Department of Biomolecular Sciences, Molecular Pathology Laboratory "PaoLa", University of Urbino "Carlo Bo", Via Arco d'Augusto 2, 61032, Fano, PU, Italy.
Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy.
Clin Epigenetics. 2018 Nov 16;10(1):143. doi: 10.1186/s13148-018-0576-y.
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.
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 (24 h, 48 h, and 72 h) 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.
EPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72 h), 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.
病理组织染色质免疫沉淀（PAT-ChIP）技术的出现，使染色质免疫沉淀（ChIP）能够从福尔马林固定石蜡包埋（FFPE）组织中进行，从而将染色质研究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 在来自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存档样本中提取染色质的效率降低是需要克服的主要障碍，尤其是在研究低丰度的表观遗传标记（如 H3K4me3）时。
我们评估了 PAT-ChIP 原始方案的不同修改，以改善从 FFPE 组织中分离染色质。为此，我们首先广泛使用正常的人结肠标本在受控条件下固定（24 小时、48 小时和 72 小时），以模拟存档样本中最常见的组织固定变异性。应用不同的超声处理方案或热介导的交联有限逆转（LRC）来测试不同的染色质提取条件。我们发现，如果与经典 PAT-ChIP 方案相比，LRC 可显著提高染色质提取效率，特别是在使用 72 小时固定的 FFPE 样本时。然后，我们使用浸润性乳腺癌的存档样本在全基因组水平上应用新的程序（增强型 PAT-ChIP，EPAT-ChIP）来研究 H3K4me3，一种低丰度的组蛋白修饰，以及 H3K27me3 和 H3K27ac，另外两种已知的组蛋白标记。
EPAT-ChIP 程序提高了从 FFPE 样本中分离染色质的效率，允许研究长时间固定的标本（72 小时），以及研究低分布的表观遗传标记（如 H3K4me3）和从低起始量的起始材料中分析多种组蛋白标记。我们相信，EPAT-ChIP 将促进染色质研究在存档的病理样本中的应用，从而有助于扩展对癌症表观基因组的现有认识，并能够确定临床上有用的肿瘤生物标志物。
