Model-based analysis of polymorphisms in an enhancer reveals cis-regulatory mechanisms

It is challenging to predict the impact of small genetic changes such as single nucleotide polymorphisms on gene expression, since mechanisms involved in gene regulation and their cis-regulatory encoding are not well-understood. Recent studies have attempted to predict the functional impact of non-coding variants based on available knowledge of cis-regulatory encoding, e.g., transcription factor (TF) motifs. In this work, we explore the relationship between regulatory variants and cis-regulatory encoding from the opposite angle, using the former to inform the latter. We employ sequence-to-expression modeling to resolve ambiguities regarding gene regulatory mechanisms using information about effects of single nucleotide variations in an enhancer. We demonstrate our methodology using a well-studied enhancer of the developmental gene intermediate neuroblasts defective (ind) in D. melanogaster. We first trained the thermodynamics-based model GEMSTAT to relate the neuroectodermal expression pattern of ind to its enhancer9s sequence, and constructed an ensemble of models that represent different parameter settings consistent with available data for this gene. We then predicted the effects of every possible single nucleotide variation within this enhancer, and compared these to SNP data recorded in the Drosophila Genome Reference Panel. We chose specific SNPs for which different models in the ensemble made conflicting predictions, and tested their effect in vivo. These experiments narrowed in on one mechanistic model as capable of explaining the observed effects. We further confirmed the generalizability of this model to orthologous enhancers and other related developmental enhancers. In conclusion, mechanistic models of cis-regulatory function not only help make specific predictions of variant impact, they may also be learned more accurately using data on variants.