H3k9me1

ChIP-seq was performed from FACS-sorted CM nuclei^{18 (link), 19 (link)}. The following antibodies were used in this study: CTCF (diagenode, C15410210-50, 4 µg/ChIP), Cohesin (anti-SMC1; biomol, A300-055A, 4 µg/ChIP), H3K9me1 (abcam, ab8896, 4 µg/ChIP), H3K9me2 (Cell Signaling Technology, #9753, 4 µg/ChIP), H3K9me3 (Diagenode, C15410193, 4 µg/ChIP). For CTCF and Cohesin immunoprecipitation, the iDeal ChIP-seq kit for Transcription Factors (Diagenode) was used with 1 µg of chromatin and for H3K9me1/2/3 the ChIP-IT High Sensitivity Kit (Active Motif) was used with 200 ng of chromatin according to manufacturer’s instructions. Sequencing libraries were prepared from the resulting DNA with the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB). To avoid over-amplification of the sequencing library, test qPCR-amplification was carried out to determine minimum cycles for final PCR.

The following antibodies were used in this study for either western blotting, ChIP-sequencing, immunohistochemistry and chromatin immunoprecipitation. SOD2 (The Binding site, PC096), Gpx1 and Gpx4 (gift from L. Chavatte), ChREBP (Novus, NB400), SCD1 (Cell Signaling, C12H5), Phf2 (Cell Signaling, D45A2), H3K9me1 (Abcam, ab8896), H3K9me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898), histone H3 (Diagenode, C15200011), p(S473)-AKT (Cell signaling, D9E), Akt (Cell Signaling, 9272), P-p70S6K (Cell Signaling, 108D2), p70S6K (Cell Signaling, 49D7), p-GSK3β (Cell Signaling, D17D2), GSK3β (Cell Signaling, 27C10), LPK (Abcam, ab6191), ACC (Cell Signaling, 3662), FAS (Cell signaling, C20G5), p62 (gift from A. F. Burnol), Nrf2 (Santa Cruz Biotechnology, 13032), G6PDH (Cell Signaling, 12263), TKT (Cell Signaling, 8616), α-SMA (Novus, NB-600-531), collagen I (Novus, NB600-408), GAPDH (Santa Cruz Biotechnology, FL-335), malic enzyme (Novus, 68578), MTTP (Novus, 62489), NQO1 (Novus, A180), RNA PolII (Santa Cruz, sc899), HSP90 (Cell Signaling, 4874), Cidec (Abcam, ab77115), Plin2 (Progen, ap125), G0S2 (Santa Cruz Biotechnology, N13), and FLAG (Sigma, F1804).

AR (Ab74272, Abcam, Waltham, MA, USA), BRD4 (A301-985A, Bethyl, Montgomery, TX, USA), H3K27ac (C15410196, DIAGENODE, Denville, NJ, USA), H3K4me1 (Abcam, ab8895), H3K4me2 (Abcam, ab7766), H3K9me1 (Abcam, ab9045), H3K9me2 (Abcam, ab1220), H3ac (Thermo Fisher, 39139), FoxA1 (Abcam, ab23738), Med1 (1710530, Sigma-Aldrich, St. Louis, MO, USA), CDK9 (Cat. sc-8338, Santa Cruz, Dallas, TX, USA), cyclin T1 (CCNT1, Santa Cruz, Cat. sc-10750), p-RNA Pol II-Ser5 (pPol2-S5, Abcam, Cat. ab5131), and IgG (2729S, Cell Signaling, Danvers, MA, USA).

For in vitro demethylation assays, SlJMJ4-GST fusion proteins were purified using glutathione sepharose 4B (GE Healthcare). Afterwards, histone demethylase activity was analyzed as previously described [18 (link)]. In brief, the purified GST-tagged SlJMJ14 (4.0 μg) was incubated with calf thymus histones (Sigma) in a reaction buffer containing 150 mM NaCl, 80 μM Fe(NH₄)₂(SO₄)₂, 50 mM Tris–HCl (pH 7.0), 1 mM α-KG, and 2 mM ascorbic acid for 6 h at 37°C. The reaction was terminated with 10 μM EDTA and subjected to western blotting analysis. For in vitro demethylation assays, histone proteins were extracted from 2-month-old leaves of SlJMJ4-OE and WT plants with the EpiQuik Total Histone Extraction Kit (Epigentek, Farmingdale, NY, USA) and analyzed by western blotting. The antibodies used in this experiment were from Abcam: H3K4me1 (ab176877, 1:3000 dilution), H3K4me2 (ab11946, 1:3000 dilution), H3K4me3 (ab8580, 1:3000 dilution), H3K9me1 (ab9045, 1:3000 dilution), H3K9me2 (ab1220, 1:1000 dilution), H3K9me3 (ab8898, 1:1000 dilution), H3K27me1 (ab115068, 1:3000 dilution), H3K27me2 (ab24684, 1:3000 dilution), H3K27me3 (ab6002, 1:3000 dilution), H3K36me1 (ab176920, 1:3000 dilution), H3K36me2 (ab176921, 1:3000 dilution), H3K36me3 (ab9050, 1:3000 dilution), and H3 (ab1791, 1:5000). H3 was used as a loading control.

Cultured cells were fixed in 4% paraformaldehyde for 15 min, washed 3× with PBS, and blocked with blocking solution (PBS + 0.5% Tween20 + 5% FBS) for 1 h at room temperature (RT). Primary antibodies were diluted in blocking solution and incubated with cells overnight at 4 °C. Primary antibodies included H3K9ac (1:1000, Active Motif, 39917), H3K9me1 (1:2000, Abcam, ab9045), H3K9me2 (1:1000, Abcam, ab1220), H3K9me3 (1:1000, Diagenode, pAb-056-050). Cells were then washed for 5 min 3× in PBS and incubated with secondary antibodies in blocking solution for 2 h at RT. Secondary antibodies included 594-conjugated donkey anti-mouse (1:1000, Life Technologies, A21203) and 594-conjugated donkey anti-rabbit (1:1000, Life Technologies, A21207). DAPI (1 μg/ml) was added with secondary antibodies. Cells were washed for 5 min 3× in PBS prior to imaging.