2-cell-like cells (2CLCs)—which comprise only ∼1% of murine embryonic stem cells (mESCs)—resemble blastomeres of 2-cell-stage embryos and are used to investigate zygotic genome activation (ZGA). Here, we discovered that TRIM66 and DAX1 function together as negative regulators of the 2C-like state in mESCs. Chimeric assays confirmed that mESCs lacking TRIM66 or DAX1 function have bidirectional embryonic and extraembryonic differentiation potential. TRIM66 functions by recruiting the co-repressor DAX1 to the Dux promoter, and TRIM66’s repressive effect on Dux is dependent on DAX1. A solved crystal structural shows that TRIM66’s PHD finger recognizes H3K4-K9me3, and mutational evidence confirmed that TRIM66’s PHD finger is essential for its repression of Dux. Thus, beyond expanding the scope of known 2CLC regulators, our study demonstrates that interventions disrupting TRIM66 or DAX1 function in mESCs yield 2CLCs with expanded bidirectional differentiation potential, opening doors for the practical application of these totipotent-like cells.

2 细胞样细胞 (2CLC) - 仅占鼠胚胎干细胞 (mESC) 的 1% - 类似于 2 细胞阶段胚胎的卵裂球，用于研究合子基因组激活 (ZGA)。在这里，我们发现 TRIM66 和 DAX1 一起作为 mESC 中 2C 样状态的负调节因子。嵌合试验证实缺乏 TRIM66 或 DAX1 功能的 mESCs 具有双向胚胎和胚胎外分化潜能。TRIM66 通过将共抑制因子 DAX1 招募到Dux启动子来发挥作用，TRIM66 对Dux的抑制作用依赖于 DAX1。已解决的晶体结构表明 TRIM66 的 PHD 指识别 H3K4-K9me3，突变证据证实 TRIM66 的 PHD 指对其抑制Dux至关重要. 因此，除了扩大已知 2CLC 调节剂的范围外，我们的研究表明，干扰 mESCs 中 TRIM66 或 DAX1 功能的干预会产生具有扩大的双向分化潜力的 2CLCs，为这些全能样细胞的实际应用打开了大门。