Generation and characterization of a zebrafish ezh2 null mutant. (A) The C to T point mutation in the ezh2 coding region was confirmed by DNA sequencing. WT, wild-type. (B) Gene structure of ezh2 and the point mutation occurs in Exon 2. (C) The C to T nonsense mutation results in a truncated peptide and losses of its major functional domains. SANT, SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains; CXC, Tesmin/TSO1-like CXC domain; SET, Su(var)3–9, Enhancer-of-zeste, Trithorax domain. (D) Images of WT and ezh2–/– mutant zebrafish from 24 hpf to 72 hpf. Note that the ezh2–/– mutant zebrafish display curved tail and thin yolk sac extension around 28 hpf and short body length and small eyes after 48 hpf. Scale bar, 0.5 mm. (E) Western Blotting shows that expression of Ezh2 is abolished and that of H3K27 mono-, di- and trimethylation are reduced in ezh2–/– mutant fish. Proteins of zebrafish larvae were extracted with RIPA buffer at 48 hpf. Western Bloting was performed with indicated antibodies. (F) Quantification of western bloting images shown in (E) with ImageJ. Student's t-test was conducted. ***P <0.001. (G, H) qRT-PCR analysis of Ezh2-targeted gene dab2ipa (G) and tmem48 (H) in 28 hpf WT and ezh2–/– mutant zebrafish. Three independent experiments were conducted. Statistical analysis was performed using student's t-test. *P < 0.05.
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