目的 观察心脏发育过程中组蛋白乙酰化酶P300、PCAF、SRC1对心脏发育核心转录因子Mef2c的动态修饰作用，为明确先天性心脏病的发生机制奠定基础。方法 选取胎龄14.5 d和16.5 d胎鼠及生后0.5 d和7 d小鼠的正常心脏组织，采用染色质免疫共沉淀技术，运用抗P300、PCAF、SRC1、乙酰化组蛋白（ac-H3）抗体免疫沉淀与其相结合的DNA，Real-Time-PCR扩增抗体所富集的DNA，观察小鼠心脏发育过程中Mef2c启动子区域组蛋白乙酰化酶的动态修饰及ac-H3水平；同时运用Real-Time-PCR检测Mef2c mRNA表达量。结果 小鼠心脏发育过程中P300、PCAF、SRC1均参与Mef2c启动子区域的乙酰化修饰，其结合水平在心脏发育的不同阶段差异均有统计学意义（均P<0.01）。Mef2c启动子区域组蛋白H3乙酰化水平及Mef2c基因mRNA表达水平在心脏发育的不同阶段差异均有统计学意义（均P<0.01）。ac-H3、Mef2c mRNA及3种组蛋白乙酰化酶表达水平均具有一定时序性，即在胎龄14.5 d时，上述指标水平均明显高于其他时间点（均P<0.01），且随心脏的发育成熟表达水平均逐渐降低，至出生后均维持在低表达水平。结论 小鼠心脏发育过程中Mef2c基因呈现动态表达，提示Mef2c基因在心脏发育中具有重要作用；同时Mef2c启动子区域乙酰化水平的变化受组蛋白乙酰化酶P300、PCAF、SRC1的动态调控。
Abstract
Objective To observe the temporal modification of transcription factor Mef2c by histone acetylases (HATs) P300, PCAF, and SRC1 during cardiogenesis and to provide a basis for investigating the pathogenesis of congenital heart disease. Methods The normal heart tissues from embryonic mice (embryonic days 14.5 and 16.5) and neonatal mice (postnatal days 0.5 and 7) were collected. The binding of P300, PCAF, and SRC1 to Mef2c gene and level of histone H3 acetylation in the promoter region of Mef2c were evaluated by chromatin immunoprecipitation assays. Meanwhile, real-time PCR was used to measure the mRNA expression of Mef2c. Results P300, PCAF, SRC1 were involved in histone acetylation in the promoter region of Mef2c during cardiogenesis in mice, and binding of P300, PCAF, and SRC1 to the promoter of Mef2c varied significantly in different stages of cardiogenesis (P<0.01). The level of histone H3 acetylation and mRNA expression of Mef2c in the promoter region of Mef2c also varied significantly in different stages of cardiac development (P<0.01). The levels of acetylated H3, Mef2c mRNA, and HATs (P300, PCAF, SRC1) changed over time. They were highest on embryonic day 14.5 (P<0.01), decreased gradually with cardiac development, and were maintained at low levels after birth. Conclusions The mRNA expression of Mef2c varies during cardiogenesis in mice, which indicates that Mef2c plays an important role in the process of cardiac development. Meanwhile, histone acetylation in the promoter region of Mef2c is regulated temporally by HATs P300, PCAF, and SRC1.
关键词
组蛋白乙酰化酶 /
心脏发育 /
转录因子 /
动态调控 /
小鼠
Key words
Histone acetylase /
Cardiac development /
Transcription factor /
Temporal regulation /
Mice
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参考文献
[1] van der Linde D, Konings EE, Slager MA, et al. Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis[J]. J Am Coll Cardiol, 2011, 58(21): 2241-2247.
[2] He Y, Wang J, Gu X, et al. Application of spatio-temporal image correlation technology in the diagnosis of fetal cardiac abnormalities[J]. Exp Ther Med, 2013, 5(6): 1637-1642.
[3] Lockhart MM, Wirrig EE, Phelps AL, et al. Mef2c regulates transcription of the extracellular matrix protein cartilage link protein 1 in the developing murine heart[J]. PLoS One, 2013, 8(2): e57073.
[4] Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair[J]. Nat Rev Mol Cell Biol, 2013, 14(8): 529-541.
[5] Voronova A, Al MA, Fischer A, et al. Gli2 and MEF2C activate each other's expression and function synergistically during cardiomyogenesis in vitro[J]. Nucleic Acids Res, 2012, 40(8): 3329-3347.
[6] Huang JB, Liu YL, Sun PW, et al. Molecular mechanisms of congenital heart disease[J]. Cardiovasc Pathol, 2010, 19(5): e183-e193.
[7] 俞立玮, 桂永浩. Gata4转录后蛋白共价修饰[J]. 中国当代儿科杂志, 2012, 14(10): 800-803.
[8] Olson EN. Gene regulatory networks in the evolution and development of the heart[J]. Science, 2006, 313(5795): 1922-1927.
[9] Vong LH, Ragusa MJ, Schwarz JJ. Generation of conditional Mef2cloxP/loxP mice for temporal-and tissue-specific analyses[J]. Genesis, 2005, 43(1): 43-48.
[10] Zeisberg EM, Ma Q, Juraszek AL, et al. Morphogenesis of the right ventricle requires myocardial expression of Gata4[J]. J Clin Invest, 2005, 115(6): 1522-1531.
[11] Qin X, Xing Q, Ma L, et al. Genetic analysis of an enhancer of the NKX2-5 gene in ventricular septal defects[J]. Gene, 2012, 508(1): 106-109.
[12] Granados-Riveron JT, Pope M, Bu'lock FA, et al. Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations[J]. Congenit Heart Dis, 2012, 7(2): 151-159.
[13] Amodio V, Tevy MF, Traina C, et al. Transactivation in Drosophila of human enhancers by human transcription factors involved in congenital heart diseases[J]. Dev Dyn, 2012, 241(1): 190-199.
[14] 夏慧苏, 余细勇. NKX2.5基因的表观遗传学改变与先天性心脏病的关系[J]. 实用医学杂志, 2009, 25(24): 4257-4258.
[15] Sadoul K, Boyault C, Pabion M, et al. Regulation of protein turnover by acetyltransferases and deacetylases[J]. Biochimie, 2008, 90(2): 306-312.
[16] 杨雪芳, 田杰, 陈国珍, 等. 小鼠胚胎心脏发育过程中组蛋白乙酰化酶亚型p300调控心脏特异转录因子的动态表达[J]. 重庆医科大学学报, 2010, 35(7): 961-965.
[17] Yang XJ, Seto E. HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention[J]. Oncogene, 2007, 26(37): 5310-5318.
基金
国家自然科学基金资助项目（81270234）；重庆市科委重点实验室专项经费资助项目。
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