The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

The non-coding genome of vertebrates harbours a variety of sequences in charge of the transcriptional control of genes. The activity of these elements is particularly important during embryonic development, when newly differentiating cell types have to switch on and off certain transcriptional programs in a very dynamic manner. The most obvious regulatory elements that come to mind are those that promote activation or repression of genes in a specific cell type at a particular time. However, these are not the only ones, and this preprint focuses on the signature of other elements that are not directly activating nor repressing transcription: poised enhancers (PEs). PEs are a subset of distal regulatory elements that are bookmarked in pluripotent cells and become fully activated upon cellular differentiation. They are associated with a unique chromatin signature that includes high accessibility, the presence of the histone modification H3K4me1 and the repressive H3K27me3 [1]. They have been identified and characterised in mouse embryonic stem cells (mESCs), which are an ideal system to efficiently assess the changes upon differentiation, induced by culturing cells in specific conditions. However, the main question that remained unanswered was if these PEs also exist and play a role in vivo. This is precisely the question that Crispatzu and colleagues address in this work.

The authors first identified PEs in different states of mouse pluripotency (naïve pluripotency, formative pluripotency, and serum+LIF conditions). Then, they asked what the epigenetic profile was (investigating chromatin accessibility by means of ATAC-seq and the profiling of H3K27me3 and H3K27ac data) of those PEs during early mouse development. They found that chromatin accessibility and H3K27me3 marks progressively increase during preimplantation development in the PEs and reach the highest levels in the postimplantation epiblast. H3K27ac is still low even in the postimplantation epiblast, meaning the PEs are not active yet, and it is only later in development (E12.5) when this histone mark is gained. Thus, the pluripotent epiblast in the mouse embryo exhibits PEs that behave similarly to what happens in vitro.

The authors then investigated if those PEs were conserved in other vertebrates. The sequence and the chromatin signature of PEs identified in the mouse were highly conserved in other mammals, but moderately conserved in non-mammalian vertebrates. However, when PEs were called de novo in human ESCs, chicken epiblast and zebrafish embryos, PEs were abundant in all of them. This indicates that, even if many PEs might be specific to a vertebrate group, all vertebrates investigated have PEs associated with developmental genes and could be involved in the regulation of developmental programs in vivo.

Beyond the mentioned epigenetic signatures, the authors also showed that PEs have particular topological properties. By means of HiChIP, a technique that combines Hi-C and ChIP, they demonstrated that PEs can physically interact with their target genes (mostly involved in developmental processes) in mESCs. Analysis of published Hi-C data in early embryos indicated that this feature is also conserved in vivo. Those interactions depend on the activity of Polycomb and Trithorax complexes as well as on architectural proteins. Contacts between PEs and target gene promoters were reduced in PRC1, PRC2 (at a lesser extent), Kmt2b and CTCF-depleted mESCs. These interactions in mESCs already promote communication between PEs and their target gene promoters before the PEs act as proper enhancers. Importantly, those interactions are maintained once PEs are activated (when they gain H3K27ac marks) and the authors demonstrate this in differentiated anterior neural progenitor cells and in E10.5 mouse brains.

Finally, the authors addressed the functional relevance of PEs during vertebrate development, in particular during brain development. To do so, they used the CRISPR/Cas9 gene editing system to delete the PE associated to Lhx5 in mouse and chicken embryos. As a result, Lhx5 expression was strongly reduced in the forebrain (where it is usually expressed in wildtype conditions, Figure 1). Equivalent results, in this case affecting eye development, were obtained when two more PEs were deleted in chicken embryos: the PE associated to SIX3 and the PE associated to SOX1. Thus, these experiments demonstrate that PEs are essential to activate transcription of their target genes in vivo.

Why I chose this preprint

When we make a reference to the non-coding genome we do not talk about “junk” DNA anymore. However, despite many years of studies focused on promoters and enhancers, we can only understand general trends of behaviours in a scenario full of exceptions. We need more characterisation of regulatory elements in different contexts and different species to better predict what is required in order to activate or repress a gene.

In vitro tools are a great model to characterise regulatory elements but I always have it in mind that we have a different context in vivo. If a certain gene can be regulated by multiple enhancers depending on the tissue or the time point, why would it be regulated in the same manner in mESCs and in the embryo? That is why I was very glad to see how the authors addressed the importance of PEs in vivo and their conservation in different vertebrates in this preprint. This type of regulatory element acquires more relevance after this study and could help to understand how certain transcriptional programs are switched on and off during embryo development.

References

1. Rada-Iglesias et al. (2011) A unique chromatin signature uncovers early developmental enhancers in human. Nature 470, 279-283.

Questions to the authors

The epigenetic features of PEs could indicate that this is a way of quick activation of their target genes. The interactions with their target genes are already established in pluripotent cells, and they only need the acquisition of H3K27ac. I wonder…:

• PEs seem to be particularly enriched in neuroectoderm associated genes. Do you think it is a feature of those lineages in particular or is it just because those tissues are differentiated early?
• Hypothetically, would you expect to also have PEs associated to genes important for brain development in species where the brain develops later or more slowly?
• In the case of the PEs of genes that are not associated to neural lineages, are those genes activated soon after gastrulation?
• I had the impression that the deletion of the PE associated to Lhx5 in mouse embryos had a stronger effect at E8.5 than at E9.5 (although still very clear). Have you checked at later stages to see if PEs could be more relevant for the onset of their target’s expression, but it then catches up?
• What would you expect if you could prevent the interactions of PEs to their target genes in the pluripotent state while the PEs remain intact? Upon differentiation, would the target gene be activated later as it is not predisposed to be fully activated?

Tags: chromatin, pluripotency, poised enhancer, vertebrates

doi: https://doi.org/10.1242/prelights.27428

Read preprint (No Ratings Yet)