In a paper published last November in PLoS Genetics, Dartmouth professor Albert Erives and graduate student Justin Crocker showed that sequential organization of enhancers is critical to gene expression through analysis of the eve Stripe 2 enhancer in Drosophila, although others have debated the functionality of this organization.
For years, researchers have investigated gene regulatory sequences and have theorized that these sequences are a target of natural selection. However, the gene regulatory sequence evolution has been considered less significant than functional protein coding evolution in relation to the evolution of the genome because the organizational aspects of each regulatory sequence are not fully understood.
Gene regulatory sequences function by displaying cis-regulatory modules, sites where DNA sequence-specific factors bind. Understanding the internal functional organization of the binding factors is important because if the way in which the binding elements position and orient themselves is understood, one can better decipher the role of gene regulatory evolution in light of the genomic sequence.
The Even Skipped (eve) Stripe 2 enhancer model of Drosophila melanogaster is the most genetically and biochemically defined out of all of the modules occurring in drosopholids. Thus, it has been the most closely studied module. Research has branched off from Drosophila into Themira, a sespid fly. Because these flies can be studied in a parallel fashion despite the lack of sequence conservation between the eve stripe 2 enhancers of Drosophila melanogaster and Themira putris. According to Crocker and Erives some have jumped to the conclusion that “complex animal regulatory sequences can tolerate nearly complete rearrangement of their transcription factor binding sites.”
Drosophila and Themira branched from a common ancestor about 110 million years ago. It has been assumed that, since so many years have passed since they shared their common ancestor, their sequences are completely scrambled in relation to each other. However, Erives and Crocker report extensive homology, particularly in well-known activator and repressor binding sites, which are claimed to be an exemplar of scrambled enhancer sequences. Therefore, Erives and Crocker concluded that the organization of eve stripe 2 enhancers in the two types of flies “have not diverged enough to rule out organized assembly of higher-order enhancesome complexes at these sequences.”
Erives and Crocker were able to determine the homology between the Themira and Drosophila stripe 2 enhancer sequences on alignment plots. They found large blocks of alignment around 600 base pairs long, almost as long as the entire enhancer. Through careful comparison of the sequences, they were able to find ordered blocks of highly conserved sequences of base pairs with multiple binding sites throughout the enhancer. They also established that the absence of extensive sequence homology is not sufficient to prove the absence of conserved organization of binding sites.
The authors have located seven large blocks of alignment between the Drosophila and Themira eve stripe 2 enhancers. Their findings refute the argument that enhancer organization is unimportant. They show that the enhancers that were said to be scrambled actually proved to be very similar. Further, even if they were scrambled, this is insufficient grounds to rule out the importance of enhancer-wide functional organization of motifs. Erives and Crocker summarize the argument by saying, “The study in question falls short in this regard for 3 reasons: 1) analysis is omitted that is necessary to prove that the sequences are in fact scrambled during evolution; 2) the data, when analyzed according to the standards of the literature, in fact says that the enhancers are highly conservative with respect to specific sites and their long-range organization; and 3) they omitted to reconcile their conclusions with extremely relevant prior literature that goes against their main conclusion.” Thus, they have found that the finer details of the DNA sequence should be scrutinized before making broad generalizations about the organization of individual enhancers.