Dissecting endogeneous genetic circuits from first principles

For the vast majority of genes in sequenced genomes, there is limited understanding of how they are regulated. Without such knowledge, it is not possible to perform a quantitative theory-experiment dialogue on how such genes give rise to physiological and evolutionary adaptation. One category of high-throughput experiments used to understand the sequence-phenotype relationship of the transcriptome is massively parallel reporter assays (MPRAs). However, to improve the versatility and scalability of MPRA pipelines, we need a “theory of the experiment” to help us better understand the impact of various biological and experimental parameters on the interpretation of experimental data. These parameters include binding site copy number, where a large number of specific binding sites may titrate away transcription factors, as well as the presence of overlapping binding sites, which may affect analysis of the degree of mutual dependence between mutations in the regulatory region and expression levels. Here, we develop a computational pipeline that makes it possible to systematically explore how each biological and experimental parameter controls measured MPRA data. Specifically, we use equilibrium statistical mechanics in conjunction with predictive base-pair resolution energy matrices to predict expression levels of genes with mutated regulatory sequences and subsequently use mutual information to interpret synthetic MPRA data including recovering the expected binding sites. Our simulations reveal important effects of the parameters on MPRA data and we demonstrate our ability to optimize MPRA experimental designs with the goal of generating thermodynamic models of the transcriptome with base-pair specificity. Further, this approach makes it possible to carefully examine the mapping between mutations in binding sites and their corresponding expression profiles, a tool useful not only for better designing MPRAs, but also for exploring regulatory evolution. Author summary With the rapid advancement of sequencing technology, there has been an exponential increase in the amount of data on the genomic sequences of diverse organisms. Nevertheless, deciphering the sequence-phenotype mapping of the genomic data remains a formidable task, especially when dealing with non-coding sequences such as the promoter and its allied regulatory architecture. In current databases, annotations on transcription factor binding sites are sorely lacking, which creates a challenge for developing a systematic theory of transcriptional regulation. To address this gap in knowledge, high-throughput methods such as massively parallel reporter assays have been employed to decipher the regulatory genome. In this work, we make use of thermodynamic models to computationally simulate MPRAs in the context of transcriptional regulation studies and produce synthetic MPRA datasets. We examine how well typical experimental and data analysis procedures of MPRAs are able to recover common regulatory architectures under different sets of experimental and biological parameters. By establishing a dialogue between high-throughput experiments and a physical theory of transcription, our efforts serve to both improve current experimental procedures and enhancing our broader understanding of the sequence-function landscape of regulatory sequences.