Role of long noncoding RNAs in human endodermal differentiation

描述(申请人提供)：数以千计的长非编码(LNC)RNA是随着人类胚胎干细胞(HESCs)的分化而诱导的，可能控制人类正常发育的许多阶段，但绝大多数这些非编码基因的功能仍不清楚。这项工作的长期目标是确定lncRNAs在内胚层分化中的作用，并利用这一见解开发新的方法来指导更有效地生产来自内胚层的人类组织。这项建议的总体目标是确定在hESCs分化为内胚层过程中诱导的两个lncRNA的功能，并在此过程中开发基因工具来更有效地评估任何lncRNA在内胚层分化过程中的功能。转化生长因子β信号转导通路是内胚层分化的关键调节因子。核心假设是，与发育调节因子共享启动子区域并被转化生长因子-β信号激活的lncRNAs是正常内胚层分化所必需的。一旦了解了关键的lncRNAs在内皮细胞分化中的功能，就有可能调节这些lncRNAs的表达，从而更有效地控制组织的分化，用于再生医学。本研究的目的是确定从ID1(lnc-id1)和GSC(lnc-GSC)差异转录的lncRNAs在内胚层分化过程中的功能。工作假设是lnc-id1和lnc-GSC都是正常内胚层分化所必需的。这一假说建立在初步数据的基础上，即LNC-ID1和LNC-GSC是由转化生长因子-β信号诱导的，并且分别与编码ID1和GSC的基因协同调节，而ID1和GSC是hESC分化的关键蛋白。基因组编辑将被用来插入截断lncRNA转录的序列，以确定内胚层分化对每个lncRNA的需求。基因组编辑也将被用来创建lncRNA诱导的hESC系，以评估每个lncRNA功能的获得对hESC状态和内胚层分化的影响。这一建议的贡献有望是确定两个lncRNAs在内胚层分化中的作用。这一贡献意义重大，因为识别调节内皮细胞分化的lncRNAs将导致更高效地生产人内皮细胞，这些细胞可用作生产胰腺、肝脏或肠道细胞的底物，用于治疗。此外，该项目中开发的遗传工具将极大地促进分析任何lncRNA在人类内胚层分化中的功能。本申请中提出的研究具有创新性，因为它代表了当前策略的一个新的重大转变，即专注于了解蛋白质编码基因的表达变化如何调节内胚层分化，而不是研究lncRNAs如何调节分化过程。

DESCRIPTION (provided by applicant): Thousands of long noncoding (lnc) RNAs are induced with differentiation of human embryonic stem cells (hESCs) and are likely to control many stages of normal human development, but the functions of the vast majority of these noncoding genes remain unknown. The long term goal of this work is to determine the role of lncRNAs in endodermal differentiation and use this insight to develop new approaches to direct more efficient production of human tissues derived from endoderm. The overall objective of this proposal is to identify the functions of two lncRNAs that are induced during differentiation of hESCs into endoderm and in doing so to develop genetic tools to more efficiently assess the function of any lncRNA during endodermal differentiation. TGF-ß signaling is the key mediator of endodermal differentiation. The central hypothesis is that the lncRNAs that share promoter regions with developmental regulators and are activated by TGF-ß signaling are required for normal endodermal differentiation. Once the functions of key lncRNAs are understood in endodermal differentiation, it will then be possible to modulate expression of these lncRNAs to more efficiently control differentiation of tissue for regenerative medicine. The aims of this proposal are to determine the functions of the lncRNAs that are divergently transcribed from ID1 (lnc-ID1) and GSC (lnc-GSC) during endodermal differentiation. The working hypothesis is that both lnc-ID1, and lnc-GSC are required for normal endodermal differentiation. This hypothesis is based on the preliminary data that lnc-ID1 and lnc-GSC are induced by TGF-ß signaling and are coordinately regulated with the genes encoding ID1 and GSC, respectively, which are key proteins in hESC differentiation. Genome editing will be used to insert sequences to truncate lncRNA transcription in order to determine the requirement for each lncRNA in endodermal differentiation. Genome editing will also be used to create lncRNA-inducible hESC lines in order to assess the effect of a gain of function of each lncRNA on hESC state and endodermal differentiation. The contribution of this proposal is expected to be the determination of the roles of two lncRNAs in endodermal differentiation. This contribution is significant because the identification of lncRNAs that regulate endodermal differentiation will lead to more efficient production of human endodermal cells that can be used as a substrate for production of pancreatic, liver or intestinal cells for therapeutics. In addition, the genetic tools developed in this project will greatly facilitate analysis of the function of any lncRNA in human endodermal differentiation. The research proposed in this application is innovative because it represents a new and significant shift from current strategies focused on understanding how changes in expression of protein coding genes regulates endodermal differentiation and instead investigates how lncRNAs regulate the differentiation process.