The developmental transitions occurring during embryogenesis involve spatial and temporal changes in gene expression patterns, which are largely dependent on a group of regulatory elements known as enhancers. Enhancers are short DNA sequences able to positively control the expression of their target genes in a distance and orientation independent manner. Previous studies uncovered a unique set of enhancers in pluripotent embryonic stem cells (ESCs), named “Poised Enhancers”. Poised enhancers are marked by repressive histone marks, like the trimethylation of histone three lysine 27, which is deposited by Polycomb repressive complex 2 (PRC2), and also are bound by co-activators like P300. The fact that poised enhancers display both activating and repressing features as well as evidences indicating that they are associated with genes involved in early organogenesis led us to suggest that these regulatory elements are already bookmarked in ESCs and thus primed for their future activation once the differentiation process starts. However, the functional relevance of poised enhancers and the role of their unique chromatin features remain unknown.
To gain insights into these major questions, first, poised enhancers were identified in mouse embryonic stem cells (mESCs) and their activation was evaluated during the establishment of anterior neural identity. Second, using CRISPR/Cas9 technology, poised enhancer candidates were deleted in mESCs, which were then differentiated into anterior neural progenitors. In general, the poised enhancer deletions resulted in severely reduced induction of the poised enhancer’s target genes. Furthermore, circularized chromosome conformation capture coupled to sequencing experiments revealed that poised enhancers and their target genes physically interacted already in ESCs, thus preceding the activation of poised enhancers and their target genes. Interestingly, these poised enhancer-target gene interactions observed in mESCs were found to be PRC2 dependent. Additionally, it was demonstrated that while PRC2 was not necessary to maintain poised enhancers in an inactive state in mESCs, was required for the induction of the poised enhancers’ target genes upon anterior neural differentiation. Finally, poised enhancers were found to frequently reside within a high CpG-dinucleotide genomic context that can directly mediate the recruitment of PRC2.
Overall, these findings demonstrate that poised enhancers are essential for the proper expression of the anterior neural developmental program. Importantly, our work illuminates an unexpected function for PRC2 in promoting neural induction. Our data demonstrates that both poised enhancers and their target genes display intrinsic sequence features that can directly mediate PRC2 recruitment. Consequently, PRC2 can bring poised enhancers and their targets into physical proximity already in mESCs, thus providing a permissive regulatory topology that we propose can facilitate the future induction of mayor anterior neural genes
