组蛋白乙酰化酶介导的组蛋白H3K9ac高乙酰化在孕期乙醇暴露致MEF2C过表达中的作用

Effects of hyperacetylation of H3K9ac mediated by histone acetylases on the overexpression of MEF2C induced by alcohol in the hearts of fetal mice during pregnancy

目的探讨组蛋白乙酰化酶介导的组蛋白H3K9ac的高乙酰化在孕期乙醇暴露致MEF2C过表达中的作用，为防治乙醇所致的心脏发育异常提供新的干预靶点。方法选取C57BL／6小鼠作为研究对象，随机分为5组：空白对照组、二甲基亚砜（DMSO）组、乙醇组、乙醇＋漆树酸组、漆树酸组。收集胎鼠心脏，采用染色质免疫共沉淀技术和Westernblot检测小鼠心肌组织中MEF2C启动子区域组蛋白乙酰化酶的结合量及组蛋白H3K9ac的乙酰化水平。运用实时荧光定量PCR检测心脏核心转录因子MERCmRNA表达水平。结果胎鼠心脏核心转录因子MEF2C启动子区域组蛋白H3K9ac乙酰化水平乙醇组（1．30±0．19）显著高于空白对照组（0．45±0．01），差异有统计学意义（P〈0．05）；且乙醇组胎鼠心脏组蛋白乙酰化酶E1A结合蛋白（0300）、p300／环磷酸腺苷反应元件结合蛋白辅助因子（PCAF）、类固醇受体辅活化子-1（SRCI）在MEF2C启动子区域的结合量（1．68±0．08、1．08±0．05、1．18±0．05）显著高于空白对照组（0．82±0．08、0．42±0．02、0．39±0．08），差异均有统计学意义（P均〈0．05）。胎鼠心脏MEF2CmRNA表达量在乙醇组（1．36±0．12）显著高于空白对照组（O．29±0．03），差异有统计学意义（P〈0．05）。组蛋白乙酰化酶抑制剂漆树酸能够显著降低MEF2C启动子区域p300、PCAF结合量（1．52±0．05比0．63±0．09，1．13±0．04比0．45±0．04）及组蛋白H3K9ac乙酰化水平（1．58±0．08比0．67±0．05），显著下调心脏核心转录因子MEF2C的表达（1．36±0．12比0．41±0．05），差异均有统计学意义（尸均〈0．05）。结论p300、PCAF介导的组蛋白H3K9ac高乙酰化可能是孕期乙醇暴露致心脏核心转录因子MEF2C过表达的重要调控因素。漆树酸能够通过抑制p300、PCAF在启动子区域的结合量显著降低乙醇所致的组蛋白H3K9ae高乙酰化，从而下调MEF2C的过表达。

Objective To investigate the effects of H3K9ac hyperacetylation mediated by histone acetylases on the overexpression of MEF2C in the hearts of fetal mice exposed to alcohol during pregnancy, and provide new interven- tion targets for prevention and treatment of cardiac dysplasia caused by alcohol exposure. Methods C57BL/6 mice were divided into 5 groups randomly, blank control group, dimethylsultbxide （DMSO） group, alcohol group, alcohol ± anacardic acid group and anacardic acid group, and then the hearts of fetal mice were collected to be analyzed. Chroma- tin immunoprecipitation and Western blot were used in assaying the binding of histone acetylases and the level of H3K9ac to the promoter of MEF2C in the hearts of fetal mice. The mRNA expression of MEF2C was tested by adopting real - time PCR. Results The level of H3K9ac in the promoter of MEF2C in the hearts of fetal mice exposed to alcohol was higher than that in the hearts of fetal mice exposed to saline （ 1.30 ±0.19 vs 0. 45 ±0.01 ） ,there was statistically significant difference （ P 〈 0.05 ）, while the binding of E1A - binding protein （ p300 ）, p300/cyclie adenosine mono- phosphate response element binding protein - associated factor （PCAF） and steroid receptor coactivator - 1 （SRCI） to the promoter of MEF2C were abnormally elevated in the hearts of fetal mice treated by alcohol （ 1.68 ± 0.08 vs 0. 82 ± 0. 08,1.08 ± 0. 05 vs 0. 42 ± 0.02,1.18 ± 0.05 vs 0. 39 ± 0. 08 ）, and there were statistically significant differences （ all P 〈 0.05 ）. The expression of MEF2C mRNA in alcohol group was higher than that in blank control group （ 1.36 ± 0. 12 vs 0. 29 ± 0.03 ）, there was statistically significant difference （ P 〈 0.05 ）. However, a pan - acetylases inhibitor, anacardic acid, could decrease significantly the binding of p300 and PCAF to the promoter of MEF2C （ 1.52 ± 0. 05 vs 0. 63 ± 0.09, 1.13 ± 0.04 vs 0. 45 ± 0.04 ）, and correct abnormal hyperacetylation of H3K9ac induced by alcohol （ 1.58 ± 0. 08 vs 0. 67 ± 0.05）, and down - regulate the over - expression of MEF2C in the hearts of fetal mice exposed to alcohol （1.36 ± 0. 12 vs O. 41 ± 0. 05 ）, and there were statistically significant differences （ all P 〈 0.05 ）.Conclusions Hyperacetylation of H3K9ac mediated by p300 and PCAF may be a key regulatory factor in the over - expression of cardiac nuclear transcription factor MEF2C in the hearts of fetal mice exposed to alcohol during pregnan- cy. Anacardic acid can significantly attenuate the level of H3K9ac through inhibiting the binding of p300 and PCAF to the promoter of MEF2C, and down -regulate the over- expression of cardiac nuclear transcription factor MEF2C in the hearts of fetal mice.