Unlocking cis-regulatory landscapes across 500 million years of evolution and disease mechanisms.

Genomic DNA encodes regulatory information that determines where, when, and to what extent genes are expressed. Theoretically, we should be able to identify these transcriptional "instructions" by examining genomic DNA sequence alone, yet this has remained challenging. Here we present the Vertebrate Regulatory MOdule Detector (VRMOD), a method that accurately predicts gene regulatory sequences using only the query genomic sequences. We applied VRMOD to 309 Ensembl genomes, generating a compendium of high-resolution, genome-position-fixed cis-regulatory modules without parameter tuning. We performed extensive computational evaluation and experimental validation of VRMOD predictions. Notably, VRMOD predicted three sub-enhancers within the human hs52 enhancer at the FTO locus from the VISTA database, including one missed by existing methods. Using a chicken embryo system and 3D tissue imaging, we showed that each sub-enhancer exhibits restricted spatiotemporal activity within specific subsets of tissues where the full enhancer is active. We further demonstrated VRMOD's utility for identifying evolutionarily non-conserved enhancers, annotating regulatory sequences in non-model organisms, and identifying candidate disease-causal variants. Collectively, VRMOD provides a universal coordinate reference system for regulatory sequences across 309 vertebrate genomes and enables genome-wide annotation of non-coding regulatory elements in any vertebrate species using genomic sequence alone.