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Fig. 5 | Genome Biology

Fig. 5

From: STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

Fig. 5

Schematic overview of the three enhancer classes and their main characteristics in 2iL- and SL-ESCs. C1 STARR-seq loci are accessible and covered by histone modifications associated with active enhancers. These loci have virtually no DNA methylation in 2iL- and low-DNA methylation in SL-ESCs. C1-loci are typically bound by well-known pluripotency factors like OCT4 (Pou5f1), SOX2, NANOG, and ESRRB in 2iL- and SL-ESCs. ZIC3 is a TF that is much more abundant in SL- compared to 2iL-ESCs and is associated with significantly stronger enhancer signals in SL-ESCs. C2 STARR-seq loci are not or very lowly covered by enhancer-associated histone modifications. C2-loci appear to be repressed in chromatin by high-DNA methylation and H3K9me3. In plasmid assays, the absence of these repressive marks causes NRF1-induced enhancer activity at a number of loci. Furthermore, enhancer associated with ERVs appear to be repressed by H3K9me3, but are active in our STARR-seq assays. A large group of C2-loci is associated with P53 motifs. P53 binding is not impeded by DNA methylation or H3K9me3 in WT ESCs, but the lack of enhancer-associated histone modifications indicate that these enhancers are silent under normal physiological conditions. A strong increase in H3K27ac after Nocodazole treatment and at intermediate-induced reprogramming timepoints shows that these enhancers can become activated in their endogenous context. C3-loci show low histone marking and accessibility associated with active regulatory elements, but no STARR-seq signal. We find C3-loci even at regions that were highly covered by our input DNA libraries. C3-loci further show very low CpG-methylation and overlap significantly with promoters classified by the Phantom5 consortium using CAGE analysis. This indicates that C3-loci largely overlap with promoters that are currently undefined in the Gencode or Refseq gene annotations. Some of the panels in these figures contain public data. These panels are annotated with [PD]. The accession numbers of public data and their corresponding panels are annotated in Additional file 2: Table S1

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