A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits
A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits
复制标题
用于研究草莓果实中组蛋白修饰的天然染色质免疫沉淀 (ChIP) 方案
DOI：
10.1186/s13007-020-0556-z
复制
发表时间：
2020-02-03
期刊：
影响因子：
5.1
通讯作者：
Li, Yi
中科院分区：
文献类型：
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作者：
Huang, Xiaorong;Pan, Qinwei;Li, Yi
背景组蛋白及其变体的共价修饰对染色质结构有很大影响，染色质结构参与基因表达的转录调控。染色质免疫沉淀（ChIP）是研究体内DNA-组蛋白相互作用的有力工具。草莓是蔷薇科和非跃变型果实的模式植物，其中组蛋白修饰已被牵连到影响果实发育和成熟。然而，一个有效的ChIP方法还没有报道，在草莓，可能是由于其高水平的多糖，影响制备的染色质的质量和效率的immunoprecipitation.ResultsWe描述了一个本地染色质免疫沉淀（N-ChIP）协议适合草莓通过优化参数的核分离，染色质提取，使用定量实时PCR（qRT-PCR）进行DNA片段化和验证分析。qRT-PCR结果表明，活性标记H3 K36 me 3和沉默标记H3 K9 me 2均有效地免疫沉淀富集区域。与X-ChIP（交联染色质，然后免疫沉淀）相比，我们优化的N-ChIP程序具有更高的信噪比和更低的背景下的活性和沉默的组蛋白修饰。此外，高通量测序后N-ChIP表明，近90%的富集H3 K9/K14 ac峰之间的生物重复重叠，表明其显着的一致性和reproducibility.ConclusionsAn N-ChIP方法适用于肉质果实组织的林地strawberryFragaria vescais在这项研究中描述。我们优化的N-ChIP方案的效率和重现性通过qRT-PCR和高通量测序进行了验证。我们的结论是，N-ChIP是一个更合适的方法，草莓果实组织相对于X-ChIP，这可以结合高通量测序，以调查组蛋白修饰的影响，在草莓和其他水果的多糖含量高。
BackgroundCovalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation.ResultsWe describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated for the enriched regions. Compared to X-ChIP (cross-linked chromatin followed by immunoprecipitation), our optimized N-ChIP procedure has a higher signal-to-noise ratio and a lower background for both the active and the silent histone modifications. Furthermore, high-throughput sequencing following N-ChIP demonstrates that nearly 90% of the enriched H3K9/K14ac peaks are overlapped between biological replicates, indicating its remarkable consistency and reproducibility.ConclusionsAn N-ChIP method suitable for the fleshy fruit tissues of woodland strawberryFragaria vescais described in this study. The efficiency and reproducibility of our optimized N-ChIP protocol are validated by both qRT-PCR and high-throughput sequencing. We conclude that N-ChIP is a more suitable method for strawberry fruit tissues relative to X-ChIP, which could be combined with high-throughput sequencing to investigate the impact of histone modifications in strawberry and potentially in other fruits with high content of polysaccharides.