Conservation of Regulatory Elements with Highly Diverged Sequences Across Large Evolutionary Distances

Embryonic gene expression is remarkably conserved across vertebrates as observed, for instance, in the developing hearts of chicken and mouse which diverged >300 million years ago. However, most cis regulatory elements (CREs) are highly divergent, which makes orthology tracing based on sequence similarity difficult, especially at larger evolutionary distances. Some evidence suggests functional conservation of CREs despite sequence divergence. However, it remains unclear how widespread such functional conservation might be. Here, we address this question by profiling the regulatory genome in the embryonic hearts of chicken and mouse at equivalent developmental stages. Gene expression and 3D chromatin structure show remarkable similiarity, while the majority of CREs are non-alignable between the two species. To identify orthologous CREs independent of sequence alignability, we introduce a synteny-based strategy called Interspecies Point Projection (IPP). Compared to alignment-based approaches, IPP identifies up to 5-fold more putative orthologs in chicken, and up to 9-fold across distantly related vertebrates. We term these sequence-diverged orthologs indirectly conserved and characterize their functional conservation compared to sequence-alignable, directly conserved CREs. Indirectly and directly conserved elements show similar enrichment of functional chromatin signatures and cell-type specific enhancer sequence composition. Yet, shared transcription factor binding sites between orthologs are more heavily rearranged in indirectly conserved elements. Finally, we validate functional conservation of indirectly conserved chicken enhancers in mouse using in vivo reporter assays. Taken together, by overcoming the limitations of alignment-based methods our results reveal functional conservation of CREs across large evolutionary distances is more widespread than previously recognized.