Deep learning reveals evolutionary conservation and divergence of sequence properties underlying gene regulatory enhancers across mammals

In mammals, genomic regions with enhancer activity turnover rapidly; in contrast, gene expression patterns and transcription factor binding preferences are largely conserved. Based on this conservation, we hypothesized that enhancers active in different mammals would exhibit conserved sequence patterns in spite of their different genomic locations. We tested this hypothesis by quantifying the conservation of sequence patterns underlying histone-mark defined enhancers across six diverse mammals in two machine learning frameworks. We first trained support vector machine (SVM) classifiers based on the frequency spectrum of short DNA sequence patterns. These classifiers accurately identified many adult liver, developing limb, and developing brain enhancers in each species. Then, we applied these classifiers across species and found that classifiers trained in one species and tested in another performed nearly as well as classifiers trained and tested on the same species. This indicates that the short sequence patterns predictive of enhancers are largely conserved. We also observed similar cross-species conservation when applying the models to human and mouse enhancers validated in transgenic assays. The sequence patterns most predictive of enhancers in each species matched the binding motifs for a common set of TFs enriched for expression in relevant tissues, supporting the biological relevance of the learned features. To test the conservation of more complex sequences patterns, we trained convolutional neural networks (CNNs) on enhancer sequences in each species. The CNNs demonstrated better performance overall, but worse cross-species generalization than SVMs, suggesting the importance of combinatorial interactions between motifs, but less conservation of these more complex sequence patterns. Thus, despite the rapid change of active enhancer locations between mammals, cross-species enhancer prediction is often possible. Furthermore, short sequence patterns encoding enhancer activity have been maintained across more than 180 million years of mammalian evolution, with evolutionary change in more complex sequence patterns.Author summaryAlterations in gene expression levels are a driving force of both speciation and complex disease; therefore, it is of great importance to understand the mechanisms underlying the evolution and function gene regulatory DNA sequences. Recent studies have revealed that while gene expression patterns and transcription factor binding preferences are broadly conserved across diverse animals, there is extensive turnover in distal gene regulatory regions, called enhancers, between closely related species. We investigate this seeming incongruence by analyzing genome-wide enhancer datasets from six diverse mammalian species. We trained two machine-learning classifiers—a k-mer spectrum support vector machine (SVM) and convolutional neural network (CNN)—to distinguish enhancers from the genomic background. The k-mer spectrum SVM models the occurrences of short sequence patterns while the CNN models both the short sequences patterns and their combinatorial patterns. Both the SVM and CNN enhancer prediction models trained in one species are able to predict enhancers in the same cellular context in other species. However, CNNs performed better at predicting enhancers in each species, but they generalize less well across species than the SVMs. This argues that the short sequence properties encoding regulatory activity are remarkably conserved across more than 180 million years of mammalian evolution with more evolutionary turnover in the more complex combinations of the conserved short sequence motifs.