Tox4 Modulates Cell Fate Reprogramming. Tox4 Modulates Cell Fate Reprogramming

Reprogramming to induced pluripotency induces the switch of somatic cell identity to induced pluripotent stem cells (iPSCs). However, the mediators and mechanisms of reprogramming remain largely unclear. To elucidate the mediators and mechanisms of reprogramming, we used a siRNA mediated knockdown approach for selected candidate genes during the conversion of somatic cells into iPSCs. We identified Tox4 as a novel factor that modulates cell fate, using reprogramming efficiency towards iPSCs as an assay. We found that Tox4 is needed early in reprogramming to efficiently generate early reprogramming intermediates, irrespective of reprogramming conditions used. Tox4 enables proper exogenous reprogramming factor expression and the closing and opening of putative somatic and pluripotency enhancers early during reprogramming, respectively. We show that TOX4 protein assembles into a high molecular form. Moreover, Tox4 is also required for the efficient conversion of fibroblasts towards the neuronal fate, suggesting a broader role of Tox4 in modulating cell fate. Our study reveals Tox4 as a novel transcriptional modulator of cell fate that mediates reprogramming from the somatic state to the pluripotent and neuronal fate. Overall design: Bulk RNA sequencing of Tox4 knockdown and control condition early during reprogramming of mouse fibroblasts towards iPSCs

将体细胞重编程至诱导多能性状态，可使其身份转换为诱导多能干细胞（induced pluripotent stem cells, iPSCs）。然而，重编程过程的介导因子与具体调控机制仍不甚明确。为阐明重编程的介导因子与调控机制，本研究在体细胞向诱导多能干细胞转化的过程中，针对筛选得到的候选基因采用了小干扰RNA（small interfering RNA, siRNA）介导的基因敲低方法。本研究以诱导多能干细胞重编程效率作为检测指标，鉴定出Tox4是一种全新的细胞命运调控因子。研究发现，无论采用何种重编程培养条件，Tox4在重编程早期均为高效生成早期重编程中间细胞所必需。Tox4可分别保障外源性重编程因子的正常表达，以及重编程早期预测的体细胞与多能性增强子的关闭与开放过程。本研究证实，TOX4蛋白可组装为高分子量形式。此外，成纤维细胞向神经元命运高效转化的过程同样依赖Tox4，这提示Tox4在调控细胞命运方面具有更为广泛的作用。本研究揭示，Tox4是一种全新的细胞命运转录调控因子，可介导体细胞向多能性及神经元命运的重编程过程。实验整体设计：在小鼠成纤维细胞向诱导多能干细胞重编程的早期阶段，对Tox4敲低组与对照组样本进行批量RNA测序。