Effect of Foxd1 knockdown in the reprogramming process

It remains unclear how the ectopic expression of defined transcription factors induces dynamic changes in gene expression profiles that establish a pluripotent state during direct cell reprogramming. In the present study, we first identified a temporal gene expression program during the reprogramming process. Promoter analyses then predicted the role of two forkhead box transcription factors, Foxd1 and Foxo1, as mediators of the gene expression program. Knockdown of Foxd1 or Foxo1 reduced the number of induced pluripotent stem cells (iPSCs). The knockout of Foxd1 prevented the downstream transcription program, including the expression of reprogramming marker genes. Interestingly, the expression level of Foxd1 was also transiently increased in a small population of cells in the middle stage of reprogramming. The presence or absence of Foxd1 expression in this stage was correlated with a future cell fate as iPSCs or non-reprogrammed cells. These results suggest that Foxd1 is a mediator and indicator of the successful progression of the gene expression program in cell reprogramming. Mouse embryonic fibroblasts (MEFs) of Foxd1+/+, Foxd1+/- or Foxd1-/- were infected with retroviruses encoding the three transcription factors (Oct4, Sox2 and Klf4) at day 0 and sampled at day 8.

目前尚不清楚，特定转录因子的异位表达如何诱导基因表达谱发生动态变化，进而在直接细胞重编程过程中建立多能性状态。本研究首先鉴定了细胞重编程过程中的时序基因表达程序。随后通过启动子分析，预测了两个叉头框转录因子Foxd1与Foxo1可作为该基因表达程序的介导因子。对Foxd1或Foxo1进行敲低，会减少诱导多能干细胞（induced pluripotent stem cells, iPSCs）的生成数量；敲除Foxd1则会阻断下游转录程序，包括重编程标记基因的表达。有趣的是，在重编程中期的少量细胞群体中，Foxd1的表达水平也会短暂升高。此阶段Foxd1的表达与否，与细胞未来的命运——形成iPSCs或未发生重编程细胞——密切相关。上述结果表明，Foxd1是细胞重编程中基因表达程序顺利推进的介导因子与指示标志物。于第0天，用编码Oct4、Sox2与Klf4三种转录因子的逆转录病毒感染Foxd1+/+、Foxd1+/-或Foxd1-/-基因型的小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs），并于第8天收集样本。