利用ChIP技术研究SAF基因编码区组蛋白修饰变化

Determining Histone Modifications on Encoded Region of Serum Amyloid A Activating Transcription Factor Gene in Inflammatory Stimulus Conditions by Chromatin Immunoprecipitation

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(Ch IP)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Ch IP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q PCR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 pol II 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 pol II转录延伸。

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.