A low-complexity domain of Tet3 dioxygenase shields the oocyte methylome from oxidative erosion [ ACE-seq]

Dynamic DNA methylation and demethylation regulate gene expression during development. While DNA methyltransferases (DNMTs) establish and maintain cytosine methylation patterns, Ten-eleven translocation (Tet) dioxygenases catalyze sequential oxidation of 5-methylcytosine (5mC) to promote demethylation. Target-specific 5mC oxidation requires precise regulation of the Tet enzymatic activity. However, the mechanism underlying their activity control remains largely unexplored. Here we show a large low-complexity domain (LCD), present within the catalytic domain of Tet3, functions to repress the dioxygenase activity. Recombinant LCD-deleted Tet3 exhibits enhanced activity to convert 5mC into oxidized species. Deletion of the Tet3 LCD in mouse oocytes renders 5mC oxidation indiscriminately across the genome, leading to most prominently the derepression of ERVK retrotransposons and upregulation of adjacent genes. The extensive 5mC oxidation is associated with impairments in oocyte development. These findings suggest an intrinsic auto-regulatory mechanism of Tet3 dioxygenase operating to ensure a tight regulation of its enzymatic activity to achieve spatiotemporal specificity of methylome reprogramming during oocyte development. Overall design: 5hmC profiling by high throughput sequencing in WT and Tet3 LCD KO GV oocytes.

动态DNA甲基化与去甲基化过程在发育进程中调控基因表达。DNA甲基转移酶（DNA methyltransferases, DNMTs）可建立并维持胞嘧啶甲基化模式，而Tet双加氧酶（Ten-eleven translocation, Tet）则通过催化5-甲基胞嘧啶（5-methylcytosine, 5mC）的连续氧化反应促进去甲基化过程。靶标特异性的5mC氧化需要对Tet酶活性进行精准调控，但目前其活性调控的核心机制仍未得到充分阐释。本研究发现，Tet3催化结构域内存在的一段低复杂度结构域（low-complexity domain, LCD）能够抑制其双加氧酶活性。缺失LCD的重组Tet3蛋白，其将5mC转化为氧化产物的活性显著增强。在小鼠卵母细胞中敲除Tet3 LCD，会导致全基因组范围内5mC氧化失去特异性，最显著的结果是ERVK逆转录转座子的去抑制以及其邻近基因的表达上调。广泛的5mC氧化会对卵母细胞发育造成损伤。上述研究结果表明，Tet3双加氧酶存在内在的自我调控机制，以确保其酶活性受到严格调控，从而在卵母细胞发育过程中实现甲基化组重编程的时空特异性。实验设计：通过高通量测序对野生型（wild type, WT）和Tet3 LCD敲除（knockout, KO）生发泡（germinal vesicle, GV）期卵母细胞进行5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）谱分析。