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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2015, Vol. 35 Issue (3): 8-17    DOI: 10.13523/j.cb.20150302
研究报告     
利用ChIP技术研究SAF基因编码区组蛋白修饰变化
曹锡梅1, 罗旭光2, 梁俊红3, 张潮3, 白丽娟3, 郭大玮3
1. 山西医科大学组织学与胚胎学教研室 太原 030001;
2. 山西医科大学微生物免疫学教研室 太原 030001;
3. 山西医科大学法医学院 太原 030001
Determining Histone Modifications on Encoded Region of Serum Amyloid A Activating Transcription Factor Gene in Inflammatory Stimulus Conditions by Chromatin Immunoprecipitation
CAO Xi-mei1, LUO Xu-guang2, LIANG Jun-hong3, ZHANG Chao3, BAI Li-juan3, GUO Da-wei3
1. Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, China;
2. Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan 030001, China;
3. Department of Forensic Science, Shanxi Medical University, Taiyuan 030001, China
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摘要:

目的:探讨血清淀粉样蛋白A转录激活因子(serum amyloid A-activating transcription factor,SAF)编码区外显子e4A和e4B区域的组蛋白表观遗传学信息,为进一步研究SAF自身的转录调控机制和该基因转录与前体mRNA剪接的关系奠定基础.方法:利用染色体免疫共沉淀实验(ChIP)分析SAF基因编码区组蛋白修饰的变化.首先收集LPS刺激不同时间的THP-1细胞,终浓度为1%甲醛交联蛋白质和DNA,利用超声波将其染色体随机断裂成适当大小的片段,选用ChIP级别兔多克隆抗组蛋白H3抗体、兔多克隆抗组蛋白H3K36三甲基化抗体、兔多克隆抗组蛋白H3K9单甲基化抗体和兔多克隆抗RNA聚合酶II CTD重复区5号丝氨酸磷酸化抗体实验,以富集得到的DNA样品为模板进行半定量PCR和定量qPCR分析.结果: LPS刺激作用下SAF基因编码区组蛋白H3富集信号减弱、外显子e4A区域单个核小体上H3K9单甲基化(H3K9me1)水平减弱、H3K36三甲基化(H3K36me3)修饰富集水平增加、RNA polII CTD重复序列第5号丝氨酸磷酸化水平降低.结论: LPS刺激导致SAF基因编码区组蛋白H3离散,该区域核小体解散、凝聚的染色体重塑呈开放构象;含SAF编码区基因信息的DNA暴露,利于RNA polII转录延伸.

关键词: 血清淀粉样蛋白A转录激活因子组蛋白修饰染色体免疫共沉淀实验THP-1细胞    
Abstract:

Objective:The aim is to probe the histone epigenetic information in encoded region of SAF gene after inflammatory cells model from human THP-1 monocytic leukemia cell line are constructed, which is expected to provide basis for the further research about the mechanism of transcriptional regulation of SAF gene. Methods:The LPS-stimulated or unstimulated THP-1 cells were colletcted and fixed by 1% formaldehyde for 10 minutes at room temperature. After that, nuclei were isolated. Chromatin was sheared by sonication, and samples were precleared for 1 hour at 4℃ with 50% protein A/G. Chromatin containing target proteins were precipitated overnight at 4℃ with 5μg antibodies or isotype-matched control IgG. Input and immunoprecipitated chromatin were incubated at 65℃ overnight to reverse crosslinks. After proteinase K digestion, DNA was extracted and purified by phenol-chloroform. The DNA was used as templates for PCR and qPCR to get the histone epigenetic signals. Antibodies included rabbit polyclonal to Histone H3, rabbit polyclonal to Histone H3 (tri methyl K36), rabbit polyclonal to Histone H3 (mono methyl K9) and rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS (phospho S5). Results:During inflammation, the total histone H3 signals in the 4A and 4B exon of SAF gene were significantly reduced. However, histone H3K36me3 didn't obviously diminish. The level of ser5 phosphorylation of the polymerase C-terminal domain (CTD) heptamer repeat was reduced in the 4A exon of SAF gene in inflammatory stimulus conditions. Expression of H3K9me1 decreased in the 4A exon of SAF gene. During inflammation, the level of H3K9me1 decreased and the enrichment of H3K36me3 increased in the single nucleosome in 4A exon of SAF gene. At the same time, the level of ser5 phosphorylation of the polymerase C-terminal domain (CTD) heptamer repeat decreased. Conclusion:The low level of histone H3 enrichment in SAF coding regions support the idea that histone H3 in SAF coding region could be evicted by LPS induction so as to pave the way for RNA polymerase II elongation. All of these effects might increase the elongation rate of the RNA polymerase II.

Key words: SAF Histone modifications    ChIP    THP-1
收稿日期: 2014-09-17 出版日期: 2015-03-25
ZTFLH:  Q-33  
基金资助:

国家自然科学基金(90606008),高等学校博士学科点专项科研基金(20111417110003),山西省自然科学基金(2006011110),山西省自然科学基金青年基金(2014021028-1)资助项目

通讯作者: 郭大玮     E-mail: guo8dawei@aliyun.com
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引用本文:

曹锡梅, 罗旭光, 梁俊红, 张潮, 白丽娟, 郭大玮. 利用ChIP技术研究SAF基因编码区组蛋白修饰变化[J]. 中国生物工程杂志, 2015, 35(3): 8-17.

CAO Xi-mei, LUO Xu-guang, LIANG Jun-hong, ZHANG Chao, BAI Li-juan, GUO Da-wei. Determining Histone Modifications on Encoded Region of Serum Amyloid A Activating Transcription Factor Gene in Inflammatory Stimulus Conditions by Chromatin Immunoprecipitation. China Biotechnology, 2015, 35(3): 8-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150302        https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I3/8


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