利用生物資訊分析小鼠胚胎發育DNA甲基化之生物標誌

表觀遺傳是研究DNA甲基化及組蛋白修飾，其為將DNA加甲基基團或組蛋白經化學基團之修飾而調節其基因抑制或活化。表觀遺傳也受到藥物、環境或飲食等因素所調控而影響基因表現。於Alexander Meissner及其同事的研究中，使用亞硫酸氫鹽測序描繪DNA甲基化圖譜，而其研究DNA甲基化變化及組蛋白與DNA甲基化關係，進而了解細胞分化過程與機制。然而，由於當時分析工具之限制，研究多能和分化細胞的表觀遺傳分析未包含相關發育基因，甚至與疾病及癌症之探討。為了尋找新的表觀遺傳標誌或其他重要標誌，本研究是採用“Illumina- Genome-Analyzer-Reduced-Representation-Bisulfite-Sequencing（RRBS）”之數據，而重新分析所有DNA甲基化之情形，其藉以找出不同的胚胎階段及分化過程可能存在的關鍵基因。首先，為了鑑定基因之位點，利用MegaBLAST將其Meissner et al, 之數據與小鼠基因組織核酸序列資料庫（其從美國國家生物技術資料中心獲取）互相比對出，而找出個序列片段之位點。第二，為了得知基因名稱，將其先前之比對數據與小鼠通用格式資料庫（gff）進行BEDtools之交集比對而產生基因名稱。再者，利用BEDtools工具進行差集之比較，挑選出細分化神經細胞或組織與胚胎幹細胞有差異性地DNA甲基化基因。最後，本研究用Metascape全面性地觀察胚胎幹細胞、分化細胞及初級組織之間所涉及生物途徑及功能途徑，進而得到15個候選基因。其中，以Sema5a和Ntrk3作為代表生物標誌物，其途徑與NANOG相關且觀察到其DNA甲基化之狀態與基因內甲基化有顯著地關係。未來，期許其生物標誌物與基因/細胞療法的臨床應用聯繫起來，並深入研究相關的蛋白質與DNA相互作用和其基因內甲基化之變化影響性。

關鍵字

甲基化； MegaBLAST ； BEDtools ； Metascape ；基因內甲基化；生物標誌

並列摘要

Epigenetics is the study of all mechanisms that DNA methylation (DNA interacts with methyl groups) and histones modification (nucleosome were modified by chemical tags in chromatin DNA) regulate what genes are activated or deactivated, whereas other factors, included drugs, environments, or diets might participate in epigenetic pattern. In 2008, Alexander Meissner and his colleagues study demonstrated that the change of DNA methylation were observed by using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing and also histone modification were related with DNA methylation patterns. However, due to the limitation of analysed tools, the whole-genome-wide epigenetic profiling of pluripotent and differentiated cells in previous studies did not comprehensively indicated in normal development, even in many pathologies or cancers. Therefore, we have established a systematic approach with three open source tools to carry numerous data from Meissner et al. and identify potential candidate methylated genes. First, to find the sequence location, we utilized MegaBLAST with faster speed and high accuracy to align data between data from Meissner et al. and referred data set. Secondly, to obtain gene name and to identify differences between each cell/tissue, BEDtools is a flexible tool for simple operations to optimize and visualize data. Finally, to interpret the interaction between known molecular pathways and potential candidate biomarkers, our genes in each sample proceeded meta-analysis with Metascape. It is significant that we provide a systematic computational approach not only to reduce the data from 10 million to thousands but also to serve as a standard protocol for comparison of genomic DNA methylation in different samples. In addition conducted 15 genes as candidate biomarkers as representative biomarkers were, Sema5a and Ntrk3 are associated with significant intragenic DNA methylation. In future, the further studies would like to link with clinical application, either for gene or cell therapy, to in-depth understand the complex protein-DNA-methylated interactions, and to widely investigate the functions and patterns of intragenic DNA methylation in genes.

並列關鍵字

methylation ； MegaBLAST ； BEDtools ； Metascape ； intrangenic methylation ； biomarker

參考文獻

1. Waddington, C.H., The epigenotype. Endeavour, 1942. 1: p. 18-20.

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2. Jones, P.A. and R. Martienssen, A blueprint for a Human Epigenome Project: the AACR Human Epigenome Workshop. Cancer Res, 2005. 65(24): p. 11241-6.