Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells
Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells
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DOI：
10.1634/stemcells.2006-0430
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发表时间：
2007-01-01
期刊：
影响因子：
5.2
通讯作者：
Collas, Philippe
中科院分区：
文献类型：
--
作者：
Dahl, John Arne;Collas, Philippe
染色质免疫沉淀（ChIP）是研究蛋白质-DNA相互作用和定位DNA上表观遗传组蛋白修饰的关键技术。目前的ChIP方案需要大量的样品处理和大量的细胞。我们开发了一种快速和定量的（Q（2））ChIP检测适合组蛋白和转录因子免疫沉淀的染色质量相当于少至100个细胞。在丁酸盐存在下悬浮液中的DNA-蛋白质交联，通过洗涤后的管移动消除背景染色质，以及交联逆转、蛋白质消化、增加的抗体-珠粒与染色质比率和DNA洗脱的组合到单个步骤中显著提高了ChIP效率并缩短了程序。我们使用Q（2）ChIP监测视黄酸介导的人胚胎癌细胞分化过程中，发育调控基因OCT 4、NANOG、LMNA和PAX 6的5'调控区组蛋白H3修饰的变化。沉淀DNA的定量聚合酶链反应分析揭示了OCT 4和NANOG上的双相异染色质组装，涉及H3赖氨酸（K）9和K27甲基化，随后是H3 K9脱乙酰化和额外的H3 K27三甲基化。H3 K4的二甲基化和三甲基化保持相对不变。相比之下，PAX 6显示了在未分化细胞中具有激活潜力的受抑制基因的组蛋白修饰特征。PAX 6在转录激活后经历H3 K9乙酰化和增强的H3 K4三甲基化。Q（2）转录因子Oct 4的ChIP证明其在分化时从NANOG启动子解离。据我们所知，这项研究是第一个揭示人类OCT 4和NANOG调控序列的组蛋白修饰变化。结果表明，有序的染色质重排的发育调控启动子分化后。
Chromatin immunoprecipitation (ChIP) is a key technique for studying protein-DNA interactions and mapping epigenetic histone modifications on DNA. Current ChIP protocols require extensive sample handling and large cell numbers. We developed a quick and quantitative (Q(2)) ChIP assay suitable for histone and transcription factor immunoprecipitation from chromatin amounts equivalent to as few as 100 cells. DNA-protein cross-linking in suspension in presence of butyrate, elimination of background chromatin through a tube shift after washes, and a combination of cross-link reversal, protein digestion, increased antibody-bead to chromatin ratio, and DNA elution into a single step considerably improve ChIP efficiency and shorten the procedure. We used Q(2)ChIP to monitor changes in histone H3 modifications on the 5' regulatory regions of the developmentally regulated genes OCT4, NANOG, LMNA, and PAX6 in the context of retinoic-acid-mediated human embryonal carcinoma cell differentiation. Quantitative polymerase chain reaction analysis of precipitated DNA unravels biphasic heterochromatin assembly on OCT4 and NANOG, involving H3 lysine (K)9 and K27 methylation followed by H3K9 deacetylation and additional H3K27 trimethylation. Di- and trimethylation of H3K4 remain relatively unaltered. In contrast, PAX6 displays histone modifications characteristic of repressed genes with potential for activation in undifferentiated cells. PAX6 undergoes H3K9 acetylation and enhanced H3K4 trimethylation upon transcriptional activation. Q(2)ChIP of the transcription factor Oct4 demonstrates its dissociation from the NANOG promoter upon differentiation. This study is, to our knowledge, the first to reveal histone modification changes on human OCT4 and NANOG regulatory sequences. The results demonstrate ordered chromatin rearrangement on developmentally regulated promoters upon differentiation.