环境与遗传因素通过以DNA甲基化为代表的表观遗传修饰，动态调控基因表达，从而影响细胞命运。DNA修饰测序技术的发展，为新颖DNA修饰的功能研究奠定了基础。然而，现有测序技术存在明显不足，严重影响了新颖DNA修饰的检测与功能研究。本项目将开发DNA修饰（5-羟甲基胞嘧啶、5-醛基胞嘧啶、5-羧基胞嘧啶及6-甲基腺嘌呤）的新型检测技术，系统研究各种DNA修饰在体细胞重编程过程中的全谱系变化（5-甲基胞嘧啶、5-强甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶），探究小分子及环境因素对DNA修饰谱系的影响。本项目开发的DNA修饰测序与检测技术、产生的DNA修饰谱系动态关联数据，也有望应用于细胞编程与重编程领域内其他以DNA甲基化为代表的表观遗传修饰研究。

Environmental and genetic factors dynamically regulate gene expression through DNA modifications exemplified by 5-methylcytosine, thereby determining cell fate. A key bottleneck to the study of these DNA modifications is the development of robust sequencing technologies towards such epigenetic modifications. Yet, unsovled limitations of the existing technologies have significantly impeded our understanding to the detecion and functional investigations of these novel modifications. Here, we propose the development of several novel sequencing technologies for the systematic detection of 5-hydroxylmethylcytosine, 5-formylcytosine, 5-carboxylcytosine, and 6-methyladenosine. Such methods will also be applied to reveal the comprehensive spectrum of these DNA modifications (5-methylcytosine, 5-hydroxylmethylcytosine, 5-formylcytosine and 5-carboxylcytosine) during somatic cell reprogramming; and the relationship between environmental factors (such as small molecules) and the DNA modifications will be profiled as well. Finally, both the sequencing and detection technologies and the dynamic modification spetrum generated by this study can be shared and applied by researchers to study other questions in cell prgramming and reprogramming.

DNA 胞嘧啶甲基化与去甲基化的动态平衡与生物的生长发育和疾病密切相关。5-羟甲基胞嘧啶（5- hydroxymethylcytosine, 5hmC）与5-醛基胞嘧啶 (5-formylcytosine, 5fC) 既是DNA主动去甲基化过程的中间产物，也是调控生命过程的重要表观遗传修饰。因此，发展高分辨率、高灵敏性的5hmC和5fC检测技术，就显得尤为重要。在本项目的资助下，我们首先开发了适用于单细胞样品的5fC全基因组单碱基分辨率的测序技术CLEVER-seq（chemical labeling enabled C-to-T conversion sequencing），绘制了小鼠受精及胚胎早期发育过程中5fC的动态谱图。我们发现，在早期发育的过程中，启动子5fC的产生早于基因表达的上调，首次提供了5fC与基因表达的时空关系。此外，我们还开发了不依赖于亚硫酸氢钠的5hmC测序技术hmC-oxCET （oxidative Cyclization-Enabled C-to-T Transition of 5hmC），实现了100-1000个细胞5hmC的全基因组单碱基分辨率测序技术。这些适合珍贵生物学样品及单细胞样品的新型表观基因组测序技术，为DNA甲基化动态调控和生物学功能研究提供了有力的工具。