Long noncoding RNAs regulating endoderm differentiation

调节内胚层分化的长非编码RNA

• 批准号: 9918169
• 负责人: ALAN C MULLEN
• 金额: $ 36.95万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-25 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918169
• 关键词: Biology; Cell Culture Techniques; Cell Differentiation process; Cell Therapy; Cells; Chromatin; Code; Coupled; Data; Defect; Development; Differentiation Therapy; Digit structure; Disease; Embryo; Endoderm; Fluorescent in Situ Hybridization; Gastrointestinal Diseases; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Human; Human Development; Injections; Intestines; Knowledge; Label; Lead; Life; Liver; Location; Lung; Messenger RNA; Mission; Mus; Mutation; Orthologous Gene; Outcome Study; Pancreas; Pathway interactions; Play; Precipitation; Proteins; Public Health; RNA; RNA Splicing; RNA-Binding Proteins; Regenerative Medicine; Research; Role; Testing; Therapeutic; Tissue Differentiation; Tissue Therapy; Tissues; Transcription Elongation; Transcription Initiation; United States National Institutes of Health; Untranslated RNA; Work; base; blastocyst; embryonic stem cell; frontier; gastrointestinal; gene induction; genome editing; human embryonic stem cell; improved; in vivo; innovation; insight; loss of function; mRNA Stability; mouse development; mouse model; novel strategies; stem cell differentiation

Project Summary Long noncoding (lnc) RNAs are a new frontier that we must explore to fully understand human development. To date, the functions of only a small number of lncRNAs have been described in human development, and the continued existence of this fundamental gap in our knowledge of lncRNA function will impede the ability to control differentiation for cell-based therapies and understand how mutations in noncoding regions of the genome lead to disease. The long-term goal of this work is to elucidate the mechanisms by which lncRNAs regulate development of human definitive endoderm (DE) and use this insight to direct more efficient differentiation of gastrointestinal tissues for regenerative medicine. We have identified the lncRNA DIGIT and show that it regulates DE differentiation of human and mouse embryonic stem cells (ESCs). The gene encoding DIGIT is divergent to the gene encoding Goosecoid (GSC) in both humans and mice. In human ESCs, we find that DIGIT controls DE differentiation through regulation of GSC. The overall objective is to determine how DIGIT regulates development. The central hypothesis is that DIGIT regulates DE differentiation through different pathways in ESC differentiation and in vivo development. The rationale for this proposal is that understanding how DIGIT controls DE differentiation will make it possible to modulate expression of this lncRNA to facilitate differentiation of tissues for regenerative medicine and provide insight into the mechanisms by which lncRNAs regulate divergent genes. This proposal will also determine how the mouse ortholog of DIGIT (Digit) regulates differentiation and whether mouse models can teach us about the function of DIGIT when hESCs are differentiated for therapy. The central hypothesis will be tested by pursuing three specific aims: (1) determine how DIGIT regulates GSC expression in hESCs, (2) determine how DIGIT and protein partners interact to regulate DE differentiation, and (3) determine the role of Digit in mouse development. In the first aim, gain and loss of function analysis coupled with RNA fluorescent in situ hybridization and chromatin precipitation will determine how DIGIT controls GSC expression. In the second aim, we will investigate how protein partners interact with DIGIT to regulate differentiation. In the third aim, we will use mouse ESC culture and in vivo development to define the role of Digit in DE differentiation. The proposed work is significant because understanding the mechanism by which lncRNAs regulate DE differentiation will lead to transform- ative strategies for the differentiation of pancreatic, liver, intestinal and lung cells for therapeutics. It will also teach us how lncRNAs regulate divergently transcribed genes and whether mouse models for this lncRNA can be used to understand hESC differentiation. This work is innovative in the focus on lncRNAs to understand mechanism of development and in how it uses genome editing approaches to disrupt and track lncRNA transcription.

项目摘要 长链非编码RNA（lnc）是一个新的前沿，我们必须探索，以充分了解人类的发展。 迄今为止，在人类发育中仅描述了少量lncRNA的功能，并且 在我们对lncRNA功能的认识中，这种基本空白的持续存在将阻碍我们研究lncRNA功能的能力。 控制细胞治疗的分化，并了解细胞非编码区的突变如何影响细胞的分化。 基因组导致疾病。这项工作的长期目标是阐明lncRNA的机制， 调节人类定形内胚层（DE）的发育，并利用这一见解指导更有效的 分化胃肠组织的再生医学。我们已经鉴定了lncRNA DIGIT， 显示其调节人类和小鼠胚胎干细胞（ESC）的DE分化。基因 在人和小鼠中，编码DIGIT的基因与编码Goosecoid（GSC）的基因不同。人 我们发现DIGIT通过调节GSC来控制DE分化。总体目标是 确定DIGIT如何规范发展。核心假设是DIGIT调节DE分化 通过ESC分化和体内发育的不同途径。这项建议的理由是 了解DIGIT如何控制DE分化将使调节这种表达成为可能。 lncRNA促进再生医学组织的分化并提供机制的见解 lncRNA通过它调节不同的基因。这项提议还将确定小鼠的直系同源物 DIGIT（Digit）调节分化以及小鼠模型是否能教会我们DIGIT的功能 当hESC分化用于治疗时。中心假设将通过追求三个具体的测试 目的：（1）确定DIGIT如何调节hESC中GSC的表达，（2）确定DIGIT和蛋白质如何调节hESC中GSC的表达。 配偶体相互作用以调节DE分化，和（3）确定Digit在小鼠发育中的作用。在 第一目标，功能获得和丧失分析，再加上RNA荧光原位杂交和染色质 沉淀将决定DIGIT如何控制GSC表达。在第二个目标中，我们将研究如何 蛋白质伴侣与DIGIT相互作用以调节分化。在第三个目标中，我们将使用小鼠ESC培养物 和体内发育以确定Digit在DE分化中的作用。拟议的工作意义重大 因为了解lncRNA调节DE分化的机制将导致转化- 用于治疗的胰腺、肝、肠和肺细胞分化的有效策略。它还将 告诉我们lncRNA是如何调节不同转录的基因，以及这种lncRNA的小鼠模型是否可以 用于了解hESC分化。这项工作在关注lncRNA以了解方面具有创新性 发展机制以及它如何使用基因组编辑方法来破坏和跟踪lncRNA 转录。