Anti h3k9ac

Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described^{22 (link),23 (link)}. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program.

ChIP experiments were conducted as described in Desvoyes et al., 2018 with minor modifications. At least 0.5g 2-week-old plants were cross-linked in ice-cold 1x PBS buffer with 1% formaldehyde by vacuum for 18min in total. 2.5ng H3K9ac antibodies (Anti-H3K9ac from Millipore, catalog number: 07-352) were added to 1mL dilution chromatin and 25μL dynabeads protein G (Invitrogen, 10004D) were used for pulling down the antibody-associated chromatin. Primers used for PCR can be found in Table S1.

Cells were grown to 80% confluence, washed twice in warm PBS, dissociated with 0.5x Accutase (Catalogue # SCR005; Millipore), and re-suspended in warm medium (DMEM F-12 Catalogue # 11320-033; Invitrogen) containing 0.1 volume crosslinking solution (Kondo, Shen, Yan, Huang & Issa, 2004 (link)). ChIP reactions were performed as described previously (Veazey et al., 2015 (link)) followed by DNA purification with a Qiaquick PCR Cleanup kit (Catalogue # 28106; QIAGEN). Antibodies used include: anti-H3K4me3 (Catalogue # 04-745; Millipore), anti- H3K27me3 (Catalogue # 39155; Active Motif), anti-H3K9ac (Catalogue # 07-352; Millipore), and anti-H3K9me2 (Catalogue # 39239; Active Motif). Antibodies for modified histones were used at 1 μg/ChIP reaction. The concentration of IgG (Catalogue # SC-2027; Santa Cruz) was also used at 1 ug/ChIP reaction. For analysis of candidate loci, real-time PCR was performed with the Dynamo Flash supermix (Catalogue # F-415XL; Thermo Scientific) according to the recommended protocol. Reactions were performed on a Bio-Rad CFX384 Touch PCR system. Data was analyzed using the formula previously described (Mukhopadhyay, Deplancke, Walhout & Tissenbaum, 2008 (link)). Primer sequences are listed in Table S1- Primer Sequences.

Petal were collected from R. chinensis ‘Old Blush’ and fixed in 1% (v/v) formaldehyde. ChIP assays were performed using anti-H3K9ac (Millipore, ref. 07-352) or anti-H3K27me3 (Millipore, ref. 07-449) antibodies according to a procedure adapted from Veluchamy et al61 (link). Library quality was assessed with Agilent 2100 Bioanalyzer (Agilent) and the libraries were subjected to high-throughput sequencing on Illumina NextSeq 500. After trimming, reads were aligned onto R. chinensis genome with bowtie262 (link) and a maximum mismatch of 1 bp and unique mapping reported. To determine the target regions of H3K9ac ChIP-seq, the Model-based Analysis of ChIP-seq (MACS2)63 (link) was used. Detection of H3K27me3 modification regions was performed using SICER64 (link). HOMER65 (link) was used to annotate H3K9ac peaks with nearby genes if peaks were located into -2k to +1kb window around the gene TSS. For H3K27me3 peaks, bedtools intersect 56 (link) was used and only genes that are overlapped with this specific modification were kept. Clustering of H3K9ac and H3K27me3 peaks was performed using SeqMINER66 (link). Rstudio, Circos67 (link) and NGSplot68 (link) were used for graphic representation of histone modifications.

Whole cell lysates/chromatin fractions were prepared and western blot analysis was performed as previously described^{12 (link)}. In brief, for chromatin extraction, cell pellets were lysed in buffer containing 10mM HEPES pH7.4, 10mM KCl, 0.05% NP-40 supplemented with a protease inhibitor cocktail (Complete EDTA-free, Roche Applied Science), 5 μM TSA, 5mM sodium butyrate, 1mM DTT, 1mM PMSF, and 0.2mM sodium orthovanadate. After incubation for 20min on ice, the lysates were centrifuged at 14,000 rpm, 10min at 4 °C. The supernatant was removed (cytosolic fraction) and the pellet (nuclei) was acid-extracted using 0.2N HCl by incubating 20min on ice. The lysate was further centrifuged at 14,000 rpm, 10min at 4 °C. The supernatant was neutralized in 1M Tris-HCl pH 8. Protein concentration was determined by Biorad protein assay. Western blots were performed using 8-15% gradient gels (Biorad). Primary antibodies were used as follows: anti-SIRT6 (Cell signaling #12486), anti-H3K9Ac (Millipore, 07-352), anti-H3K56Ac (Abcam, ab76307), anti-total H3 (Abcam, ab1791), anti-GLUT1 (Abcam, ab40084), anti-PDK1 (Cell signaling, #3820), anti-LDHA (Cell signaling, #2012S), anti-phospho-PDH (Abcam, ab92696), and anti-β-actin (Sigma, A5316). All uncropped and unprocessed scans are available in Source Data Figures 1-4.