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1. Conserved and novel enhancers in the Aedes aegypti single-minded locus recapitulate embryonic ventral midline gene expression

   https://doi.org/10.1371/journal.pgen.1010891  

   Schember, Isabella; Reid, William; Sterling-Lentsch, Geyenna; Halfon, Marc S (April 2024, PLOS Genetics)

   Cadigan, Ken M (Ed.)

   Transcriptional cis-regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A.aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves. 

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2. A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer

   https://doi.org/10.1371/journal.pone.0311120  

   Waite, Jack B; Boytz, RuthMabel; Traeger, Alexis R; Lind, Torrey M; Lumbao-Conradson, Koya; Torigoe, Sharon E (September 2024, PLOS ONE)

   Vall-llosera_Camps, Miquel (Ed.)

   Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene,Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the threeKlf4enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site inKlf4enhancer E2 rescued enhancer function andKlf4expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities ofKlf4enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency. 

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3. Differential modularity of the mammalian Engrailed 1 enhancer network directs sweat gland development

   https://doi.org/10.1371/journal.pgen.1010614  

   Aldea, Daniel; Kokalari, Blerina; Atsuta, Yuji; Dingwall, Heather L.; Zheng, Ying; Nace, Arben; Cotsarelis, George; Kamberov, Yana G. (February 2023, PLOS Genetics)

   Beier, David R. (Ed.)

   Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of Engrailed 1(EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species’ uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits. 

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4. A cis ‐regulatory change underlying the motor neuron‐specific loss of Ebf expression in immotile tunicate larvae

   https://doi.org/10.1111/ede.12364  

   Lowe, Elijah K.; Racioppi, Claudia; Peyriéras, Nadine; Ristoratore, Filomena; Christiaen, Lionel; Swalla, Billie J.; Stolfi, Alberto (December 2020, Evolution & Development)

   Abstract Many species in the tunicate family Molgulidae have independently lost their swimming larval form and instead develop as tailless, immotile larvae. These larvae do not develop structures that are essential for swimming such as the notochord, otolith, and tail muscles. However, little is known about neural development in these nonswimming larvae. Here, we studied the patterning of the Motor Ganglion (MG) ofMolgula occulta, a nonswimming species. We found that spatial patterns of MG neuron regulators in this species are conserved, compared with species with swimming larvae, suggesting that the gene networks regulating their expression are intact despite the loss of swimming. However, expression of the key motor neuron regulatory geneEbf (Collier/Olf/EBF)was reduced in the developing MG ofM. occultawhen compared with molgulid species with swimming larvae. This was corroborated by measuring allele‐specific expression ofEbfin hybrid embryos from crosses ofM. occultawith the swimming speciesM. oculata. Heterologous reporter construct assays in the model tunicate speciesCiona robustarevealed a specificcis‐regulatory sequence change that reduces expression ofEbfin the MG, but not in other cells. Taken together, these data suggest that MG neurons are still specified inM. occultalarvae, but their differentiation might be impaired due to reduction ofEbfexpression levels. 

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5. Repeated shifts in sociality are associated with fine-tuning of highly conserved and lineage-specific enhancers in a socially flexible bee

   https://doi.org/10.1101/2024.08.15.608154  

   Jones, Beryl M; Webb, Andrew E; Geib, Scott M; Sim, Sheina; Schweizer, Rena M; Branstetter, Michael G; Evans, Jay D; Kocher, Sarah D (August 2024, bioRxiv)

   Comparative genomic studies of social insects suggest that changes in gene regulation are associated with evolutionary transitions in social behavior, but the activity of predicted regulatory regions has not been tested empirically. We used STARR-seq, a high-throughput enhancer discovery tool, to identify and measure the activity of enhancers in the socially variable sweat bee,Lasioglossum albipes. We identified over 36,000 enhancers in theL. albipesgenome from three social and three solitary populations. Many enhancers were identified in only a subset ofL. albipespopulations, revealing rapid divergence in regulatory regions within this species. Population-specific enhancers were often proximal to the same genes across populations, suggesting compensatory gains and losses of regulatory regions may preserve gene activity. We also identified 1182 enhancers with significant differences in activity between social and solitary populations, some of which are conserved regulatory regions across species of bees. These results indicate that social trait variation inL. albipesis driven both by the fine-tuning of ancient enhancers as well as lineage-specific regulatory changes. Combining enhancer activity with population genetic data revealed variants associated with differences in enhancer activity and identified a subset of differential enhancers with signatures of selection associated with social behavior. Together, these results provide the first empirical map of enhancers in a socially flexible bee and highlight links between cis-regulatory variation and the evolution of social behavior. 

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