethanol) at 4°C. The homogenate was filtered through one layer of Miracloth (EMD Millipore, Billerica, MA, United States) and centrifuged at 1,500 × g for 10 min at 4°C to pellet the nuclei. The pellet was washed two to four times in nuclei resuspension buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, and 2.5 mM MgCl₂) with 0.2% Triton X-100 until the pellet became white or gray, then the pellet was resuspended gently in nuclei resuspension buffer without Triton X-100 and centrifuged at 1,500 × g for 10 min at 4°C to pellet the nuclei. The nuclei pellet was treated for 3 h with 0.4 N H₂SO₄ and the proteins were precipitated with 25% trichloroacetic acid for 1–2 h. The precipitated proteins were then washed two times with acetone, air-dried, and resuspended in 4 M urea. Equal amounts of histone were subjected to SDS-PAGE, transferred to a PVDF membrane, and probed with anti-ubiquitin (sc-8017; Santa Cruz Biotechnology, Santa Cruz, CA, United States), -H2Aub (8240; Cell Signaling Technology, Danvers, MA, United States), or -H3 antibodies (06-755; EMD Millipore), respectively.