Bradysia (Sciara) coprophila larvae up-regulate DNA repair pathways and down-regulate developmental regulators in response to ionizing radiation.

The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera.

Transvection between nonallelic genomic positions in Drosophila.

In Drosophila, pairing of maternal and paternal homologous chromosomes can permit trans-interactions between enhancers on one homolog and promoters on another, an example of transvection. Although trans-interactions have been observed at many loci in the Drosophila genome and in other organisms, the parameters that govern enhancer action in trans remain poorly understood. Using a transgenic reporter system, we asked whether enhancers and promoters at nonallelic, but nearby, genomic positions can communication in trans. Using one transgenic insertion carrying the synthetic enhancer GMR and another nearby insertion carrying the hsp70 promoter driving a fluorescent reporter, we show that transgenes separated by 2.6 kb of linear distance can support enhancer action in trans at the 53F8 locus. Furthermore, transvection between the nonallelic insertions can be augmented by a small deletion flanking one insert, likely via changes to the paired configuration of the homologs. Subsequent analyses of other insertions in 53F8 that carry different transgenic sequences demonstrate that the capacity to support transvection between nonallelic sites varies greatly, suggesting that factors beyond the linear distance between insertion sites play an important role. Finally, analysis of transvection between nearby nonallelic sites at other genomic locations shows evidence of position effects, where one locus supported GMR action in trans over a linear distance of over 10 kb, whereas another locus showed no evidence of transvection over a span <200 bp. Overall, our data demonstrate that transvection between nonallelic sites represents a complex interplay between genomic context, interallelic distance, and promoter identity.

Altering enhancer-promoter linear distance impacts promoter competition in cis and in trans.

In Drosophila, pairing of maternal and paternal homologs can permit trans-interactions between enhancers on one homolog and promoters on another, an example of a phenomenon called transvection. When chromosomes are paired, promoters in cis and in trans to an enhancer can compete for the enhancer's activity, but the parameters that govern this competition are as yet poorly understood. To assess how the linear spacing between an enhancer and promoter can influence promoter competition in Drosophila, we employed transgenic constructs wherein the eye-specific enhancer GMR is placed at varying distances from a heterologous hsp70 promoter driving a fluorescent reporter. While GMR activates the reporter to a high degree when the enhancer and promoter are spaced by a few hundred base pairs, activation is strongly attenuated when the enhancer is moved 3 kb away. By examining transcription of endogenous genes near the point of transgene insertion, we show that linear spacing of 3 kb between GMR and the hsp70 promoter results in elevated transcription of neighboring promoters, suggesting a loss of specificity between the enhancer and its intended transgenic target promoter. Furthermore, increasing spacing between GMR and hsp70 by just 100 bp can enhance transvection, resulting in increased activation of a promoter on a paired homolog at the expense of a promoter in cis to the enhancer. Finally, cis-/trans-promoter competition assays in which one promoter carries mutations to key core promoter elements show that GMR will skew its activity toward a wild-type promoter, suggesting that an enhancer is in a balanced competition between its potential target promoters in cis and in trans.

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila.

Three-dimensional eukaryotic genome organization provides the structural basis for gene regulation. In Drosophila melanogaster, genome folding is characterized by somatic homolog pairing, where homologous chromosomes are intimately paired from end to end; however, how homologs identify one another and pair has remained mysterious. Recently, this process has been proposed to be driven by specifically interacting 'buttons' encoded along chromosomes. Here, we turned this hypothesis into a quantitative biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We tested our model using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. We show solid agreement between model predictions and experiments in the pairing dynamics of individual homologous loci. Our results strongly support a button-based mechanism of somatic homolog pairing in Drosophila and provide a theoretical framework for revealing the molecular identity and regulation of buttons.

Homolog Pairing at the Push of a Button.

Homologous chromosomes pair in somatic cells in Drosophila, but how this occurs is poorly understood. In this issue of Developmental Cell, Viets et al. (2019) show that proteins and chromatin structure mediate pairing and argue against a DNA sequence-based mechanism.

Position Effects Influence Transvection in Drosophila melanogaster.

Transvection is an epigenetic phenomenon wherein regulatory elements communicate between different chromosomes in trans, and is thereby dependent upon the three-dimensional organization of the genome. Transvection is best understood in Drosophila, where homologous chromosomes are closely paired in most somatic nuclei, although similar phenomena have been observed in other species. Previous data have supported that the Drosophila genome is generally permissive to enhancer action in trans, a form of transvection where an enhancer on one homolog activates gene expression from a promoter on a paired homolog. However, the capacity of different genomic positions to influence the quantitative output of transvection has yet to be addressed. To investigate this question, we employed a transgenic system that assesses and compares enhancer action in cis and in trans at defined chromosomal locations. Using the strong synthetic eye-specific enhancer GMR, we show that loci supporting strong cis-expression tend to support robust enhancer action in trans, whereas locations with weaker cis-expression show reduced transvection in a fluorescent reporter assay. Our subsequent analysis is consistent with a model wherein the chromatin state of the transgenic insertion site is a primary determinant of the degree to which enhancer action in trans will be supported, whereas other factors such as locus-specific variation in somatic homolog pairing are of less importance in influencing position effects on transvection.

Two modes of transvection at the eyes absent gene of Drosophila demonstrate plasticity in transcriptional regulatory interactions in cis and in trans.

For many genes, proper gene expression requires coordinated and dynamic interactions between multiple regulatory elements, each of which can either promote or silence transcription. In Drosophila, the complexity of the regulatory landscape is further complicated by the tight physical pairing of homologous chromosomes, which can permit regulatory elements to interact in trans, a phenomenon known as transvection. To better understand how gene expression can be programmed through cis- and trans-regulatory interactions, we analyzed transvection effects for a collection of alleles of the eyes absent (eya) gene. We find that trans-activation of a promoter by the eya eye-specific enhancers is broadly supported in many allelic backgrounds, and that the availability of an enhancer to act in trans can be predicted based on the molecular lesion of an eya allele. Furthermore, by manipulating promoter availability in cis and in trans, we demonstrate that the eye-specific enhancers of eya show plasticity in their promoter preference between two different transcriptional start sites, which depends on promoter competition between the two potential targets. Finally, we show that certain alleles of eya demonstrate pairing-sensitive silencing resulting from trans-interactions between Polycomb Response Elements (PREs), and genetic and genomic data support a general role for PcG proteins in mediating transcriptional silencing at eya. Overall, our data highlight how eya gene regulation relies upon a complex but plastic interplay between multiple enhancers, promoters, and PREs.

The Capacity to Act in Trans Varies Among Drosophila Enhancers.

The interphase nucleus is organized such that genomic segments interact in cis, on the same chromosome, and in trans, between different chromosomes. In Drosophila and other Dipterans, extensive interactions are observed between homologous chromosomes, which can permit enhancers and promoters to communicate in trans Enhancer action in trans has been observed for a handful of genes in Drosophila, but it is as yet unclear whether this is a general property of all enhancers or specific to a few. Here, we test a collection of well-characterized enhancers for the capacity to act in trans Specifically, we tested 18 enhancers that are active in either the eye or wing disc of third instar Drosophila larvae and, using two different assays, found evidence that each enhancer can act in trans However, the degree to which trans-action was supported varied greatly between enhancers. Quantitative analysis of enhancer activity supports a model wherein an enhancer's strength of transcriptional activation is a major determinant of its ability to act in trans, but that additional factors may also contribute to an enhancer's trans-activity. In sum, our data suggest that a capacity to activate a promoter on a paired chromosome is common among Drosophila enhancers.

Transvection-based gene regulation in Drosophila is a complex and plastic trait.

Transvection, a chromosome pairing-dependent form of trans-based gene regulation, is potentially widespread in the Drosophila melanogaster genome and varies across cell types and within tissues in D. melanogaster, characteristics of a complex trait. Here, we demonstrate that the trans-interactions at the Malic enzyme (Men) locus are, in fact, transvection as classically defined and are plastic with respect to both genetic background and environment. Using chromosomal inversions, we show that trans-interactions at the Men locus are eliminated by changes in chromosomal architecture that presumably disrupt somatic pairing. We further show that the magnitude of transvection at the Men locus is modified by both genetic background and environment (temperature), demonstrating that transvection is a plastic phenotype. Our results suggest that transvection effects in D. melanogaster are shaped by a dynamic interplay between environment and genetic background. Interestingly, we find that cis-based regulation of the Men gene is more robust to genetic background and environment than trans-based. Finally, we begin to uncover the nonlocal factors that may contribute to variation in transvection overall, implicating Abd-B in the regulation of Men in cis and in trans in an allele-specific and tissue-specific manner, driven by differences in expression of the two genes across genetic backgrounds and environmental conditions.

Captured segment exchange: a strategy for custom engineering large genomic regions in Drosophila melanogaster.

Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition-site insertions can be "captured" as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50-kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition-site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.

A genome-wide screen identifies genes that affect somatic homolog pairing in Drosophila.

In Drosophila and other Dipterans, homologous chromosomes are in close contact in virtually all nuclei, a phenomenon known as somatic homolog pairing. Although homolog pairing has been recognized for over a century, relatively little is known about its regulation. We performed a genome-wide RNAi-based screen that monitored the X-specific localization of the male-specific lethal (MSL) complex, and we identified 59 candidate genes whose knockdown via RNAi causes a change in the pattern of MSL staining that is consistent with a disruption of X-chromosomal homolog pairing. Using DNA fluorescent in situ hybridization (FISH), we confirmed that knockdown of 17 of these genes has a dramatic effect on pairing of the 359 bp repeat at the base of the X. Furthermore, dsRNAs targeting Pr-set7, which encodes an H4K20 methyltransferase, cause a modest disruption in somatic homolog pairing. Consistent with our results in cultured cells, a classical mutation in one of the strongest candidate genes, pebble (pbl), causes a decrease in somatic homolog pairing in developing embryos. Interestingly, many of the genes identified by our screen have known roles in diverse cell-cycle events, suggesting an important link between somatic homolog pairing and the choreography of chromosomes during the cell cycle.

Comparing enhancer action in cis and in trans.

Studies from diverse systems have shown that distinct interchromosomal interactions are a central component of nuclear organization. In some cases, these interactions allow an enhancer to act in trans, modulating the expression of a gene encoded on a separate chromosome held in close proximity. Despite recent advances in uncovering such phenomena, our understanding of how a regulatory element acts on another chromosome remains incomplete. Here, we describe a transgenic approach to better understand enhancer action in trans in Drosophila melanogaster. Using phiC31-based recombinase-mediated cassette exchange (RMCE), we placed transgenes carrying combinations of the simple enhancer GMR, a minimal promoter, and different fluorescent reporters at equivalent positions on homologous chromosomes so that they would pair via the endogenous somatic pairing machinery of Drosophila. Our data demonstrate that the enhancer GMR is capable of activating a promoter in trans and does so in a variegated pattern, suggesting stochastic interactions between the enhancer and the promoter when they are carried on separate chromosomes. Furthermore, we quantitatively assessed the impact of two concurrent promoter targets in cis and in trans to GMR, demonstrating that each promoter is capable of competing for the enhancer's activity, with the presence of one negatively affecting expression from the other. Finally, the single-cell resolution afforded by our approach allowed us to show that promoters in cis and in trans to GMR can both be activated in the same nucleus, implying that a single enhancer can share its activity between multiple promoter targets carried on separate chromosomes.

Simplified Insertion of Transgenes Onto Balancer Chromosomes via Recombinase-Mediated Cassette Exchange.

Balancer chromosomes are critical tools for Drosophila genetics. Many useful transgenes are inserted onto balancers using a random and inefficient process. Here we describe balancer chromosomes that can be directly targeted with transgenes of interest via recombinase-mediated cassette exchange (RMCE).

The twin spot generator for differential Drosophila lineage analysis.

In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.

A genomewide survey argues that every zygotic gene product is dispensable for the initiation of somatic homolog pairing in Drosophila.

Studies from diverse organisms show that distinct interchromosomal interactions are associated with many developmental events. Despite recent advances in uncovering such phenomena, our understanding of how interchromosomal interactions are initiated and regulated is incomplete. During the maternal-to-zygotic transition (MZT) of Drosophila embryogenesis, stable interchromosomal contacts form between maternal and paternal homologous chromosomes, a phenomenon known as somatic homolog pairing. To better understand the events that initiate pairing, we performed a genomewide assessment of the zygotic contribution to this process. Specifically, we took advantage of the segregational properties of compound chromosomes to generate embryos lacking entire chromosome arms and, thus, all zygotic gene products derived from those arms. Using DNA fluorescence in situ hybridization (FISH) to assess the initiation of pairing at five separate loci, this approach allowed us to survey the entire zygotic genome using just a handful of crosses. Remarkably, we found no defect in pairing in embryos lacking any chromosome arm, indicating that no zygotic gene product is essential for pairing to initiate. From these data, we conclude that the initiation of pairing can occur independently of zygotic control and may therefore be part of the developmental program encoded by the maternal genome.

A simple polymerase chain reaction-based method for the construction of recombinase-mediated cassette exchange donor vectors.

Here we describe a simple method for generating donor vectors suitable for targeted transgenesis via recombinase-mediated cassette exchange (RMCE) using the PhiC31 integrase. This PCR-based strategy employs small attB "tails" on the primers used to amplify a sequence of interest, permitting the rapid creation of transgenes for in vivo analysis.

Disruption of topoisomerase II perturbs pairing in drosophila cell culture.

Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc167 cells into G1, S, and G2 subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing.

DNA replication and models for the origin of piRNAs.

The piRNA class of small RNAs are distinct from other small RNAs by their approximately 26-31 nucleotide size, single-strandedness and strand-specificity as well as by the clustered arrangement of their origins. Here, we highlight how these features are reminiscent of the mechanisms of DNA replication, and then present three models suggesting that the origin of piRNAs may be mechanistically similar to key processes in DNA replication.

Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.