内源性表位标记的H1FX细胞系构建及其染色质分布图谱

doi:10.6040/j.issn.1671-7554.0.2022.1302

摘要/Abstract

摘要： 目的利用成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)基因组编辑技术对内源性H1FX进行表位标记来执行染色质免疫共沉淀测序(ChIP-seq),探究前列腺癌22Rv1细胞中H1FX在基因组上的结合位点及分布规律。方法以质粒pX330-U6-Chimeric_BB-CBh-hSpCas9为载体,设计特异性向导RNA(gRNA),构建pX330-H1FX重组质粒,引导Cas9核酸酶至接近H1FX终止密码子的位置处进行切割。以含有3×FLAG表位标签、自剪切多肽2A和遗传霉素耐药基因序列的质粒pFETCh_Donor为载体,序列两侧添加与双链断裂两侧区域同源的同源臂(HOMO),构建pFETCh_Donor-HOMO重组质粒。将pX330-H1FX重组质粒和pFETCh_Donor-HOMO重组质粒共转染到前列腺癌22Rv1细胞中,切割DNA并通过同源重组修复的方式整合表位标签。48 h后使用含遗传霉素的培养基筛选细胞。筛选出内源性表位标记的H1FX-FLAG细胞系后使用FLAG抗体进行ChIP。对ChIP富集的DNA和给料对照(Input)DNA进行建库,建库合格后送测序。后续对H1FX ChIP-seq数据与基因表达数据进行整合分析。结果 Western blotting结果显示,筛选到只表达H1FX-FLAG融合蛋白的细胞系;聚合酶链反应结果显示,FLAG表位标签整合至基因组正确的位置;测序结果显示,插入序列及接头处序列正确,内源性表位标记的H1FX-FLAG细胞系构建成功。H1FX ChIP-seq数据与基因表达数据整合分析发现,高表达基因的启动子区域更倾向于缺乏H1FX的结合。结论成功构建了内源性表位标记的H1FX-FLAG细胞系,初步分析了前列腺癌22Rv1细胞中H1FX在基因组上的结合位点及分布规律,为进一步研究H1FX在前列腺癌中的作用提供参考。

关键词: 成簇规律间隔短回文重复序列, H1FX, 连接组蛋白, 同源重组, 表观基因组学, 前列腺癌

Abstract: Objective To tag endogenous H1FX with epitope sequences for chromatin immunoprecipitation sequencing(ChIP-seq)using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)genome editing technology, and to investigate the genomic binding sites and distribution of H1FX in prostate cancer 22Rv1 cells. Methods Specific guide RNA(gRNA)was designed and inserted into the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 to construct the pX330-H1FX recombinant plasmid. Cas9 nuclease was guided to the position close to the H1FX stop codon for cleavage. With plasmid pFETCh_Donor containing the 3×FLAG epitope tag, self-cleaving peptide 2A and geneticin resistance gene sequence as the vector, the recombinant plasmid pFETCh_Donor-HOMO was constructed by adding homology arms(HOMO)that were homologous to the region on both sides of the double strand break. PX330-H1FX recombinant plasmid and pFETCh_Donor-HOMO recombinant plasmid were co-transfected into prostate cancer 22Rv1 cells. DNA was cleaved and epitope tags were integrated by homologous recombination repair. Cells were selected using medium containing geneticin after 48 hours. ChIP was performed on endogenous epitope-tagged H1FX-FLAG cell lines using FLAG antibody. ChIP-enriched DNA and input DNA libraries were constructed and sent for sequencing after the librarys were qualified. Subsequently, H1FX ChIP-seq data and gene expression data were integrated and analyzed. Results The results of Western blotting showed that the cell line only expressing H1FX-FLAG fusion protein was screened. The results of polymerase chain reaction showed that the FALG epitope tag was integrated into the correct position of the genome. Sequencing results showed that the insertion sequence and junction sequence were correct, and the H1FX-FLAG cell line tagged with endogenous epitopes was successfully constructed. The integration analysis of H1FX ChIP-seq data and gene expression data showed that the promoter regions of highly expressed genes were more likely to lack H1FX binding. Conclusion The endogenous epitope-tagged H1FX-FLAG cell lines were successfully constructed. The genomic binding sites and distribution of H1FX in 22Rv1 prostate cancer cells were preliminarily analyzed, which laid a foundation for further study on the role of H1FX in prostate cancer.

Key words: Clustered regularly interspaced short palindromic repeats, H1FX, Linker histone, Homologous recombination, Epigenomics, Prostate cancer

中图分类号:

R737.25

徐雅宁,张祥林,刘晓雨,郭海洋. 内源性表位标记的H1FX细胞系构建及其染色质分布图谱[J]. 山东大学学报 (医学版), 2023, 61(8): 1-9.

XU Yaning, ZHANG Xianglin, LIU Xiaoyu, GUO Haiyang. Construction of endogenous epitope-tagged H1FX cell lines and the chromatin distribution mapping[J]. Journal of Shandong University (Health Sciences), 2023, 61(8): 1-9.

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