DNA methylation enables recurrent endogenisation of giant viruses in an animal relative. DNA methylation enables recurrent endogenisation of giant viruses in an animal relative

5-methylcytosine (5mC) is a widespread silencing mechanism that controls genomic parasites. However, in many eukaryotes 5mC has gained complex roles in gene regulation beyond parasite control. Animals are a paradigmatic case for 5mC evolution, as they show widespread variability across lineages, ranging from gene regulation and transposable element control to loss of this base modification. Here we show that the protist animal relative Amoebidium appalachense displays both transposon and gene body methylation, a pattern reminiscent of invertebrates and plants. Unexpectedly, large hypermethylated regions of the Amoebidium genome derive from viral insertions, including hundreds of endogenised giant viruses contributing 14% of the encoded genes. Using a combination of inhibitors and functional genomic assays, we demonstrate that 5mC silences these giant virus insertions. Moreover, alternative Amoebidium isolates show polymorphic giant virus insertions, highlighting a dynamic process of infection, endogenisation and purging. Therefore we propose that 5mC is critical for the controlled co-existence of newly acquired viral DNA into eukaryotic genomes, making Amoebidium a unique model to understand the hybrid origins of eukaryotic genomes. Overall design: Profiling of cytosine methylation for Amoebidium appalachense and other ichthyosporeans using Enzymatic Methyl-seq, as well as 5-Azacytidine treatment vs control RNA-seq and Enzymatic Methyl-seq.

5-甲基胞嘧啶（5-methylcytosine，5mC）是一种广泛存在的表观遗传沉默机制，可调控基因组内的寄生遗传元件。然而，在众多真核生物中，5mC已演化出超越寄生序列调控的复杂基因调控功能。动物是5mC演化研究的范式案例：其在不同演化支系中呈现广泛差异，功能涵盖基因调控、转座因子（transposable element）抑制，乃至完全丧失该碱基修饰。本研究显示，作为动物近亲的原生生物阿巴拉契亚变形虫（Amoebidium appalachense）同时存在转座子甲基化与基因体甲基化（gene body methylation）模式，该特征与无脊椎动物和植物相似。出乎意料的是，该变形虫基因组中的大片高甲基化区域源自病毒插入序列，其中包含数百株内源性巨型病毒，其所编码的基因占总编码基因的14%。我们通过抑制剂处理结合功能基因组实验，证实5mC可沉默这些巨型病毒插入序列。此外，不同的阿巴拉契亚变形虫分离株呈现出巨型病毒插入序列的多态性，这凸显了病毒感染、内源性整合与宿主清除的动态过程。因此我们提出，5mC对于真核基因组中新获得的病毒DNA的可控共存至关重要，这使得阿巴拉契亚变形虫成为研究真核基因组混合起源的独特模型。实验整体设计：采用酶法甲基化测序（Enzymatic Methyl-seq）对阿巴拉契亚变形虫及其他鱼孢虫类（ichthyosporeans）进行胞嘧啶甲基化谱分析，同时设置5-氮胞苷（5-Azacytidine）处理组与对照组，开展RNA测序及酶法甲基化测序。