Justin Crocker

Evolution Acts on Enhancer Organization to Fine-Tune Gradient Threshold Readouts

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

TALE-mediated modulation of transcriptional enhancers in vivo

We tested whether transcription activator–like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

Dynamic evolution of precise regulatory encodings creates the clustered site signature of enhancers

Concentration gradients of morphogenic proteins pattern the embryonic axes of Drosophila by activating different genes at different concentrations. The neurogenic ectoderm enhancers (NEEs) activate different genes at different threshold levels of the Dorsal (Dl) morphogen, which patterns the dorsal/ventral axis. NEEs share a unique arrangement of highly constrained DNA-binding sites for Dl, Twist (Twi), Snail (Sna) and Suppressor of Hairless (Su(H)), and encode the threshold variable in the precise length of DNA that separates one well-defined Dl element from a Twi element. However, NEEs also possess dense clusters of variant Dl sites. Here, we show that these increasingly variant sites are eclipsed relic elements, which were superseded by more recently evolved threshold encodings. Given the divergence in egg size during Drosophila lineage evolution, the observed characteristic clusters of divergent sites indicate a history of frequent selection for changes in threshold responses to the Dl morphogen gradient and confirm the NEE structure/function model.

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.

The Soft Touch: Low-Affinity Transcription Factor Binding Sites in Development and Evolution.

Transcription factor proteins regulate gene expression by binding to specific DNA regions. Most studies of transcription factor binding sites have focused on the highest affinity sites for each factor. There is abundant evidence, however, that binding sites with a range of affinities, including very low affinities, are critical to gene regulation. Here, we present the theoretical and experimental evidence for the importance of low-affinity sites in gene regulation and development. We also discuss the implications of the widespread use of low-affinity sites in eukaryotic genomes for robustness, precision, specificity, and evolution of gene regulation.

The Soft Touch

Transcription factor proteins regulate gene expression by binding to specific DNA regions. Most studies of transcription factor binding sites have focused on the highest affinity sites for each factor. There is abundant evidence, however, that binding sites with a range of affinities, including very low affinities, are critical to gene regulation. Here, we present the theoretical and experimental evidence for the importance of low-affinity sites in gene regulation and development. We also discuss the implications of the widespread use of low-affinity sites in eukaryotic genomes for robustness, precision, specificity, and evolution of gene regulation.

Nuclear microenvironments modulate transcription from low-affinity enhancers

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

Genetic and Transgenic Reagents for Drosophila simulans , D. mauritiana , D. yakuba , D. santomea and D. virilis

Species of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. However, studies in these species have been limited by a paucity of genetic and transgenic reagents. Here, we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein (EYFP) gene expressed in the eyes and a ϕC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

Accurate and Sensitive Quantification of Protein-DNA Binding Affinity

Significance One-tenth of human genes produce proteins called transcription factors (TFs) that bind to our genome and read the local DNA sequence. They work together to regulate the degree to which each gene is expressed. The affinity with which DNA is bound by a particular TF can vary more than a thousand-fold with different DNA sequences. This study presents the first computational method able to quantify the sequence-affinity relationship almost perfectly over the full affinity range. It achieves this by analyzing data from experiments that use massively parallel DNA sequencing to comprehensively probe protein–DNA interactions. Strikingly, it can accurately predict the effect in vivo of DNA mutations on gene expression levels in fly embryos even for very-low-affinity binding sites. Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Visualizing long-range enhancer–promoter interaction

A new study uses multicolor live imaging to simultaneously visualize enhancer–promoter interaction and transcription in Drosophila embryos.