Highlights d Poised enhancers are necessary for the induction of major anterior neural genes d Poised enhancers physically contact their target genes in embryonic stem cells d The interaction between poised enhancers and their target genes is PRC2 dependent d PRC2 provides major anterior neural loci with a permissive regulatory topology

CpG islands (CGIs) represent a widespread feature of vertebrate genomes, being associated with ~70% of all gene promoters. CGIs control transcription initiation by conferring nearby promoters with unique chromatin properties. In addition, there are thousands of distal or orphan CGIs (oCGIs) whose functional relevance is barely known. Here we show that oCGIs are an essential component of poised enhancers (PEs) that augment their long-range regulatory activity and control the responsiveness of their target genes. Using a knock-in strategy in mouse embryonic stem cells (ESCs), we introduced PEs with or without oCGIs within topologically associating domains (TADs) harboring genes with different types of promoters. Analysis of the resulting cell lines revealed that oCGIs act as tethering elements that promote the physical and functional communication between PEs and distally located genes, particularly those with large CGI clusters in their promoters. Therefore, by acting as genetic determinants of gene-enhancer compatibility, CGIs can contribute to gene expression control under both physiological and potentially pathological conditions.

Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.

During neurogenesis, dynamic developmental cues, transcription factors and histone modifying enzymes regulate the gene expression programs by modulating the activity of neural-specific enhancers. How transient developmental signals coordinate transcription factor recruitment to enhancers and to which extent chromatin modifiers contribute to enhancer activity is starting to be uncovered. Here, we take advantage of neural stem cells as a model to unravel the mechanisms underlying neural enhancer activation in response to the TGFβ signaling. Genome-wide experiments demonstrate that the proneural factor ASCL1 assists SMAD3 in the binding to a subset of enhancers. Once located at the enhancers, SMAD3 recruits the histone demethylase JMJD3 and the remodeling factor CHD8, creating the appropriate chromatin landscape to allow enhancer transcription and posterior gene activation. Finally, to analyze the phenotypical traits owed to cis-regulatory regions, we use CRISPR–Cas9 technology to demonstrate that the TGFβ-responsive Neurog2 enhancer is essential for proper neuronal polarization.

Cellular heterogeneity within embryonic and adult tissues is involved in multiple biological and pathological processes. Here, we present a simple epigenomic strategy that allows the functional dissection of cellular heterogeneity. By integrating H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data, we demonstrate that the presence of broad H3K27me3 domains at transcriptionally active genes reflects the heterogeneous expression of major cell identity regulators. Using dorsoventral patterning of the spinal neural tube as a model, the proposed approach successfully identifies the majority of previously known dorsoventral patterning transcription factors with high sensitivity and precision. Moreover, poorly characterized patterning regulators can be similarly predicted, as shown for ZNF488, which confers p1/p2 neural progenitor identity. Finally, we show that, as our strategy is based on universal chromatin features, it can be used to functionally dissect cellular heterogeneity within various organisms and tissues, thus illustrating its potential applicability to a broad range of biological and pathological contexts.