H3k27me1

For all HMTase assays, 446 nM of mononucleosomes or 223 nM of dinucleosomes were incubated with indicated amounts of the different PRC2 complexes, in a reaction buffer containing 20 mM HEPES pH 7.8, 50 mM NaCl, 2.5 mM MgCl₂, 5% glycerol, 0.25 mM EDTA, 0.5 mM DTT and 80 μM S-adenosylmethionine (SAM). Reactions were allowed to proceed for 90 min at RT before quenching by the addition of 1x (final concentration) SDS loading buffer and heat inactivation at 95°C for 5 min. Proteins were separated by electrophoresis on a 16% (w/v) SDS gel, transferred to a nitrocellulose membrane and probed with antibodies against H3K27me3 (Millipore, 07–449), H3K27me1 (Millipore, 07–448) and H4 (Abcam, ab10158). For quantification, HMTase reactions and the corresponding western blots on D.m. unmodified, H3Kc36me3, H3^K36A/R mononucleosomes were performed in triplicates and subjected to densitometric analysis (Chemiluminescence signal, ImageQuant LAS 4000). The integrated densitometric signal (band) in each lane was background corrected against the control lane (lane 1, no PRC2 in the reaction) and normalized with respect to the lane containing the highest amount (i.e. 100%) of PRC2 on unmodified nucleosomes (lane 4). The relative amounts of trimethylation/monomethylation for all other lanes were calculated with respect to lane 4. Graphical representations were made with Prism 8 (GraphPad).

Immunostaining experiments were performed according to Latrasse et al^{65 (link)}. Briefly, fourth leaves of 4-week-old tomato plants were fixed and then nuclei were isolated, placed on a poly-lysine slide, and incubated overnight at 4 °C with primary antibodies (400X diluted) of H3K9me2 (Abcam, ab1220), H3K9ac (Millipore, 07–352), H3K27me1 (Millipore, 07–448) and RNAPII (Abcam, ab26721). Slides were washed and then incubated for 1 h at room temperature in the dark with Goat anti-Rabbit Alexa Fluor Plus 488 (A11034 Invitrogen) and Goat anti-Mouse Alexa Fluor Plus 555 (A32727 Invitrogen) or with Goat anti-Rabbit Alexa Fluor Plus 555 (A32732 Invitrogen) and Goat anti-Mouse Alexa Fluor Plus 488 (A32723 Invitrogen) secondary antibodies (400X diluted). DNA was counterstained with 4,6 diamidino-2-phenylindole (DAPI) in SlowFade Diamond Antifade mounting media. Slides were directly imaged on a confocal microscope (Zeiss Microsystems).

Normal rabbit IgG (12–370) and normal mouse IgG (12–371) were purchased from Millipore/Upstate. H3K79me3 (17–10,130), H3K36me3 (17–10,032), H3K27me1 (17–643), H3K4me3 (07–473), Acetyl-Histone H4 (17–630) and H3K9ac (17–658) antibodies were procured from Millipore. All of the above antibodies were used for ChIP analysis at 5 μg antibody per 30 μg chromatin. Oct-3/4 (sc-8628) and Sox2 (sc-17,319) antibodies were procured from Santa Cruz Biotechnology. Anti-Nanog (AF1997) was purchased from R&D Systems. Anti-H3 (ab1791) was procured from Abcam. All of the above antibodies were used for immunoblot analysis at 1:1000 dilution.

The EZ-Magna ChIP kit (EMD Millipore) was used to conduct the chromatin immunoprecipitation (ChIP) assays in accordance with the manufacturer’s protocol. After carrying out the above-mentioned cell treatments, the cells were lysed with Cell Lysis Buffer and Nuclear Lysis Buffer and sonicated to obtain chromatin fragments. Next, the lysates were immunoprecipitated with Magnetic Protein A Beads conjugated with Histone3 (Millipore) or IgG (Millipore) as a control. Western blot assay was performed to detect the enrichment of LSD1, H3K27me1 (Millipore), H3K4me2 (Millipore), and DNMT1 (Millipore) in the precipitated protein complex.

Human primary mammary cells were purchased from Chi Scientific and cultured in DMEM/F12 (10 % FBS, with addition of insulin, hydrocortisone, and EGF) at 37 °C with 5 % CO₂. MCF7 was purchased from Cell Bank of Chinese Academy and cultured in DMEM with FBS, insulin (10 μg/mL), sodium pyruvate (Invitrogen), and nonessential amino acid (Invitrogen); T47D is a gift from Dr. Yong-Feng Shang of Tianjin Medical University and cultured in RPMI1640 with FBS and insulin. Both the primary cells and cancer cell lines were sub-cultured 1:4 on reaching confluence; each passage was considered two PD.
Antibodies were purchased from the indicated companies: H3K9ac, hTERT, KRT14, and ACTG (Epitomics); KRT18 (ProteinTech), JMJD1B, JMJD2B, JMJD2A, H4K20me3, H3K79me2, E-cadherin, and RAS (CST); H3K4me1, H3K4me3, H3K27me3, H3K27me1, H3K27me2, and H3K36me2 (Millipore); H3, H3K9me1, H3K4me2, and H3K36me3 (Abcam); H3K9me2, H3K9me3, GAPDH, EHMT2, SUV39H1, CBX5, DNMT3A, DNMT3B, DNMT1, and KDM1A (Abclonal); KDM3A/JMJD1A (Abclonal for western and Millipore for immunostaining). The information of primers and siRNAs are listed in Additional file 2: Table S16.