Modification of cis-regulatory sequences is an important means by which developmental processes can evolve and generate changes in patterning and morphology. Understanding how these cis-regulatory enhancer sequences control gene expression is critical not only for understanding the mechanistic basis of development, but also for understanding how the systems are modified or evolve over time.
In this work I describe the discovery and characterization of many new enhancers that regulate genes for which a similar enhancer driving a similar expression pattern was previously known; these are "shadow" enhancers. The genes examined are critical developmental control genes responsible for the patterning and subdivision of the early Drosophila embryo. At first glance, many of these enhancers seem redundant, but are found to be evolutionarily conserved, suggesting a role in fitness and specific developmental function.
Sufficiently controlled tests of shadow enhancer function required the development and use of techniques such as bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging. Recombineering allowed the precise modification of large genomic regions and an unprecedented level of control in evaluating enhancer function when combined with the use of reporters and genetic rescue experiments. The ability to accurately quantify gene expression was aided by the development of a reporter gene containing an intron, allowing clear visualization of sites of active transcription. These tools facilitated the evaluation of shadow enhancer function.
Evidence is presented which shows that in some cases one enhancer of the shadow enhancer pair can be removed without disrupting core function of the associated gene under normal developmental conditions. The system breaks down, however, under stress such as elevated temperatures during development, but only when one enhancer is missing. These results suggest that shadow enhancers represent a novel mechanism for the canalization of gene expression in varying environmental conditions and genetic backgrounds.
It is shown that in several cases shadow enhancers help ensure complete patterns of transcriptional activation across domains of gene expression. We present a mechanistic model in which increasing the number of enhancers for a given pattern helps ensure proper transcriptional state even allowing for stochastic interruptions of underlying molecular interactions such as enhancer/promoter looping or activator/repressor binding. It is suggested that shadow enhancers play a key role in ensuring robustness and reliability of gene expression patterns in development.
We propose a model whereby cryptic duplication events lead to the birth of shadow enhancers. Such enhancers can provide immediate value for fitness, for example, by ensuring robustness in response to environmental or genetic variations. The second enhancer might facilitate the evolution of novel patterns of gene expression, or become incorporated into the core developmental machinery to produce precise and rapid patterns of gene expression.