Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology
Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology
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DOI：
10.1186/s13148-018-0576-y
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发表时间：
2018-11-16
影响因子：
5.7
通讯作者：
Fanelli, Mirco
中科院分区：
文献类型：
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作者：
Amatori, Stefano;Persico, Giuseppe;Fanelli, Mirco
背景：病理组织染色质免疫沉淀 (PAT-ChIP) 是一种允许对福尔马林固定石蜡包埋 (FFPE) 组织进行染色质免疫沉淀 (ChIP) 的技术，其引入将染色质研究的应用扩展到临床患者样本。然而，在临床标本的常规组织固定过程中引入的广泛交联可能会阻碍 PAT-ChIP 在存档组织样本的全基因组研究 (PAT-ChIP-Seq) 中的应用。从过度固定的福尔马林档案样本中提取染色质的效率降低是需要克服的主要障碍，特别是在研究低丰度表观遗传标记（例如 H3K4me3）时。 结果：我们评估了原始 PAT-ChIP 方案的不同修改，以改善从 FFPE 组织中分离染色质。为了实现这一目标，我们首先广泛使用在受控条件（24小时、48小时和72小时）下固定的正常人类结肠标本来模拟存档样本中最常见的组织固定的可变性。应用不同的超声处理方案或热介导的有限交联逆转 (LRC) 测试了不同的染色质提取条件。我们发现，与规范的 PAT-ChIP 方案相比，LRC 大大提高了染色质提取效率，特别是在使用 72 小时固定 FFPE 样品时。我们将这种新程序命名为增强型 PAT-ChIP (EPAT-ChIP)，然后在全基因组水平上应用浸润性乳腺癌档案样本来研究 H3K4me3（一种低丰度组蛋白修饰）以及 H3K27me3 和 H3K27ac（另外两种众所周知的组蛋白标记）。 结论：EPAT-ChIP 程序提高了从 FFPE 样本中分离染色质的效率，从而可以研究长时间固定的样本(72h)，以及低分布表观遗传标记（例如 H3K4me3）的研究以及来自少量起始材料的多个组蛋白标记的分析。我们相信 EPAT-ChIP 将促进染色质研究在存档病理样本中的应用，从而有助于扩展目前对癌症表观基因组的理解，并能够识别临床上有用的肿瘤生物标志物。
Background: The introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. However, extensive crosslinking introduced during routine tissue fixation of clinical specimens may hamper the application of PAT-ChIP to genome-wide studies (PAT-ChIP-Seq) from archived tissue samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated.Results: We evaluated different modifications of the original PAT-ChIP protocol to improve chromatin isolation from FFPE tissues. With this aim, we first made extensive usage of a normal human colon specimen fixed at controlled conditions (24h, 48h, and 72h) to mimic the variability of tissue fixation that is most frequently found in archived samples. Different conditions of chromatin extraction were tested applying either diverse sonication protocols or heat-mediated limited reversal of crosslinking (LRC). We found that, if compared with canonical PAT-ChIP protocol, LRC strongly increases chromatin extraction efficiency, especially when 72-h fixed FFPE samples are used. The new procedure, that we named enhanced PAT-ChIP (EPAT-ChIP), was then applied at genome-wide level using an archival sample of invasive breast carcinoma to investigate H3K4me3, a lowly abundant histone modification, and H3K27me3 and H3K27ac, two additional well-known histone marks.Conclusions: EPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72h), as well as the investigation of low distributed epigenetic marks (e.g., H3K4me3) and the analysis of multiple histone marks from low amounts of starting material. We believe that EPAT-ChIP will facilitate the application of chromatin studies to archived pathology samples, thus contributing to extend the current understanding of cancer epigenomes and enabling the identification of clinically useful tumor biomarkers.