H4k12ac

For RNA pull‐down, the procedure was performed with minor modifications.²⁰ Briefly, biotin‐labeled RP11‐367G18.1 variant 2 was synthesis by in vitro transcription and then purified. The biotinylated RP11‐367G18.1 variant 2 was incubated with cell lysates at 4°C for 6 h. The streptavidin agarose beads were added into the mixture and incubated for 1 h. The beads were washed and boiled for western blot analysis. As previously described,¹⁸ proteins and histones extracted from the cells were analyzed by SDS–PAGE using antibodies against HIF‐1α, N‐cadherin (BD Biosciences), H4K5Ac, H4K8Ac, H4K12Ac, H4K20me3, H3K56Ac, histone H4, p300, Tip60, HBO1, HAT1, CBP (Abcam), E‐cadherin, histone H3 (Cell Signaling Technology), plakoglobin, H3K4me2, H3K9Ac, H4K16Ac (Millipore), vimentin (Sigma‐Aldrich), and β‐actin (Genetex).

Immunofluorescent staining was conducted according
to a previously published study (27 (link)). The experiment was
performed three times. Here, 20 oocytes at MII stage,
were washed in PBS containing 3 mg/ml PVP (PBS/
PVP), fixed for 1 hour in 4% paraformaldehyde in PBS,
and permeabilized by incubation with 0.2% Triton X-100
in PBS for 30 minutes at room temperature. The cells were
blocked in PBS/5% bovine serum albumin (BSA) for 45
minutes and incubated with primary antibody against antiacetyl
H4/K12 (H4K12ac, Abcam, UK) (1:200 dilution)
and H3/K9 (H3K9ac, Abcam, UK) (1:500 dilution) at
4°C overnight. The following day, after washing three
times with PBS/PVA for 5 minutes, the oocytes were
incubated with the secondary antibody conjugated with
FITC for 1 hour at 37°C. Then, DNA was incubated with
3 mg/ml 4, 6-diamidino-2- phenylindole (DAPI) for 20
minutes, and the cells were mounted on glass slides with
etched rings to prevent them from being ruptured by the
cover slip. The slides were imaged using a fluorescent
microscope. Fluorescence intensity was determined by
ImageJ software.

In total, 10 ml of yeast culture was collected for each condition. Cell pellets were resuspended in 300 μl lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 7 mM EDTA, 5 mM DTT, cOmplete mini protease inhibitor (Roche 4693124001)). The samples were then vortexed with glass beads (Biospec 11079105) at 4 °C for 20 min. Samples were centrifuged and the supernatant collected for analysis. Samples were loaded onto NuPAGE Novex 4 to 12% Bis-Tris protein gels (Invitrogen, NP0322). Gels were transferred to nitrocellulose membrane by wet transfer. Membranes were incubated with antibodies in PBS-0.2% tween with 5% milk. The following primary antibodies were used at the specified concentrations: H3 (CST 9715, 1:10,000), H3K4ac (Millipore 07-539, 1:1000), H3K9ac (Millipore 07352, 1:5000), H3K14ac (Millipore 07-353, 1:10,000), H3K18ac (Millipore 07-354, 1:5000), H3K23ac (Millipore 07-355, 1:10,000), H3K27ac (Millipore 07-360, 1:15,000), H3K56ac (Millipore 07-677, 1:1000), H3 pan-acetyl (Millipore 06-599, 1:500), H4 (Millipore 05-858, 1:1000), H4K5ac (Millipore 07-327, 1:2000), H4K8ac (Abcam ab15823, 1:1000), H4K12ac (Abcam ab1761, 1:5000), H4 pan-acetyl (Millipore 05-858, 1:1000), and pSTAIR loading control (Sigma-Aldrich P7962, 1:4000). Membranes were developed using ECL films (GE Healthcare Life Sciences, 28906836) and a XOGRAF Compact X4 film processor.