Synthetic enhancer design by in silico compensatory evolution reveals flexibility and constraint in cis-regulation

Kenneth A Barr,Ah-Ram Kim,Alexandre F Ramos,John Reinitz,Carlos Martinez,Jennifer R Moran

doi:10.1186/s12918-017-0485-2

Abstract

BackgroundModels that incorporate specific chemical mechanisms have been successful in describing the activity of Drosophila developmental enhancers as a function of underlying transcription factor binding motifs. Despite this, the minimum set of mechanisms required to reconstruct an enhancer from its constituent parts is not known. Synthetic biology offers the potential to test the sufficiency of known mechanisms to describe the activity of enhancers, as well as to uncover constraints on the number, order, and spacing of motifs.ResultsUsing a functional model and in silico compensatory evolution, we generated putative synthetic even-skipped stripe 2 enhancers with varying degrees of similarity to the natural enhancer. These elements represent the evolutionary trajectories of the natural stripe 2 enhancer towards two synthetic enhancers designed ab initio. In the first trajectory, spatially regulated expression was maintained, even after more than a third of binding sites were lost. In the second, sequences with high similarity to the natural element did not drive expression, but a highly diverged sequence about half the length of the minimal stripe 2 enhancer drove ten times greater expression. Additionally, homotypic clusters of Zelda or Stat92E motifs, but not Bicoid, drove expression in developing embryos.ConclusionsHere, we present a functional model of gene regulation to test the degree to which the known transcription factors and their interactions explain the activity of the Drosophila even-skipped stripe 2 enhancer. Initial success in the first trajectory showed that the gene regulation model explains much of the function of the stripe 2 enhancer. Cases where expression deviated from prediction indicates that undescribed factors likely act to modulate expression. We also showed that activation driven Bicoid and Hunchback is highly sensitive to spatial organization of binding motifs. In contrast, Zelda and Stat92E drive expression from simple homotypic clusters, suggesting that activation driven by these factors is less constrained. Collectively, the 40 sequences generated in this work provides a powerful training set for building future models of gene regulation.

Highlights

Models that incorporate specific chemical mechanisms have been successful in describing the activity of Drosophila developmental enhancers as a function of underlying transcription factor binding motifs
We showed that homotypic arrays of the activators Zelda (Zld) and Stat92E (Dst) are able to drive expression, but activation driven by Bcd and Hb is sensitive to the spacing, affinity, and orientation of sites
The embryos were subsequently imaged in nuclear cycle 14 (C14), timeclass 6 (T6) [16, 17] for nuclei, lacZ and Eve protein (Fig. 1b)

Summary

Introduction

Models that incorporate specific chemical mechanisms have been successful in describing the activity of Drosophila developmental enhancers as a function of underlying transcription factor binding motifs. The minimum set of mechanisms required to reconstruct an enhancer from its constituent parts is not known. Known as cis-regulatory modules (CRMs), are DNA segments that recruit sets of sequence-specific transcription factors (TFs) in order to control the spatiotemporal expression of genes. These elements are critical in controlling cell fate in development [1] and are under selection [2,3,4]. Deletions of sequences outside the 12 footprinted sites all led to changes in function and additional TFs are required to prevent aberrant expression driven by this enhancer [8]

Methods

Results

Discussion

Conclusion