What does it take to evolve an enhancer?A simulation-based study of factors influencing the emergence of combinatorial regulation

There is widespread interest today in understanding enhancers, which are regulatory elements typically harboring several transcription factor binding sites and mediating the combinatorial effect of transcription factors on gene expression. The evolution of enhancers poses interesting unanswered questions, for example, the evolutionary time taken for a typical enhancer to emerge or the factors shaping its evolution. Existing approaches to cis-regulatory evolution have often ignored the combinatorial nature and varied biochemical mechanisms of gene regulation encoded in enhancers. We report on our investigation of enhancer evolution through the use of PEBCRES, a framework for evolutionary simulation of enhancers that employs amechanistic and well-supported sequence-to-expressionmodel to assign fitness to the evolving enhancer genotype.Weestimated the time necessary to evolve, from genomic background, enhancers capable of driving complex gene expression patterns similar to those involved in early development in Drosophila.Wefound the time-to-evolve to range between 0.5 and 10 Myr, and to vary greatly with the target expression pattern, complexity of the real enhancer known to encode that pattern, and the strength of input fromspecific transcription factors. To our knowledge, this is the first estimate ofwaiting times for realistic enhancers to evolve. The in silico evolved enhancers had, with a few interesting exceptions, site compositions similar to those seen in real enhancers for the same patterns. Our simulations also revealed that certain features of an enhancer might evolve not due to their biological function but as AIDS to the evolutionary process itself.