Klimczak Marta, Czerwińska Patrycja, Mazurek Sylwia, Sozańska Barbara, Biecek Przemysław, Mackiewicz Andrzej, Wiznerowicz Maciej
Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan, Poland; Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland; The International Institute of Molecular and Cell Biology, Warsaw, Poland.
Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan, Poland; Department of Cancer Immunology, Poznan University of Medical Sciences, Poznan, Poland.
Stem Cell Res. 2017 Aug;23:163-172. doi: 10.1016/j.scr.2017.07.012. Epub 2017 Jul 15.
Cellular reprogramming proceeds in a stepwise pathway initiated by binding and transcription of pluripotency factors followed by genome-wide epigenetic changes. Priming events, such as erasure of DNA methylation and chromatin remodeling determines the success of pluripotency acquisition later. Therefore, growing efforts are made to understand epigenetic regulatory network that makes reprogramming possible and efficient. Here, we analyze the role of transcriptional corepressor TRIM28, involved in heterochromatin formation, during the process of reprogramming of mouse somatic cells into induced pluripotent stem cells (iPS cells). We demonstrate that Trim28 knockdown (Trim28 KD) causes that emerging iPS cells differentiate immediately back into MEFs therefore they fail to yield stable iPS cell colonies. To better comprehend the mechanism of TRIM28 action in reprogramming, we performed a reverse-phase protein array (RPPA) using in excess of 300 different antibodies and compared the proteomic profiles of wild-type and Trim28 KD cells during reprogramming. We revealed the differences in the dynamics of reprogramming of wild-type and Trim28 KD cells. Interestingly, proteomic profile of Trim28 KD cells at the final stage of reprogramming resembled differentiated state rather than maintenance of pluripotency and self-renewal, strongly suggesting spontaneous differentiation of Trim28 KD cells back to their parental cell type. We also observed that action of TRIM28 in reprogramming is accompanied by differential enrichment of proteins involved in cell cycle, adhesion and stemness. Collectively, these results suggest that regulation of epigenetic modifications coordinated by TRIM28 plays a crucial role in reprogramming process.
细胞重编程通过一个逐步的途径进行，该途径由多能性因子的结合和转录启动，随后是全基因组的表观遗传变化。引发事件，如DNA甲基化的消除和染色质重塑，决定了随后多能性获得的成功。因此，人们越来越努力去理解使重编程成为可能且高效的表观遗传调控网络。在这里，我们分析了参与异染色质形成的转录共抑制因子TRIM28在小鼠体细胞重编程为诱导多能干细胞（iPS细胞）过程中的作用。我们证明，Trim28基因敲低（Trim28 KD）导致新出现的iPS细胞立即分化回成纤维细胞，因此它们无法产生稳定的iPS细胞集落。为了更好地理解TRIM28在重编程中的作用机制，我们使用超过300种不同的抗体进行了反向蛋白质阵列（RPPA），并比较了重编程过程中野生型和Trim28 KD细胞的蛋白质组学图谱。我们揭示了野生型和Trim28 KD细胞重编程动力学的差异。有趣的是，Trim28 KD细胞在重编程最后阶段的蛋白质组学图谱类似于分化状态，而不是多能性和自我更新的维持，这强烈表明Trim28 KD细胞自发分化回其亲代细胞类型。我们还观察到，TRIM28在重编程中的作用伴随着参与细胞周期、黏附和干性的蛋白质的差异富集。总的来说，这些结果表明，由TRIM28协调的表观遗传修饰调控在重编程过程中起着关键作用。
