Predicting the DNA binding specificity of mutated transcription factors using family-level biophysically interpretable machine learning.

Sequence-specific interactions of transcription factors (TFs) with genomic DNA underlie many cellular processes. High-throughput in vitro binding assays coupled with computational analysis have made it possible to accurately define such sequence recognition in a biophysically interpretable yet mechanism-agonistic way for individual TFs. The fact that such sequence-to-affinity models are now available for hundreds of TFs provides new avenues for predicting how the DNA binding specificity of a TF changes when its protein sequence is mutated. To this end, we developed an analytical framework based on a tetrahedron embedding that can be applied at the level of a given structural TF family. Using bHLH as a test case, we demonstrate that we can systematically map dependencies between the protein sequence of a TF and base preference within the DNA binding site. We also develop a regression approach to predict the quantitative energetic impact of mutations in the DNA binding domain of a TF on its DNA binding specificity, and perform SELEX-seq assays on mutated TFs to experimentally validate our results. Our results point to the feasibility of predicting the functional impact of disease mutations and allelic variation in the cell-wide TF repertoire by leveraging high-quality functional information across sets of homologous wild-type proteins.

Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors.

In eukaryotes, members of transcription factor families often exhibit similar DNA binding properties in vitro, yet orchestrate paralog-specific gene regulatory networks in vivo. The serially homologous first (T1) and third (T3) thoracic legs of Drosophila, which are specified by the Hox proteins Scr and Ubx, respectively, offer a unique opportunity to address this paradox in vivo. Genome-wide analyses using epitope-tagged alleles of both Hox loci in the T1 and T3 leg imaginal discs, the precursors to the adult legs and ventral body regions, show that ~8% of Hox binding is paralog-specific. Binding specificity is mediated by interactions with distinct cofactors in different domains: the Hox cofactor Exd acts in the proximal domain and is necessary for Scr to bind many of its paralog-specific targets, while in the distal leg domain, the homeodomain protein Distal-less (Dll) enhances Scr binding to a different subset of loci. These findings reveal how Hox paralogs, and perhaps paralogs of other transcription factor families, orchestrate alternative downstream gene regulatory networks with the help of multiple, context-specific cofactors.

An Atlas of Transcription Factors Expressed in Male Pupal Terminalia of Drosophila melanogaster.

During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have identified network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (https://flyterminalia.pitt.edu/) as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

The evolutionary origination of a novel expression pattern through an extreme heterochronic shift.

The evolutionary origins of morphological structures are thought to often depend upon the redeployment of old genes into new developmental settings. Although many examples of cis-regulatory divergence have shown how pre-existing patterns of gene expression have been altered, only a small number of case studies have traced the origins of cis-regulatory elements that drive new expression domains. Here, we elucidate the evolutionary history of a novel expression pattern of the yellow gene within the Zaprionus genus of fruit flies. We observed a unique pattern of yellow transcript accumulation in the wing disc during the third larval instar, a stage that precedes its typical expression pattern associated with cuticular melanization by about a week. The region of the Zaprionus wing disc that expresses yellow subsequently develops into a portion of the thorax, a tissue for which yellow expression has been reported for several fruit fly species. Tests of GFP reporter transgenes containing the Zaprionus yellow regulatory region revealed that the wing disc pattern arose by changes in the cis-regulatory region of yellow. Moreover, the wing disc enhancer activity of yellow depends upon a short conserved sequence with ancestral thoracic functions, suggesting that the pupal thorax regulatory sequence was genetically reprogrammed to drive expression that commences much earlier during development. These results highlight how novel domains of gene expression may arise by extreme shifts in timing during the origins of novel traits.

Co-option of an Ancestral Hox-Regulated Network Underlies a Recently Evolved Morphological Novelty.

The evolutionary origins of complex morphological structures such as the vertebrate eye or insect wing remain one of the greatest mysteries of biology. Recent comparative studies of gene expression imply that new structures are not built from scratch, but rather form by co-opting preexisting gene networks. A key prediction of this model is that upstream factors within the network will activate their preexisting targets (i.e., enhancers) to form novel anatomies. Here, we show how a recently derived morphological novelty present in the genitalia of D. melanogaster employs an ancestral Hox-regulated network deployed in the embryo to generate the larval posterior spiracle. We demonstrate how transcriptional enhancers and constituent transcription factor binding sites are used in both ancestral and novel contexts. These results illustrate network co-option at the level of individual connections between regulatory genes and highlight how morphological novelty may originate through the co-option of networks controlling seemingly unrelated structures.

Assessing constraints on the path of regulatory sequence evolution.

Structural and functional constraints are known to play a major role in restricting the path of evolution of protein activities. However, constraints acting on evolving transcriptional regulatory sequences, e.g. enhancers, are largely unknown. Recently, we elucidated how a novel expression pattern of the Neprilysin-1 (Nep1) gene in the optic lobe of Drosophila santomea evolved via co-option of existing enhancer activities. Drosophila santomea, which has diverged from Drosophila yakuba by approximately 400 000 years has accumulated four fixed mutations that each contribute to the full activity of this enhancer. Recreating and testing the optic lobe enhancer of the ancestor of D. santomea and D. yakuba revealed that the strong D. santomea enhancer activity evolved from a weak ancestral activity. Because each mutation on the path from the D. yakuba/santomea ancestor to modern-day D. santomea contributes to the newly derived optic lobe enhancer activity, we sought here to use this system to study the path of evolution of enhancer sequences. We inferred likely paths of evolution of this enhancer by observing the transcriptional output of all possible intermediate steps between the ancestral D. yakuba/santomea enhancer and the modern D. santomea enhancer. Many possible paths had epistatic and cooperative effects. Furthermore, we found that several paths significantly increased ectopic transcriptional activity or affected existing enhancer activities from which the novel activity was co-opted. We suggest that these attributes highlight constraints that guide the path of evolution of enhancers.

Partial agonist, but not pure antiestrogens stimulate doming, a marker of normal differentiation, in the MCF-7 breast cancer cell line.

UNLABELLED: Abstract Background: The mechanisms by which tamoxifen inhibits breast tumor growth are not completely understood. Partial agonist antiestrogens such as tamoxifen may cause the estrogen receptor (ER) to interact with genes different from those activated by ER bound to estradiol. Doming is a property often associated with, and considered a marker of, differentiation in mammary epithelial cells in culture. This study compared the ability of pure and partial agonist antiestrogens to stimulate doming. MATERIALS AND METHODS: MCF-7 cells grown in medium with 10% calf serum were treated with antiestrogens. Domes were counted in three rows (width of the 4× field) across the flask. RESULTS: Three partial agonist antiestrogens [4-hydroxytamoxifen (OHT), H1285 and RU 39,411] caused dome formation. None of the pure antiestrogens tested (ICI 164,384, ICI 182,780 and RU 58,668) caused doming. Doming was stimulated in a dose-dependent manner starting at 1 nM OHT with maximum stimulation at 10-100 nM. Estradiol did not stimulate doming, but blocked doming at 1%-10% of the OHT concentration. Trichostatin A (TSA) reduced the level of estrogen receptor alpha (ERα) and adding it 24 h before adding OHT prevented dome formation. CONCLUSIONS: OHT and the other partial agonist antiestrogens appear to act through the ER to stimulate doming. The ability of tamoxifen to induce a marker of differentiation may play a role in its inhibition of breast tumors. If so, then the fact that other partial agonist antiestrogens share this ability, but that pure antiestrogens lack it, may be an important consideration in developing new antiestrogens for breast cancer therapy.