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

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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.