Klagkou Eleftheria, Valakos Dimitrios, Foutadakis Spyros, Polyzos Alexander, Papadopoulou Angeliki, Vatsellas Giannis, Thanos Dimitris
Biomedical Research Foundation, Academy of Athens (BRFAA), 4 Soranou Efesiou St., 11527 Athens, Greece.
Section of Biochemistry and Molecular Biology, Department of Biology, School of Science, National and Kapodistrian University of Athens (NKUA), Panepistimiopolis, Zografou, 15772 Athens, Greece.
Int J Mol Sci. 2024 Dec 6;25(23):13128. doi: 10.3390/ijms252313128.
Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the gene fused to and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.
将体细胞重编程为诱导多能性是一个由转录因子Oct4、Sox2、Klf4和c-Myc（OSKM）介导的多步骤随机过程，这些转录因子协调全局表观遗传和转录变化。我们对整合的ChIP-seq、ATAC-seq和RNA-seq数据进行了大规模分析，揭示了重编程过程中OSKM DNA结合的时空高度动态模式。我们发现OSKM与不同类别的多能性相关增强子的结合呈现出不同的时间模式。参与重编程的基因受多个增强子协同活性的调控，OSKM会依次结合这些增强子以进行严格的转录控制。基于这些发现，我们开发了一种无偏方法来鉴定重编程诱导增强子（RIE），构建增强子陷阱并实时分离正在进行重编程的细胞。我们使用了一个来自与 融合基因的代表性RIE，并在重编程过程的不同时间点分离细胞，发现它们具有独特的发育能力，因为它们由于独特的分子特征而高效重编程。总之，我们的实验开发了一种无偏方法，通过利用OSKM的功能动态实时鉴定和分离可重编程细胞，这些细胞可作为有效的重编程生物标志物。
