Cryptic splice sites generate heterogeneous EZH2 isoforms with distinct functions in embryonic development [CUT&Tag]

A novel Ezh2 variant was identified, we named Ezh2b, which is lost 27-nt region in the end of exon3, has different functions on embryonic development in mouse compared to Ezh2 with 27-nt region (namely as Ezh2a). Here, we apply transcriptome sequencing to explore the underlying molecular mechanisms. Overall design: The embryonic fibroblasts (MEF) were isolated from Ezh2amut, Ezh2bmut and respective littermate wild-type control mice. The MEFs were cultured in DMEM high-glucose medium . DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 µg/ml streptomycin. Utilizing the Hyperactive® Universal CUT&Tag Assay Kit for Illumina (TD903), we performed CUT&Tag experiments as follows: MEFs were harvested at room temperature and incubated with ConA Beads for 10 minutes. They were then incubated with anti-EZH2 (CST, 5246), anti-H3K27me3 (CST, #9733) antibodies overnight at 4°C. On the next day, the samples were incubated with a secondary antibody at room temperature for 1h, followed by incubation with pA/G-Tnp for 1h. After DNA purification with the MinElute kit, and the PCR was performed to amplify the library for 12 cycles and libraries were purified with the MinElute kit. Amplified libraries were sequenced on the Illumina Novaseq 6000 platform.