Mechanistic and functional characterization of poised enhancers during the induction of major anterior neural loci using in vitro and in vivo developmental models.

使用体外和体内发育模型诱导主要前神经位点过程中平衡增强子的机制和功能表征。

• 批准号: 251811628
• 负责人: Dr. Alvaro Rada-Iglesias
• 金额: --
• 依托单位: Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251811628?language=en
• 关键词: Mechanistic; functional; characterization; poised; enhancers

We previously identified poised enhancers in human embryonic stem cells (ESC) as a limited set of cis-regulatory sequences displaying a unique chromatin signature, which included binding by the co-activator p300 and enrichment in H3K27me3, a histone modification mediated by polycomb-group proteins. We originally proposed that the poised enhancer chromatin signature could bookmark these sequences in pluripotent cells to facilitate their timely activation upon ESC differentiation. However, the functional relevance of poised enhancers was only supported by correlative observations. Using mouse ESC as a genetically tractable model, we have now used genetic deletions to demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, poised enhancers already established physical interactions with their target genes in ESC in a Polycomb repressive complex 2 (PRC2) dependent manner. Loss of PRC2 led to neither the activation of poised enhancers nor the induction of their putative target genes in undifferentiated ESC. In contrast, loss of PRC2 severely and specifically compromised the induction of major anterior neural genes representing poised enhancer targets. Hence, our recent work illuminates a novel function for polycomb proteins, which we propose facilitate neural induction by providing major anterior neural loci with a permissive regulatory topology.We now propose a number for experiments that aim at elucidating two major open questions regarding poised enhancers: (i) Are poised enhancers functionally relevant in vivo?; (ii) Which are the mechanisms whereby poised enhancers become activated during the establishment of anterior neural identity?. To answer the first question we will evaluate whether poised enhancers display their unique chromatin signature in pluripotent cells in vivo (i.e. mouse embryonic epiblast). Furthermore, we will generate mouse embryos with poised enhancer deletions to determine whether these regulatory sequences are important for the activation of their target genes and, thus, for forebrain development in vivo. Finally, we will investigate the importance of ZIC2 as a novel mediator of poised enhancer activation during the establishment of anterior neural identity, which we believe can provide major insights into the mechanisms controlling the regulatory activity of poised enhancers. Overall, the proposed work will conclusively evaluate the importance of poised enhancers as regulators of anterior neural genes, thereby potentially illuminating some of the genetic and epigenetic mechanisms controlling neural induction.

我们以前确定了人胚胎干细胞（ESC）中的平衡增强子作为一组有限的顺式调节序列，显示出独特的染色质特征，其中包括辅激活因子p300的结合和H3K27me3的富集，这是一种由多梳组蛋白介导的组蛋白修饰。我们最初提出，平衡的增强子染色质签名可以在多能细胞中标记这些序列，以促进它们在ESC分化时的及时激活。然而，平衡增强子的功能相关性仅得到相关观察结果的支持。使用小鼠ESC作为遗传学上易处理的模型，我们现在已经使用遗传缺失来证明，平衡的增强子是诱导主要的前神经调节因子所必需的。有趣的是，平衡增强子已经以Polycomb抑制复合物2（PRC2）依赖的方式与ESC中的靶基因建立了物理相互作用。PRC2的缺失既不导致平衡增强子的激活，也不导致未分化ESC中推定的靶基因的诱导。相比之下，PRC2的缺失严重且特异性地损害了代表稳定增强子靶点的主要前神经基因的诱导。因此，我们最近的工作阐明了一个新的功能polycomb蛋白，我们建议促进神经诱导提供主要的前神经基因座与一个许可的监管topology.We现在提出了一些实验，旨在阐明两个主要的开放问题，关于平衡增强子：（i）平衡增强子在体内功能相关？(ii)在前神经元身份的建立过程中，哪些机制是平衡的增强子被激活的？为了回答第一个问题，我们将评估平衡增强子是否在体内多能细胞（即小鼠胚胎外胚层）中显示其独特的染色质特征。 此外，我们将产生具有增强子缺失的小鼠胚胎，以确定这些调控序列是否对靶基因的激活以及前脑的体内发育很重要。最后，我们将调查的重要性ZIC 2作为一种新的调解人的平衡增强子激活在建立前神经身份，我们相信可以提供重要的见解的机制控制平衡增强子的调节活性。总体而言，拟议的工作将最终评估作为前神经基因的调节剂的平衡增强子的重要性，从而可能阐明一些控制神经诱导的遗传和表观遗传机制。