A Closer Look at the eve Stripe 2 Enhancers of Drosophila and Themira

Gene regulatory sequences have been investigated and/or proposed to be important targets of natural selection during animal evolution [1]–[14]. However, much controversy has been generated by the contention that they are not likely to be as important as functional protein-coding evolution given the low number of such examples established to date [15],[16]. However, an important obstacle in identifying such sequences is our lack of understanding of the organizational basis for such sequences. Such an understanding could allow the rapid identification and annotation of gene regulatory functions in sequenced genomes. Gene regulatory sequences function by displaying clusters of sites for DNA sequence-specific binding factors. Such clusters are called cis-regulatory modules (CRMs), of which the transcriptional enhancers constitute a large and important class. The degree to which the constituent binding elements of enhancers are necessarily organized by position, orientation, and relative spacing in order to function will dictate the constraints governing enhancer evolution. Thus, the internal functional organization of enhancers is important for understanding the mode and tempo of gene regulatory evolution as well as for deciphering and annotating genomic sequences. Arguably, no other metazoan cis-regulatory module has yet been as genetically and biochemically defined as the even-skipped (eve) stripe 2 enhancer module of Drosophila melanogaster [17]–[23]. For this reason, this module has been intensely studied from a phylogenetic perspective amongst drosophilids [24]–[27]. These phylogenetic studies of the eve stripe 2 enhancer have now been extended into Themira, a sepsid fly [28]. This latest study is used to make a central claim that a lack of sequence conservation between the eve stripe 2 enhancers of Drosophila melanogaster and Themira putris suggests that “complex animal regulatory sequences can tolerate nearly complete rearrangement of their transcription factor binding sites”. Thus, this study is being interpreted to reach conclusions addressing an important ongoing debate on the degree of functional organization of enhancers [29]. The results of this debate therefore impact the much larger discussion on the genetic loci of evolution [15],[16]. Both Drosophila and Themira are acalyptrate flies and last shared a common ancestor at least ∼110 Mya, and so it is suggested that this distance is long enough for the sequences to be completely scrambled in divergent organisms still sharing a similar embryonic patterning system. While the sepsid study presents an informative taxonomic collection of an evolving enhancer, this study falls short in critically testing the claim that enhancer organization is not important. Moreover, here we report that we find extensive homology in what is claimed to be an exemplar of scrambled enhancer sequences. As explained below, these ordered blocks of homology encompass well-known activator and repressor binding sites. Thus, the organization of Acalyptratae eve stripe 2 enhancers has not diverged enough to rule out organized assembly of higher-order enhancesome complexes at these sequences. Extensive Homology in the eve Stripe 2 Enhancers of Drosophila and Themira We first began by graphing the Themira and Drosophila stripe 2 enhancer sequences on two-dimensional sequence alignment plots (Figure 1). Such a dot plot or graphic matrix shows all regions of similarity between two sequences [30]. Such an alignment is helpful for visualizing possible insertions, deletions, rearrangements, inversions, repeats, and overall homology, without being constrained by global alignments. We also computed the same dot plot using the reverse complement of one of the sequences (Figure 1B and 1E). In addition to showing similar sequences that happen to occur in the opposite orientation, graphing the reverse complement serves as an internal negative control for conservation of serial blocks of sequence. Here, we report that when we graph the eve stripe 2 enhancers in parallel orientations, we see large blocks of alignment spanning ∼600 bp, almost the entire length of the enhancer (Figure 1A). These blocks are larger and more numerous compared to the number and types of alignable blocks achieved when we align them in anti-parallel orientation, i.e., when we plot against the reverse complement of one of the sequences (compare Figure 1A and 1D versus 1B and 1E, or see score distributions in 1C and 1F, respectively). We made such plots for two different thresholds that correspond to an ∼14 bp length of alignment that would encompass most binding sites (Figure 1A–1C) as well as a more extensive ∼20 bp length of alignment (Figure 1D–1F). At the more stringent level, most of the alignments in the anti-parallel direction are lost (Figure 1E). However, a clear identity line of ordered blocks of conservation is visible in the parallel alignment (Figure 1D). Thus, there exists ordered blocks of highly conserved sequence of a length consistent with multiple binding sites spanning the length of the enhancer. Figure 1 Two-dimensional dot plots of the eve stripe 2 enhancers of Drosophila and Themira. The Drosophila/Themira study of an embryonic enhancer of the anterior posterior (A/P) axis could have been better informed by considering the Drosophila/Anopheles study of an embryonic enhancer of the dorsal/ventral axis (D/V) [31]. This study analyzed homologous vnd neuroectodermal enhancers from both Drosophila and the mosquito Anopheles, which last shared a common ancestor at least ∼250 Mya (Figure 2A). This study shows that core cis-elements are organized in a similar architectural plan (Figure 2B). Moreover, this conserved organization was present in non-homologous neuroectodermal enhancers that had evolved in parallel at other loci [31],[32]. However, the Drosophila and Anopheles vnd enhancers are so scrambled that it is difficult to find any evidence of serial sequence homology unlike the Drosophila/Themira pair (Figure 2C and 2D). This is consistent with the additional ∼140 My of divergence between Acalyptratae and mosquitoes on top of the ∼110 My of divergence between the Drosophila and Themira (Figure 2A). Figure 2 Evolutionary scrambling at the Drosophila and Anopheles vnd neuroectoderm enhancers (NEEs). The lesson in the mosquito example that should have informed the sepsid eve stripe 2 study is that the absence of extensive sequence homology is not indicative of the absence of conserved organization of binding sites. Therefore, a simple claim that an enhancer is scrambled is insufficient grounds to rule out functional organization of sites. However, in this particular case, the sepsid enhancer is actually more conserved than the Anopheles enhancer relative to each of their Drosophila orthologs (compare graphs and score in Figures 1 and ​and2).2). Below we show that these blocks of alignment in Acalyptratae sequences correspond to known transcription factor binding sites.