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Understanding the evolutionary potential of mutations in gene regulatory networks is essential to furthering the study of evolution and development. However, in multicellular systems, genetic manipulation of regulatory networks in a targeted and high-throughput way remains challenging. In this study, we designed TF-High-Evolutionary (HighEvo), a transcription factor (TF) fused with a base editor (activation-induced deaminase), to continuously induce germline mutations at TF-binding sites across regulatory networks in Drosophila. Populations of flies expressing TF-HighEvo in their germlines accumulated mutations at rates an order of magnitude higher than natural populations. Importantly, these mutations accumulated around the targeted TF-binding sites across the genome, leading to distinct morphological phenotypes consistent with the developmental roles of the tagged TFs. As such, this TF-HighEvo method allows the interrogation of the mutational space of gene regulatory networks at scale and can serve as a powerful reagent for experimental evolution and genetic screens focused on the regulatory genome., Competing Interests: Conflict of Interest The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated., Competing Interests: Conflict of Interest. The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Treatment options for multiple myeloma (MM) at 1st relapse are expanding. The current study compared common 2nd line regimens administered in a real-world setting. MM patients registered in Maccabi health care services and treated with second line therapy during 2014-2020 were evaluated, analyzing factors affecting time to third line therapy (TT3T). The study included 500 MM patients, previously treated with proteasome inhibitor (PI)-based induction. Median age at second line treatment was 68.5 years (IQR: 61.6-76.4). Most patients received a triplet based induction composed of PI (n = 471, 94.2%), with (n = 71) or without IMID (n = 400), followed by second line treatment composed of lenalidomide-dexamethasone (RD) (n = 225, 45%) or lenalidomide-dexamethasone-daratumumab (RD-Dara (n = 104, 20.8%)). Multivariable analysis confirmed treatment type (RD-Dara vs. IMID) to be associated with a lower risk to progress to third line therapy; (HR = 0.5, 95% CI 0.3-0.86, p = 0.012). Within a median follow-up period of 22.5 months (intraquartile range 11.1-39.4 m), median TT3T was not reached in patients receiving RD-Dara vs. 32.4 months (95% CI 18.0-46.8 m) with IMID, 18 months (95% CI 10.4-25.6 m) with IMID-PI and 12.1 months (95% CI 5.6-18.7 m) with PI-based regimen. In contrast, PI vs. IMID-based therapy and increased body weight were associated with a higher likelihood of progression (HR = 2.56 (95% CI 1.49-4.42); HR = 1.43, (95% CI 0.96-2.14), p = 0.08). To conclude, second line therapy with RD-Dara was associated with a significantly longer TT3T compared with IMID-based regimen, longer than obtained with PI-IMID and PI-based regimens, in patients treated outside clinical studies and previously exposed to bortezomib., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cactophilic species of the Drosophila buzzatii cluster (repleta group) comprise an excellent model group to investigate genomic changes underlying adaptation to extreme climate conditions and host plants. In particular, these species form a tractable system to study the transition from chemically simpler breeding sites (like prickly pears of the genus Opuntia) to chemically more complex hosts (columnar cacti). Here, we report four highly contiguous genome assemblies of three species of the buzzatii cluster. Based on this genomic data and inferred phylogenetic relationships, we identified candidate taxonomically restricted genes (TRGs) likely involved in the evolution of cactophily and cactus host specialization. Functional enrichment analyses of TRGs within the buzzatii cluster identified genes involved in detoxification, water preservation, immune system response, anatomical structure development, and morphogenesis. In contrast, processes that regulate responses to stress, as well as the metabolism of nitrogen compounds, transport, and secretion were found in the set of species that are columnar cacti dwellers. These findings are in line with the hypothesis that those genomic changes brought about key mechanisms underlying the adaptation of the buzzatii cluster species to arid regions in South America., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Animal genomes are compartmentalized into insulated regulatory units named topology-associated domains (TADs). TADs insulate gene promoters from enhancers that occupy neighboring TADs. Chromosomal rearrangements that disrupt TAD structure can generate new regulatory interactions between enhancers and promoters that were once separated into different TADs, which might lead to new gene expression patterns. On the one hand, TAD rearrangements are known to cause deleterious phenotypes, but, on the other hand, rearrangements can also create novel expression patterns that may be selected during evolution because they generate advantageous phenotypes. Here, we review recent studies that explore the effects of chromosomal rearrangements and genetic perturbations on TAD structure and gene regulation in the context of development and evolution. We discuss the possible contribution of evolutionary breakpoints (EBRs) that affect TAD structure to the evolution of gene regulation and the phenotype., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Enhancers are regulatory elements of genomes that determine spatio-temporal patterns of gene expression. The human genome contains a vast number of enhancers, which largely outnumber protein-coding genes. Historically, enhancers have been regarded as highly tissue-specific. However, recent evidence has demonstrated that many enhancers are pleiotropic, with activity in multiple developmental contexts. Yet, the extent and impact of pleiotropy remain largely unexplored. In this study we analyzed active enhancers across human organs based on the analysis of both eRNA transcription (FANTOM5 consortium data sets) and chromatin architecture (ENCODE consortium data sets). We show that pleiotropic enhancers are pervasive in the human genome and that most enhancers active in a particular organ are also active in other organs. In addition, our analysis suggests that the proportion of context-specific enhancers of a given organ is explained, at least in part, by the proportion of context-specific genes in that same organ. The notion that such a high proportion of human enhancers can be pleiotropic suggests that small regions of regulatory DNA contain abundant regulatory information and that these regions evolve under important evolutionary constraints., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The cuticle of insects is decorated with non-sensory hairs called trichomes. A few Drosophila species independently lost most of the dorso-lateral trichomes on first instar larvae. Genetic experiments revealed that this naked cuticle phenotype was caused by the evolution of enhancer function at the ovo/shavenbaby (ovo/svb) locus. Here we explore how this discovery catalyzed major new insights into morphological evolution in different developmental contexts, enhancer pleiotropy in gene regulation and the functionality and evolution of the Svb gene regulatory network (GRN). Taken together this highlights the importance of understanding the architecture and evolution of gene regulatory networks in detail and the great potential for further study of the Svb GRN., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Escherichia coli dynamics in urban watersheds are affected by a complex balance among external inputs, niche modulation and genetic variability. To explore the ecological processes influencing E. coli spatial patterns, we analyzed its abundance and phylogenetic structure in water samples from a stream network with heterogeneous urban infrastructure and environmental conditions. Our results showed that environmental and infrastructure variables, such as macrophyte coverage, DIN and sewerage density, mostly explained E. coli abundance. Moreover, main generalist phylogroups A and B1 were found in high proportion, which, together with an observed negative relationship between E. coli abundance and phylogroup diversity, suggests that their dominance might be due to competitive exclusion. Lower frequency phylogroups were associated with sites of higher ecological disturbance, mainly involving simplified habitats, higher drainage infrastructure and septic tank density. In addition to the strong negative relationship between phylogroup diversity and dominance, the occurrence of these phylogroups would be associated with increased facilitated dispersal. Nutrients also contributed to explaining phylogroup distribution. Our study proposes the differential contribution of distinct ecological processes to the patterns of E. coli in an urban watershed, which is useful for the monitoring and management of fecal pollution., (© 2021. The Author(s).)

Introduction: Older adults with cancer are at increased risk of delirium due to age, comorbidities, medications, cognitive impairment, and possibly cancer treatments. However, there is scant information on the risks of delirium with chemotherapy and approaches to prevent or treat it. We performed a systematic review and meta-analysis to summarize available evidence., Materials and Methods: We systematically searched peer-reviewed journal articles in English, French, German, and Dutch from five databases from 1990 to May 2019 to identify studies examining delirium in adult patients receiving chemotherapy. We also attempted to identify delirium risk prediction models and prevention or treatment trials. All reviews and data extraction were performed by two independent reviewers. Summary estimates were derived from random effects models., Results: A total of 23,389 titles and abstracts were screened, and 1272 full-text articles were reviewed. Nineteen articles reported on delirium using an acceptable diagnostic standard. Sample sizes varied from 7 to 324. The incidence of delirium ranged from 0 to 51% (weighted mean 9%, 95% confidence interval 5-16%). In a sensitivity analysis including 122 studies that used terminology suggestive of delirium but did not meet our inclusion criteria, the weighted incidence of delirium was 10% (95% confidence interval 8-12%). Age was not consistently associated with increased delirium risk. No intervention studies to prevent or treat delirium were identified., Conclusions: Delirium may occur in 1 in 11 older adults receiving chemotherapy; however, there were substantial limitations in reported studies. This systemic review highlights key gaps in knowledge, particularly regarding risk factors, prevention, and treatments., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Despite considerable progress in recent decades in dissecting the genetic causes of natural morphological variation, there is limited understanding of how variation within species ultimately contributes to species differences. We have studied patterning of the non-sensory hairs, commonly known as "trichomes," on the dorsal cuticle of first-instar larvae of Drosophila. Most Drosophila species produce a dense lawn of dorsal trichomes, but a subset of these trichomes were lost in D. sechellia and D. ezoana due entirely to regulatory evolution of the shavenbaby (svb) gene. Here, we describe intraspecific variation in dorsal trichome patterns of first-instar larvae of D. virilis that is similar to the trichome pattern variation identified previously between species. We found that a single large effect QTL, which includes svb, explains most of the trichome number difference between two D. virilis strains and that svb expression correlates with the trichome difference between strains. This QTL does not explain the entire difference between strains, implying that additional loci contribute to variation in trichome numbers. Thus, the genetic architecture of intraspecific variation exhibits similarities and differences with interspecific variation that may reflect differences in long-term and short-term evolutionary processes., (© 2020 The Authors. Evolution © 2020 The Society for the Study of Evolution.)

The current paradigm in the field of gene regulation postulates that regulatory information for generating gene expression is organized into modules (enhancers), each containing the information for driving gene expression in a single spatiotemporal context. This modular organization is thought to facilitate the evolution of gene expression by minimizing pleiotropic effects. Here we review recent studies that provide evidence of quite the opposite: (i) enhancers can function in multiple developmental contexts, implying that enhancers can be pleiotropic, (ii) transcription factor binding sites within pleiotropic enhancers are reused in different contexts, and (iii) pleiotropy impacts the structure and evolution of enhancers. Altogether, this evidence suggests that enhancer pleiotropy is pervasive in animal genomes, challenging the commonly held view of modularity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. We compared the expression and function of components between the larval and leg trichome GRNs to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. We found key differences between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. Our work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, our findings have important implications for understanding the pathways and predictability of evolution.

Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. Here, we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. We explored how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 ϕ C31 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., (Copyright © 2017 Stern et al.)

Novel body structures are often generated by the redeployment of ancestral components of the genome. In this issue of Developmental Cell, Glassford et al. (2015) present a thorough analysis of the co-option of a gene regulatory network in the origin of an evolutionary novelty., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The stochastic nature of biochemical processes is a source of variability that influences developmental stability. Developmental instability (DI) is often estimated through fluctuating asymmetry (FA), a parameter that deals with within-individual variation in bilateral structures. A relevant goal is to shed light on how environment, physiology and genotype relate to DI, thus providing a more comprehensive view of organismal development. Using Drosophila melanogaster isogenic lines, we investigated the effect of parental age, parental diet and offspring heterozygosity on DI. In this work, we have uncovered a clear relationship between parental age and offspring asymmetry. We show that asymmetry of the progeny increases concomitantly with parental age. Moreover, we demonstrate that enriching the diet of parents mitigates the effect of age on offspring symmetry. We show as well that increasing the heterozygosity of the progeny eliminates the effect of parental age on offspring symmetry. Taken together, our results suggest that diet, genotype and age of the parents interact to determine offspring DI in wild populations. These findings provide us with an avenue to understand the mechanisms underlying DI., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)