Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.
Genomics Research Center, Academia Sinica, Taipei, Taiwan.
FASEB J. 2019 Sep;33(9):10577-10592. doi: 10.1096/fj.201800220RR. Epub 2019 Jun 26.
We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.
我们通过高通量筛选揭示，激活转录因子 1（ATF1）是人类胚胎干细胞（hESC）中的一种新型多能调控因子。ATF1 表达的敲低显著上调神经外胚层（NE）基因，但不上调中胚层、内胚层和滋养外胚层基因。值得注意的是，短发夹 RNA（shRNA）、小干扰 RNA（siRNA）或成簇规律间隔短回文重复（CRISPR）/CRISPR 相关蛋白 9（Cas9）下调或敲除 ATF1 足以在未分化或分化条件下上调性别决定区 Y 框（SOX）2 和配对盒 6（PAX6）的表达，而 ATF1 的过表达则抑制 NE 分化。神经诱导后第 1-3 天，内源性 ATF1 自发下调。通过双敲低实验，发现 SOX2 的上调对于靶向 hESC 的 shRNA 中 PAX6 和 SOX1 表达的增加至关重要。通过荧光素酶报告基因检测，我们确定 ATF1 是基因表达的负转录调节因子。发现了 ATF1 的一个新功能，这些发现有助于更广泛地理解 hESC 中调节 NE 分化的最初步骤。
