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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.)