Role of long noncoding RNAs in human endodermal differentiation

描述（申请人提供）：人类胚胎干细胞（hESC）分化诱导了数千个长非编码（lnc）RNA，可能控制人类正常发育的许多阶段，但这些非编码基因中绝大多数的功能仍然未知。这项工作的长期目标是确定lncRNA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源自内胚层的人体组织。该提案的总体目标是鉴定在hESC分化成内胚层期间诱导的两种lncRNA的功能，并且在这样做的过程中开发遗传工具以更有效地评估内胚层分化期间任何lncRNA的功能。TGF-β信号传导是内胚层分化的关键介质。中心假设是，与发育调节因子共享启动子区域并由TGF-β信号传导激活的lncRNA是正常内胚层分化所需的。一旦了解了关键lncRNA在内胚层分化中的功能，就有可能调节这些lncRNA的表达，以更有效地控制再生医学的组织分化。本研究的目的是确定在内胚层分化过程中从ID 1（lnc-ID 1）和GSC（lnc-GSC）分化转录的lncRNA的功能。工作假设是lnc-ID 1和lnc-GSC两者都是正常内胚层分化所需的。这一假设基于以下初步数据：lnc-ID 1和lnc-GSC由TGF-β信号传导诱导，并且分别与编码ID 1和GSC的基因协调调节，ID 1和GSC是hESC分化中的关键蛋白。基因组编辑将用于插入序列以截短lncRNA转录，以确定内胚层分化中对每种lncRNA的需求。基因组编辑也将用于创建lncRNA诱导型hESC系，以评估每种lncRNA的功能获得对hESC状态和内胚层分化的影响。这一建议的贡献预计将是确定两个lncRNA在内胚层分化中的作用。这一贡献是重要的，因为鉴定调节内胚层分化的lncRNA将导致更有效地产生人内胚层细胞，所述人内胚层细胞可用作产生用于治疗的胰腺、肝或肠细胞的底物。此外，本项目中开发的遗传工具将大大有助于分析任何lncRNA在人类内胚层分化中的功能。本申请中提出的研究是创新的，因为它代表了一个新的和重大的转变，从目前的策略集中在理解蛋白质编码基因的表达变化如何调节内胚层分化，而是研究lncRNA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.