Centre for Regenerative Medicine, Institute for Regenaration and Repair, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh, Scotland, UK.
Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.
Nat Commun. 2023 Jan 30;14(1):488. doi: 10.1038/s41467-023-36097-9.
Induced pluripotent stem cell (iPSC) reprogramming is inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 24 reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhances murine iPSC generation in several reprogramming settings, emerging as the most robust roadblock. We show that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2, and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with co-activator domains converts ZFP266 from an inhibitor to a potent facilitator of iPSC reprogramming. We propose that the SINE-KRAB-ZFP interaction is a critical regulator of chromatin accessibility at regulatory elements required for efficient cellular identity changes. In addition, this work serves as a resource to further illuminate molecular mechanisms hindering reprogramming.
诱导多能干细胞（iPSC）重编程效率低下，理解导致这种低效的分子机制是成功控制细胞身份的关键。在这里，我们通过 CRISPR/Cas9 介导的全基因组敲除（KO）筛选报告了 24 个重编程障碍基因。在这些基因中，预测的 KRAB 锌指蛋白（KRAB-ZFP）Zfp266 的耗竭强烈且一致地增强了几种重编程环境中的鼠 iPSC 生成，成为最强大的障碍。我们表明 ZFP266 结合短散布核元件（SINEs），这些元件位于先驱因子 OCT4（POU5F1）、SOX2 和 KLF4 的结合位点附近，并阻碍染色质开放。用共激活结构域替换 KRAB 共抑制子将 ZFP266 从 iPSC 重编程的抑制剂转换为有效的促进剂。我们提出 SINE-KRAB-ZFP 相互作用是调控元件染色质可及性的关键调节剂，这些调控元件是有效细胞身份改变所必需的。此外，这项工作为进一步阐明阻碍重编程的分子机制提供了资源。
