Medical Genomics, UCL Cancer Institute, University College London, London, UK.
Gigascience. 2012 Jul 12;1(1):3. doi: 10.1186/2047-217X-1-3.
Methylated DNA immunoprecipitation (MeDIP) is a popular enrichment based method and can be combined with sequencing (termed MeDIP-seq) to interrogate the methylation status of cytosines across entire genomes. However, quality control and analysis of MeDIP-seq data have remained to be a challenge.
We report genome-wide DNA methylation profiles of wild type (wt) and mutant mouse cells, comprising 3 biological replicates of Thymine DNA glycosylase (Tdg) knockout (KO) embryonic stem cells (ESCs), in vitro differentiated neural precursor cells (NPCs) and embryonic fibroblasts (MEFs). The resulting 18 methylomes were analysed with MeDUSA (Methylated DNA Utility for Sequence Analysis), a novel MeDIP-seq computational analysis pipeline for the identification of differentially methylated regions (DMRs). The observed increase of hypermethylation in MEF promoter-associated CpG islands supports a previously proposed role for Tdg in the protection of regulatory regions from epigenetic silencing. Further analysis of genes and regions associated with the DMRs by gene ontology, pathway, and ChIP analyses revealed further insights into Tdg function, including an association of TDG with low-methylated distal regulatory regions.
We demonstrate that MeDUSA is able to detect both large-scale changes between cells from different stages of differentiation and also small but significant changes between the methylomes of cells that only differ in the KO of a single gene. These changes were validated utilising publicly available datasets and confirm TDG's function in the protection of regulatory regions from epigenetic silencing.
甲基化 DNA 免疫沉淀（MeDIP）是一种常用的富集方法，可与测序（称为 MeDIP-seq）相结合，以检测整个基因组中胞嘧啶的甲基化状态。然而，MeDIP-seq 数据的质量控制和分析仍然是一个挑战。
我们报告了野生型（wt）和突变型小鼠细胞的全基因组 DNA 甲基化图谱，包括胸腺嘧啶 DNA 糖基化酶（Tdg）敲除（KO）胚胎干细胞（ESCs）、体外分化的神经前体细胞（NPCs）和胚胎成纤维细胞（MEFs）的 3 个生物学重复。使用 MeDUSA（用于序列分析的甲基化 DNA 实用程序）对 18 个甲基组进行分析，MeDUSA 是一种新的 MeDIP-seq 计算分析管道，用于鉴定差异甲基化区域（DMRs）。在 MEF 启动子相关 CpG 岛中观察到的超甲基化增加支持了 Tdg 在保护调节区域免受表观遗传沉默中的作用的先前假设。通过基因本体、途径和 ChIP 分析对与 DMRs 相关的基因和区域的进一步分析揭示了 Tdg 功能的进一步见解，包括 TDG 与低甲基化的远端调节区域的关联。
我们证明 MeDUSA 能够检测出来自不同分化阶段的细胞之间的大规模变化，也能够检测出仅在单个基因 KO 上存在差异的细胞之间的小但显著的变化。这些变化利用公开可用的数据集进行了验证，并证实了 TDG 在保护调节区域免受表观遗传沉默中的作用。
