Epigenetic dynamics, such as DNA methylation and chromatin accessibility, have been extensively explored in human preimplantation embryos. However, the active demethylation process during this crucial period remains largely unexplored. In this study, we use single-cell chemical-labeling-enabled C-to-T conversion sequencing (CLEVER-seq) to quantify the DNA 5-formylcytosine (5fC) levels of human preimplantation embryos. We find that 5-formylcytosine phosphate guanine (5fCpG) exhibits genomic element-specific distribution features and is enriched in L1 and endogenous retrovirus-K (ERVK), the subfamilies of repeat elements long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs), respectively. Unlike in mice, paired pronuclei in the same zygote present variable difference of 5fCpG levels, although the male pronuclei experience stronger global demethylation. The nucleosome-occupied regions show a higher 5fCpG level compared with nucleosome-depleted ones, suggesting the role of 5fC in organizing nucleosome position. Collectively, our work offers a valuable resource for ten-eleven translocation protein family (TET)-dependent active demethylation-related study during human early embryonic development.

表观遗传动力学，例如 DNA 甲基化和染色质可及性，已在人类植入前胚胎中得到广泛探索。然而，这一关键时期的主动去甲基化过程在很大程度上仍未被探索。在这项研究中，我们使用单细胞化学标记 C-T 转换测序 (CLEVER-seq) 来量化人类植入前胚胎的 DNA 5-甲酰胞嘧啶 (5fC) 水平。我们发现5-甲酰基胞嘧啶磷酸鸟嘌呤（5fCpG）表现出基因组元件特异性分布特征，并且富含L1和内源性逆转录病毒-K（ERVK）、重复元件长散布核元件（LINE）和长末端重复序列（LTR）的亚家族）， 分别。与小鼠不同，尽管雄性原核经历了更强的整体去甲基化，但同一受精卵中的配对原核呈现出 5fCpG 水平的可变差异。与核小体耗尽的区域相比，核小体占据的区域显示出更高的 5fCpG 水平，表明 5fC 在组织核小体位置中的作用。总的来说，我们的工作为人类早期胚胎发育过程中依赖于 10-11 易位蛋白家族 (TET) 的主动去甲基化相关研究提供了宝贵的资源。