Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
Changes in the activity of transcriptional enhancers have been shown to influence phenotypic variation across species. However, it remains challenging to identify the sequence features associated with species differences in enhancer activity. Here we assessed the contribution of orphan CpG islands (oCGIs), which are regions enriched in CpG dinucleotides that are known to recruit chromatin modifiers and components of the transcriptional machinery. We integrated oCGIs in nine mammals with previously ascertained maps of chromatin modifications across multiple tissues, and found that species-specific oCGIs significantly co-localize with species-specific histone modification peaks. We focused on one enhancer of interest (hs754) which contains an oCGI in human but not in mouse, and overlaps an H3K27ac peak in the developing human brain, but not in mouse. We generated a humanized mouse model and profiled the locus using ChIP-seq, finding increased deposition of the histone modifications H3K27ac and H3K4me3 and gained CTCF binding sites in the humanized enhancer. ChIP-seq data for H3K27me3 was also generated and was not included in the final study, but is deposited here. We also measured gene expression in the humanized mouse and did not find any changes associated with the humanized enhancer, although using the datasets described above we found that genes near enhancers with species-specific oCGIs and species-specific activity show a species bias in expression. This work identifies oCGI turnover as a mechanism affecting mammalian enhancer evolution.
Overall design
ChIP-seq (H3K27ac, H3K4me3, H3K27me3, and CTCF) and RNA-seq were performed using developing diencephalon tissue (E11.5 and E17.5) from wild type and hs754-humanized mouse lines. The number of replicates for each genotype are as follows: 3 replicates for E17.5 ChIP-seq; 2 replicates for E11.5 ChIP-seq; 4 replicates for E11.5 and E17.5 RNA-seq.
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