Self-Renewal and Differentiation: Molecular Events that Commit ES Cells to Exit t

自我更新和分化：使 ES 细胞退出的分子事件

• 批准号: 8381275
• 负责人: James Alexander Thomson
• 金额: $ 36.96万
• 依托单位: MORGRIDGE INSTITUTE FOR RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8381275
• 关键词: Activins; Attention; BMP4; Binding Sites; Brachyury protein; Cell Differentiation process; Cells; Chorionic Gonadotropin; Commit; DNA Binding; Development; Endoderm; Event; Excision; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Maps; Measures; Mediating; Mediator of activation protein; Mesoderm; Molecular; Outcome; Phosphorylation; Placenta; Population; Proteins; RNA Interference; Signal Transduction; Time; chromatin immunoprecipitation; clinically relevant; embryonic stem cell; human embryonic stem cell; overexpression; pluripotency; poly(L-glutamic acid(60)-L-alanine(30)-L-tyrosine(10)); promoter; response; self-renewal; transcription factor; trophoblast

Project 2: Self-Renewal and Differentiation: Molecular events that commit ES cells to exit the pluripotent state. (James Thomson, PI) A. SPECIFIC AIMS In low (4 ng/ml) or absent exogenous bFGF, BMP4 induces human ES cells to form a homogenous population of trophoblast, the outer layer of the placenta. However, we have recently found that in the presence of high (100 ng/ml) concentrations of bFGF, BMP4 instead induces human ES cells to form a population of cells that no longer express trophoblast markers, but instead transiently express brachyury, a mesoderm or mesendoderm marker, and subsequently express a mixture of endoderm and mesoderm markers. In this project, we will study how BMPs induce human ES cells to exit the pluripotent state and commit to differentiation, and study how FGF mediates these divergent developmental outcomes in response to the same inducer. Understanding how ES cells exit the pluripotent state and why this exit is generally irreversible is central to achieving efficient reprogramming (Project 3), and understanding these key early lineage decisions will allow more efficient differentiation to specific clinically-relevant lineages. We will accomplish the following aims: Aim 1. We will establish a detailed time course of gene expression in human ES cells upon BMP4- induced differentiation, both in the presence (brachyury positive result) and absence (chorionic gonadotropin positive result) of bFGF, and correlate these changes with the commitment to exit the pluripotent state. Commitment will be measured by adding BMP4 for successively longer time periods, removing BMP4, and then examining how many cells retain markers of pluripotency several days later. The hypothesis of this aim is that the quantitative commitment curve will be most closely correlated with the expression levels of the genes that directly control these differentiation events. We will subsequently overexpress the transcription factors most closely associated with commitment, and we will downregulate genes by RNA interference that are downregulated during commitment to identify those which are sufficient to mediate differentiation to a brachyury-positive population. Preliminary results demonstrate that GATA2 and GATA3 are both individually sufficient to mediate trophoblast differentiation in the absence of bFGF, so we are optimistic that a single transcription factor will be sufficient to mediate human ES cell differentiation to a brachyury positive population in the presence of bFGF. Aim 2. We will use whole genome chromatin immunoprecipitation on chip (ChlP-chip) to map the genomic binding sites of TGFp/Activin-activated Smad 2/3 and BMP-activated Smad 1/5/8 during BMP4- induced differentiation. The central hypothesis of this aim is that Smad 2/3 directly activates the expression of key pluripotency factors in human ES cells, directly suppresses the expression of genes that would otherwise promote differentiation, and competes at the same promoters with Smad 1/5/8 which has the opposing effects. A second hypothesis is that bFGF will change the DNA binding sites of Smad 1/5/8 during BMP-induced differentiation, leading to the different developmental outcomes observed. Aim 3. We will use whole genome chromatin immunoprecipitation on chip (ChlP-chip) to map genomic binding sites of GATA2 and GATA3 during BMP4-induced differentiation. The hypothesis of this aim is that GATA2 and GAT A3 directly negatively regulate the transcription of key pluripotency genes, and directly positively regulate their own transcription, so that once they are induced by Smads, GAT A expression becomes self-sustaining and BMP-independent, and thus differentiation continues even upon BMP removal. A second hypothesis is that bFGF will change the DNA binding sites of GATA2/GATA3 during BMP-induced differentiation, leading to the different developmental outcomes observed. Aim 4. We will identify proteins that are differentially phosphorylated between BMP4-induced trophoblast differentiation (no bFGF) and BMP-induced brachyury positive cellular differentiation (high bFGF) to identify mediators of FGF signaling that cause the switch between these divergent developmental outcomes. The hypothesis of this aim is that differential phosphorylation of a limited number of transcription factors is casually related to the different developmental outcomes observed after BMP induction in the presence or absence of bFGF. Differentiation commitment curves will again become key for focusing attention to relevant phosphorylation events.

项目2：自我更新和分化：使ES细胞退出的分子事件 多能状态。 （詹姆斯·汤姆森（James Thomson），PI） A.具体目标 在低（4 ng/ml）或不存在外源性BFGF中，BMP4诱导人ES细胞形成同质种群 滋养细胞，胎盘的外层。但是，我们最近发现，在较高的情况下 （100 ng/ml）BFGF的浓度，BMP4诱导人ES细胞形成一个细胞群， 不复 中胚层标记，随后表达内胚层和中胚层标记的混合物。在这个 项目，我们将研究BMP如何诱导人ES细胞退出多能状态并致力于 分化，并研究FGF如何响应于相同的这些不同的发育结果 诱导者。了解ES细胞如何退出多能状态以及为什么该退出通常不可逆 实现有效重编程（项目3）的中心，并理解这些关键的早期血统决策 将允许更有效地分化与特定临床相关的谱系。 我们将实现以下目标： 目标1。我们将在BMP4-上建立人ES细胞中基因表达的详细时间过程 在存在的情况下（Brachyury阳性结果）和不存在（绒毛膜）诱导的分化 BFGF的促性腺激素阳性结果，并将这些变化与退出的承诺相关联 多能状态。承诺将通过添加BMP4在较长的时间段内衡量， 去除BMP4，然后检查几天后有多少细胞保留多能性的标记。这 该目标的假设是定量承诺曲线将与该曲线密切相关 直接控制这些分化事件的基因的表达水平。随后我们将过表达 转录因子与承诺最紧密相关，我们将通过 在承诺识别足以进行介导的人的承诺过程中被下调的RNA干扰 与臂阳性阳性人群的区分。初步结果表明GATA2和GATA3是 两者都足以在没有BFGF的情况下介导滋养细胞的分化，因此我们很乐观 单个转录因子足以介导人类ES细胞分化为Brachyury 在BFGF存在下人口阳性。 AIM 2。我们将在CHIP（CHLP-CHIP）上使用整个基因组染色质免疫沉淀来绘制 在BMP4-期间，TGFP/激活素激活SMAD 2/3和BMP激活的SMAD的基因组结合位点在BMP4-- 诱导的分化。该目标的中心假设是SMAD 2/3直接激活表达 人类ES细胞中关键多能因素的构成，直接抑制基因的表达 否则促进差异化，并与SMAD 1/5/8在同一启动子竞争 相反的效果。第二个假设是，BFGF将在1/5/8的DNA结合位点更改 BMP诱导的分化，导致观察到不同的发展结果。 AIM 3。我们将在芯片上使用整个基因组染色质免疫沉淀来绘制基因组 BMP4诱导的分化过程中GATA2和GATA3的结合位点。这个目标的假设是 GATA2和GAT A3直接对密钥多能基因的转录进行负调节，并直接调节 积极调节自己的转录，以便一旦被Smads诱导，gat表达式 变得自我维持和独立于BMP，因此即使去除BMP，分化也会继续。一个 第二个假设是，BFGF将在BMP诱导的过程中更改GATA2/GATA3的DNA结合位点 分化，导致观察到不同的发展结果。 AIM 4。我们将鉴定BMP4诱导的蛋白质差异化的蛋白质 滋养细胞的分化（无BFGF）和BMP诱导的跑型甲状腺阳性细胞分化（高 BFGF）识别引起这些发散之间切换的FGF信号传导的介体 发展结果。该目标的假设是有限数量的差异磷酸化 转录因子的随便与BMP后观察到的不同发展结果相关 在存在或不存在BFGF的情况下诱导。差异化承诺曲线将再次成为关键 将注意力集中在相关的磷酸化事件上。