Changes in locus wide repression underlie the evolution of Drosophila abdominal pigmentation.

Changes in gene regulation represent an important path to generate developmental differences affecting anatomical traits. Interspecific divergence in gene expression often results from changes in transcription-stimulating enhancer elements. While gene repression is crucial for precise spatiotemporal expression patterns, the relative contribution of repressive transcriptional silencers to regulatory evolution remains to be addressed. Here, we show that the Drosophila pigmentation gene ebony has mainly evolved through changes in the spatial domains of silencers patterning its abdominal expression. By precisely editing the endogenous ebony locus of D. melanogaster, we demonstrate the requirement of two redundant abdominal enhancers and three silencers that repress the redundant enhancers in a patterned manner. We observe a role for changes in these silencers in every case of ebony evolution observed to date. Our findings suggest that negative regulation by silencers likely has an under-appreciated role in gene regulatory evolution.

A novel role for trithorax in the gene regulatory network for a rapidly evolving fruit fly pigmentation trait.

Animal traits develop through the expression and action of numerous regulatory and realizator genes that comprise a gene regulatory network (GRN). For each GRN, its underlying patterns of gene expression are controlled by cis-regulatory elements (CREs) that bind activating and repressing transcription factors. These interactions drive cell-type and developmental stage-specific transcriptional activation or repression. Most GRNs remain incompletely mapped, and a major barrier to this daunting task is CRE identification. Here, we used an in silico method to identify predicted CREs (pCREs) that comprise the GRN which governs sex-specific pigmentation of Drosophila melanogaster. Through in vivo assays, we demonstrate that many pCREs activate expression in the correct cell-type and developmental stage. We employed genome editing to demonstrate that two CREs control the pupal abdomen expression of trithorax, whose function is required for the dimorphic phenotype. Surprisingly, trithorax had no detectable effect on this GRN's key trans-regulators, but shapes the sex-specific expression of two realizator genes. Comparison of sequences orthologous to these CREs supports an evolutionary scenario where these trithorax CREs predated the origin of the dimorphic trait. Collectively, this study demonstrates how in silico approaches can shed novel insights on the GRN basis for a trait's development and evolution.

Widespread cis- and trans-regulatory evolution underlies the origin, diversification, and loss of a sexually dimorphic fruit fly pigmentation trait.

Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait-building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis-regulatory elements (CREs) that drive their expression (cis-evolution) or within genes for CRE-interacting transcription factors (trans-evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. We investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. We show these CREs to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male-specific pigmentation. We evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. We show that the transgene host frequently determines CRE activity, implicating trans-evolution as a significant factor for this trait's diversity. We validated the gain of dimorphic Bab transcription factor expression as a trans-change contributing to the dimorphic trait. Our findings suggest an amenability to change for the landscape of trans-regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs.

The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSX(F) activate bab expression whereas in males DSX(M) directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

Augmentation of a wound response element accompanies the origin of a Hox-regulated Drosophila abdominal pigmentation trait.

A challenge for evolutionary research is to uncover how new morphological traits evolve the coordinated spatial and temporal expression patterns of genes that govern their formation during development. Detailed studies are often limited to characterizing how one or a few genes contributed to a trait's emergence, and thus our knowledge of how entire GRNs evolve their coordinated expression of each gene remains unresolved. The melanic color patterns decorating the male abdominal tergites of Drosophila (D.) melanogaster evolved in part by novel expression patterns for genes acting at the terminus of a pigment metabolic pathway, driven by cis-regulatory elements (CREs) with distinct mechanisms of Hox regulation. Here, we examined the expression and evolutionary histories of two important enzymes in this pathway, encoded by the pale and Ddc genes. We found that while both genes exhibit dynamic patterns of expression, a robust pattern of Ddc expression specifically evolved in the lineage of fruit flies with pronounced melanic abdomens. Derived Ddc expression requires the activity of a CRE previously shown to activate expression in response to epidermal wounding. We show that a binding site for the Grainy head transcription factor that promotes the ancestral wound healing function of this CRE is also required for abdominal activity. Together with previous findings in this system, our work shows how the GRN for a novel trait emerged by assembling unique yet similarly functioning CREs from heterogeneous starting points.

Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species.

The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting "enhancer" elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony's transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer's activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.

The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. Here, we investigate how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. Our data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-à-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

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.

Generation of a novel wing colour pattern by the Wingless morphogen.

The complex, geometric colour patterns of many animal bodies have important roles in behaviour and ecology. The generation of certain patterns has been the subject of considerable theoretical exploration, however, very little is known about the actual mechanisms underlying colour pattern formation or evolution. Here we have investigated the generation and evolution of the complex, spotted wing pattern of Drosophila guttifera. We show that wing spots are induced by the Wingless morphogen, which is expressed at many discrete sites that are specified by pre-existing positional information that governs the development of wing structures. Furthermore, we demonstrate that the elaborate spot pattern evolved from simpler schemes by co-option of Wingless expression at new sites. This example of a complex design developing and evolving by the layering of new patterns on pre-patterns is likely to be a general theme in other animals.

Recurrent modification of a conserved cis-regulatory element underlies fruit fly pigmentation diversity.

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages.

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation.

Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. Here, we greatly expand this network through an RNAi-screen of 558 transcription factors. We identified 28 genes, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. We show that many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. We found the pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution.

HLA-B*57 elite suppressor and chronic progressor HIV-1 isolates replicate vigorously and cause CD4+ T cell depletion in humanized BLT mice.

UNLABELLED: Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design. IMPORTANCE: Elite suppressors are HIV-1-infected patients who have undetectable levels of viremia despite not being on antiviral drugs. One of the most fundamental questions about this phenomenon involves the mechanism of control. To address this question, we isolated virus from elite suppressors and from HIV-1-infected patients who have the usual progressive disease course. We compared how well the isolates from the two groups of patients replicated in culture and in humanized mice. Our results suggest that elite suppressors are capable of controlling HIV-1 due to the possession of unique host factors rather than infection with defective virus.

Genetic and molecular insights into the development and evolution of sexual dimorphism.

Sexual dimorphism is common throughout the animal kingdom. However, a molecular understanding of how sex-specific traits develop and evolve has been elusive. Recently, substantial progress has been made in elucidating how diverse sex-determination systems are integrated into developmental gene networks. One common theme from these studies is that sex-limited traits and gene expression are produced by the combined action of transcriptional effectors of sex-determination pathways and other transcription factors on target gene cis-regulatory elements. Sex-specific traits evolve by the gain, loss or modification of linkages in the genetic networks regulated by sex-determination transcription factors.

A genetic screen of transcription factors in the Drosophila melanogaster abdomen identifies novel pigmentation genes.

Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical school to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the Spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course, that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources.

The evolution of Bab paralog expression and abdominal pigmentation among Sophophora fruit fly species.

The evolution of gene networks lies at the heart of understanding trait divergence. Intrinsic to development is the dimension of time: a network must be altered during the correct phase of development to generate the appropriate phenotype. One model of developmental network evolution is the origination of dimorphic (male-specific) abdomen pigmentation in the fruit fly subgenus Sophophora. In Drosophila (D.) melanogaster, dimorphic pigmentation is controlled by the dimorphic expression of the paralogous Bab1 and Bab2 transcription factors that repress pigmentation. These expression patterns are thought to have evolved from a monomorphic ancestral state. Here we show that the spatial domain and contrast in dimorphic Bab expression increases during the latter half of pupal development, and this late pupal expression is necessary and sufficient to suppress pigmentation. Late pupal Bab expression was monomorphic for species from basal clades exhibiting monomorphic pigmentation, though dimorphic expression was observed in D. pseudoobscura that represents an intermediate-branching monomorphic clade. Among species from the dimorphic Sophophora clades, Bab expression was dimorphic, but a poor correlation was found between the domains of expression and male pigmentation. Lastly, while Bab paralog co-expression was generally observed, an instance of paralog-specific expression was found, indicating more complex regulatory mechanisms and mutational effects have shaped the evolution of the bab locus. These results highlight the importance of the time and place of Bab expression for pigmentation development and evolution, and suggest that dimorphism evolved early in Sophophora, but diversity in male pigmentation was not further shaped by alterations in Bab expression.

Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations.

Rare human immunodeficiency virus 1-infected individuals, termed elite suppressors (ES), maintain plasma virus levels of <50 copies/ml and normal CD4 counts without therapy. The major histocompatibility complex class I allele group human histocompatibility leukocyte antigen (HLA)-B*57 is overrepresented in this population. Mutations in HLA-B*57-restricted epitopes have been observed in ES, but their significance has remained unclear. Here we investigate the extent and impact of cytotoxic T lymphocyte (CTL) escape mutations in HLA-B*57+ ES. We provide the first direct evidence that most ES experience chronic low level viremia. Sequencing revealed a striking discordance between the genotypes of plasma virus and archived provirus in resting CD4+ T cells. Mutations in HLA-B*57-restricted Gag epitopes were present in all viruses from plasma but were rare in proviruses, suggesting powerful selective pressure acting at these epitopes. Surprisingly, strong CD8+ T cell interferon-gamma responses were detected against some mutant epitopes found in plasma virus, suggesting the development of de novo responses to viral variants. In some individuals, relative CD8+ T cell interleukin-2 responses showed better correlation with the selection observed in vivo. Thus, analysis of low level viremia reveals an unexpectedly high level of CTL escape mutations reflecting selective pressure acting at HLA-B*57-restricted epitopes in ES. Continued viral suppression probably reflects CTL responses against unmutated epitopes and residual or de novo responses against epitopes with escape mutations.

Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila.

Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene.

The independent evolution of morphological similarities is widespread. For simple traits, such as overall body colour, repeated transitions by means of mutations in the same gene may be common. However, for more complex traits, the possible genetic paths may be more numerous; the molecular mechanisms underlying their independent origins and the extent to which they are constrained to follow certain genetic paths are largely unknown. Here we show that a male wing pigmentation pattern involved in courtship display has been gained and lost multiple times in a Drosophila clade. Each of the cases we have analysed (two gains and two losses) involved regulatory changes at the pleiotropic pigmentation gene yellow. Losses involved the parallel inactivation of the same cis-regulatory element (CRE), with changes at a few nucleotides sufficient to account for the functional divergence of one element between two sibling species. Surprisingly, two independent gains of wing spots resulted from the co-option of distinct ancestral CREs. These results demonstrate how the functional diversification of the modular CREs of pleiotropic genes contributes to evolutionary novelty and the independent evolution of morphological similarities.

A case of seronegative HIV-1 infection.

Patients infected with human immunodeficiency virus type 1 (HIV-1) typically seroconvert within weeks of primary infection. In rare cases, patients do not develop antibodies against HIV-1 despite demonstrable infection. We describe here a human leukocyte antigen (HLA)-B*5802-positive individual who presented with acquired immune deficiency syndrome despite repeatedly negative HIV-1 antibody screening test results. Phylogenetic analysis of env clones revealed little sequence diversity, and weak HIV-1-specific CD8(+) T cell responses were present to Gag epitopes. The patient seroconverted after immune reconstitution during receipt of highly active antiretroviral therapy. Lack of an antibody response to HIV-1 is rare and appears to be due to a defect in HIV-1-specific immunity rather than infection with attenuated virus.