通过工程化改造Oct4研究体细胞重编程过程的基因组调控机制

项目名称： 通过工程化改造Oct4研究体细胞重编程过程的基因组调控机制

项目编号： No.31471238

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 遗传学与生物信息学、细胞生物学

项目作者： Ralf Jauch

作者单位： 中国科学院广州生物医药与健康研究院

项目金额： 85万元

中文摘要： Oct4（也称POU5F1）是POU转录因子家族中唯一已知能介导体细胞重编程建立多能性的成员。Oct4对于体细胞重编程是必须的，但其作用机制及在重编程不同时期的功能差异尚不清楚。本课题组利用DNA结合定量分析技术，检测到Oct因子识别DNA序列的不同结构域，并通过工程化互换Oct4和Oct6的这些结构域，发现改造型Oct因子与其野生型对重编程有着显著不同的作用：改造后，Oct6能诱导重编程而Oct4影响后期多能性的维持并趋向分化。本研究在前期工作基础上，通过建立二次诱导系统，对Oct因子的调控模式进行深入研究：利用二次诱导系统在重编程细胞中分别表达野生及改造型Oct4，结合染色质免疫共沉淀-高通量测序技术（ChIP-seq），获得Oct因子在重编程起始、成熟、稳定期的全基因组调控信息，从而阐明Oct4在重编程过程中决定性的作用机制，进一步研发出安全有效的工程化重编程因子。

中文关键词： Oct4；重编程；诱导多能干细胞

英文摘要： Oct4 (octamer binding transcription factor 4) belongs to the Pit-Oct-Unc (POU)transcription factor (TF) family. Only Oct4, but no other POU protein, can support the induction of pluripotency from somatic cells. If Oct4 is replaced by, for example, the PouIII factor Brn4, reprogrammed cells are committing to a neural cell fate. Efforts to study the dynamic changes during the reprogramming process suggested that Oct4 plays different, albeit essential, roles during the early and late stages of reprogramming. To reveal the basis for the unique roles of Oct4, we conducted quantitative DNA binding assays and detected structural elements that allow Oct4 to discriminate between composite DNA binding sites in vitro. We swapped those elements between Oct4 and Oct6 to engineer synthetic proteins with altered preferences for DNA sequences. As a consequence, these synthetic Oct factors exhibited profound differences in reprogramming assays as compared to their wild-type counterparts. For example, a doubly mutated Oct6 protein is now gains the ability pluripotency induction. Moreover, a mild point mutation of Oct4 termed Oct4M impairs Oct4's ability to support pluripotency maturation. Rather, Oct4M pauses at D40 of reprogramming and re-differentiates. Apparently, Oct4M can support initial phases of reprogramming but lost the function to support pluripotency maturation. Here we propose to expand these studies to resolve fundamental questions in the field of cellular reprogramming. What is the molecular function of Oct4 at various reprogramming stages? How do the subtly different DNA motif preferences between Oct4, Oct6 and synthetic factors change the inductions of cell fate decisions? Can we rationally engineer synthetic TF proteins that execute gene expression programs and direct cell fates in a controlled manner and thereby exclude harmful oncogenic properties of the resulting cells? To answer these questions, we will set up a secondary inducible system that allows comparing the reprogramming of mouse and human fibroblasts induced by Oct4, PouIII members and at least of four reengineered versions thereof. Next, we will perform chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) to carry out a time resolved analysis at 3 time points to compare of their genomic binding profiles. This work will clarify and separate the roles of Oct4 at different stages of the reprogramming process, explain its uniqueness compared to other POU factors and facilitate the rational design of synthetic proteins to produce safe tissues suitable for cell therapies.

英文关键词： Oct4;reprogramming;iPSC