Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity.

Eileen Em Furlong,Charles Girardot,E Hilary Gustafson,Pierre Khoueiry,Lucia Ciglar,Saurabh Sinha,Pei-Chen Peng

doi:10.7554/elife.28440

Abstract

Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.

Highlights

Transcription factors (TFs) act largely through enhancer elements - modular sequences that instruct genes when and where to be expressed (Levine and Davidson, 2005; Slattery et al, 2014; Spitz and Furlong, 2012) and provide robustness and precision to developmental programs (Cannavoet al., 2016; Frankel et al, 2010; Perry et al, 2010)
We recently proposed the TF collective model, where TFs bind to enhancers cooperatively through a combination of protein::DNA and protein::protein interactions allowing for a flexible combination of TF’s binding sites (TFBSs)
The evolutionary properties of TF function can be considered at two levels; (1) the relationship between sequence divergence and TF occupancy and (2) the relationship between TF occupancy divergence and enhancer activity

Summary

Introduction

Transcription factors (TFs) act largely through enhancer elements - modular sequences that instruct genes when and where to be expressed (Levine and Davidson, 2005; Slattery et al, 2014; Spitz and Furlong, 2012) and provide robustness and precision to developmental programs (Cannavoet al., 2016; Frankel et al, 2010; Perry et al, 2010). The loss or gain of TF binding sites in the otx enhancer in ascidians (Oda-Ishii et al, 2005), the Endo 16 enhancer in sea urchins (Balhoff and Wray, 2005), and the eve enhancer in Drosophila (Ludwig et al, 1998), for example, have only marginal effects on enhancer activity. This often-surprising gap between changes in genotype and phenotype in developmental contexts highlights the current challenge in understanding both enhancer function and evolutionary constraints

Methods

Results

Conclusion