Finally, we leverage naturally occurring genetic variation in a panel of human pluripotent stem cell lines to identify sequence determinants and candidate transcription factors that may play crucial roles in establishing the primed state at key enhancers for human neural development.

Surprisingly, we find that epigenetic priming also occurs at enhancers that become active at much later developmental stages. In some cases, epigenetic priming can already be detected in the epiblast at neural enhancers that are specific for mouse E11.5 and 7 weeks post conception in humans.

We focussed on ‘primed’ enhancers, which harbour specific chromatin signatures: ATAC-seq positive, DNA hypomethylation & H3K4me1 positive. But importantly, they lack the active enhancer mark H3K27ac, and are thought to become active only later in development during cell lineage diversification.

Finally, we asked whether these putative non-canonical enhancers are pluripotent stem cell specific. Mining publicly available genome-wide STARR-seq data sets, we found STARR-seq+ enhancers with similar chromatin profiles in human cancer cell lines, mouse ESCs and a Drosophila cell line.

But had we simply re-discovered non-canonical enhancers previously described in mouse embryonic stem cells discovered by the lab of ‪@wbickmor.bsky.social‬ (Pradeepa Nature Genetics 2026)? Our chromatin profiling suggests no (no enrichment for H3K122ac, but instead weak enrichment for H4K12ac).‬

In one case, remarkably CRISPRi perturbation of primed enhancer function led to changes of pluripotent stem cell characteristics, including loss of PSC colony morphology, reduced stem cell marker expression and aberrant expression of differentiation markers.

We tested this hypothesis directly using CRISPR epigenome engineering to interfere with enhancer function. Recruitment of dCas9-KRAB (CRISPRi) to primed enhancers resulted in reduced expression levels of target genes. Conversely, CRISPR activation led to increased expression levels of target genes.

Further, we found that inter-individual sequence variants in primed enhancers correlated with the expression levels of target genes, and with the chromatin accessibility of the primed enhancers we assessed. Very small n numbers admittedly but nonetheless intriguing?

In some cases – yes. But intriguingly, the majority of primed STARR-seq enhancers did NOT acquire the active enhancer mark H3K27ac in the more specialised cell types examined (neural progenitor cells (NPCs), mesendoderm cells (ME), mesenchymal stem cells (MSCs), trophoblast-like stem cells (TBL)).

Using Promoter Capture Hi-C, we linked STARR-seq enhancers to their putative target genes. This revealed that genes interacting with active enhancers had the highest expression levels, followed by genes interacting with primed enhancers, followed by genes interacting with poised enhancers.

Generating the first STARR-seq map in a non-cancer human cell line, we classified the identified STARR-seq+ enhancers into:
- Active (ATAC-seq+; H3K4me1+; H3K27ac+)
- Primed/Inactive (ATAC-seq+; H3K4me1+; H3K27ac-)
- Poised (ATAC-seq+; H3K27me3+)
- Chromatin repressed (ATAC-seq-)

I very much look forward to @mosterwalder.bsky.social's talk on "Functional architecture of cardiac TF regulatory landscapes in control of mammalian heart development" in 'The 3D Regulatory Genome', @enhancedgenomics.bsky.social's virtual seminar series.

Join us at:
us02web.zoom.us/webinar/regi...

I am very excited to host Dr Sarah Marzi @sj-marzi.bsky.social in our virtual seminar series 'The 3D Regulatory Genome' for her talk on "Epigenetic regulation of environmental and genetic risk in neurodegenerative diseases"

📅 March 13th 2025
⏰ 4 pm GMT

Join us at: us02web.zoom.us/webinar/regi...