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

我们之前在人类胚胎干细胞（ESC）中发现了一组有限的顺式调控序列，显示出独特的染色质特征，包括与共激活子p300的结合和H3K27me3的富集，H3K27me3是一种由polycomb-group蛋白介导的组蛋白修饰。我们最初提出，平衡增强子染色质特征可以在多能细胞中标记这些序列，以促进它们在ESC分化时及时激活。然而，平衡增强子的功能相关性仅得到相关观察的支持。使用小鼠ESC作为遗传可处理的模型，我们现在使用遗传缺失来证明平衡增强子对于诱导主要的前神经调节是必要的。有趣的是，平衡增强子已经以Polycomb抑制复合体2 （PRC2）依赖的方式与ESC中的靶基因建立了物理相互作用。在未分化的ESC中，PRC2的缺失既不会激活平衡增强子，也不会诱导其假定的靶基因。相比之下，PRC2的缺失严重且特异性地损害了代表平衡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多梳蛋白的新功能，我们提出通过为主要的前神经位点提供允许的调节拓扑来促进神经诱导。我们现在提出了一些旨在阐明关于平衡增强子的两个主要开放问题的实验：(i)平衡增强子在体内是否具有功能相关性？（ii）在前神经同一性建立过程中，平衡增强子被激活的机制是什么？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，我们将产生具有平衡增强子缺失的小鼠胚胎，以确定这些调控序列是否对其靶基因的激活以及对体内前脑发育重要。最后，我们将研究ZIC2在前神经身份建立过程中作为平衡增强子激活的新介质的重要性，我们相信这可以为控制平衡增强子调节活性的机制提供重要的见解。总的来说，这项工作将最终评估平衡增强子作为前神经基因调节因子的重要性，从而潜在地阐明控制神经诱导的一些遗传和表观遗传机制。

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.