Pronounced specificity of the TET enzyme catalytic domain guides cellular function. Pronounced specificity of the TET enzyme catalytic domain guides cellular function

Epigenetic memory in the form of cytosine methylation is essential for vertebrate development and the formation of cellular identity. Active removal of DNA methylation by the action of the TET hydroxylase family helps enable developmental potency, both in vivo and during creation of induced pluripotent stem cells. Despite this, little is known about how TET proteins are targeted to DNA. We report that mammalian TET enzymes show strong preference (>200 fold) for oxidising certain CG-containing hexamers in vitro, and during global methylation reprogramming in cultured cells and during embryogenesis. These preferred sequences, and also the most poorly targeted motifs, constitute recognition sites for developmental transcription factors whose binding activity is sensitive to DNA methylation. X-ray structural analysis and molecular dynamics simulations suggest that TET proteins use indirect readout to sense the sequence context flanking CG sites. These results are significant for understanding epigenetic reprogramming during development and the biological role TET enzymes play in modulating epigenetic memory. Overall design: An ESC line with TET1-3 genetically inactivated (TET-TKO) was rescued using an inducible PiggyBAC transposon based vector containing the catalytic domain from mouse TET3 induced by doxicycline and transcriptionally linked to an mCherry reporter gene. Cells without doxycicline treatment were used as control. Wild-type V6.5 hybrid embryonic stem cells (i.e. C57BL/6 X 129/sv cross) were treated with decitabine (5-aza-2’-deoxycytidine). Genome-wide bisulfite sequencing was used to uncover the rate at which DNA demethylation occurred in these samples.

以胞嘧啶甲基化形式存在的表观遗传记忆，对于脊椎动物发育以及细胞身份的建立至关重要。通过TET羟化酶家族的介导作用主动去除DNA甲基化，有助于激活发育潜能，这一过程既存在于体内，也发生在诱导多能干细胞的构建过程中。尽管如此，目前学界对TET蛋白如何靶向结合DNA的机制仍知之甚少。本研究发现，哺乳动物TET酶在体外环境、培养细胞的全基因组甲基化重编程过程以及胚胎发育阶段，对特定含CG六聚体的氧化偏好性极强，偏好程度超过200倍。这些偏好序列以及靶向性最低的基序，均为发育相关转录因子的识别位点，且这类转录因子的结合活性对DNA甲基化敏感。X射线晶体结构分析与分子动力学模拟结果显示，TET蛋白通过间接读取机制感知CG位点侧翼的序列环境。上述研究结果对于理解发育过程中的表观遗传重编程机制，以及TET酶在调控表观遗传记忆中发挥的生物学功能，具有重要的学术价值。整体实验设计：将经遗传灭活TET1-3基因的胚胎干细胞（Embryonic Stem Cell, ESC）系（TET-TKO），通过诱导型PiggyBAC转座子载体进行拯救，该载体包含由多西环素（doxycycline）诱导的小鼠TET3催化结构域，并与mCherry报告基因转录偶联。以未施加多西环素处理的细胞作为对照。对野生型V6.5杂交胚胎干细胞（C57BL/6与129/sv杂交株）施加地西他滨（5-aza-2’-脱氧胞苷）处理。采用全基因组亚硫酸氢盐测序技术，解析上述样本中DNA去甲基化的发生速率。