Biology subjects

Eisen, M. B.

Publications and source records attributed to Eisen, M. B..

13 recordsLinked to original sources

bioRxiv2018

Effects of the maternal factor Zelda on zygotic enhancer activity in the Drosophila embryo

The maternal factor Zelda is broadly bound to zygotic enhancers during early fly embryogenesis, and has been shown to be important for the expression of a large number of genes. However, its function remains poorly understood. Here, we carried out detailed analysis of the functional role of Zelda on the activities of a group of enhancers that drive patterned gene expression along the anterior -posterior axis. We found that among these enhancers, only one lost its activity entirely when all its Zelda bind sites were mutated. For all others, mutations of all of their Zelda binding sites only had limited effect, which varied temporally and spatially. These results suggest that Zld may exert a quantitative effect on a broad range of enhancers, which presumably is critical to generate highly diverse spatial and temporal expression patterns for different genes in the developmental gene network in fly embryo. Lastly, we found that the observed effect of Zelda site mutations was much stronger when a mutant enhancer was tested using a BAC based reporter construct than a simple reporter construct, suggesting that the effect of Zld is dependent on chromatin environment.

developmental biology↗

bioRxiv2018

Zelda potentiates transcription factor binding to zygotic enhancers by increasing local chromatin accessibility during early Drosophila melanogaster embryogenesis.

The maternally deposited transcription factor Zelda binds to and is required for the activation of a large number of genes in early Drosophila development, and has been suggested to act as a pioneer factor. In this study, we investigated the temporal dynamics of Zelda binding along with the maternal patterning factors Dorsal and Caudal during early embryogenesis. We found in regions bound by Zelda and either Dorsal or Caudal, Zelda binding was detected, and reached maximum levels, earlier than Caudal and Dorsal, providing support of its role as a pioneer factor. We found that Dorsal and Caudal binding correlated strongly with Zelda binding at mitotic cycle 12, suggesting that Zelda is important for early binding by these factors and early onset of their target gene expression. At the same time, we show that among Dorsal target enhancers, the dorsal and ventral ectoderm enhancers are much more strongly associated with Zelda than mesoderm enhancers, revealing an additional function of Zelda in coordinating spatial activity of enhancers. We have also investigated the role of Zelda on chromatin structure. We found that in early embryos, before Dorsal and Caudal are bound at significant levels, Zelda binding is associated with histone acetylation and local histone depletion. These chromatin associated changes accompanied with increased local chromatin accessibility were also detected around Zelda peaks in coding sequences that do not appear to play a role in subsequent transcription factor binding. These findings suggest that Zelda binding itself can lead to chromatin structural changes. Finally, we found that Zelda motifs, both bound and unbound, tend to be associated with positioned nucleosomes, which we suggest may be important for the regulatory specificity of enhancers.

developmental biology↗

bioRxiv2018

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

The regulation of transcription requires the coordination of numerous activities on DNA, yet it remains poorly understood how transcription factors facilitate these multiple functions. Here we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key patterning factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid dependent transcription. Based on our observations we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.

developmental biology↗

bioRxiv2018

Entomophthovirus: An insect-derived iflavirus that infects a behavior manipulating fungal pathogen of dipterans

We discovered a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Entomophthovirus, is a capsid-forming, positive-strand RNA virus in the viral family iflaviridae, whose known members almost exclusively infect insects. We show that the virus RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and that virus particles are present in E. muscae. Two close relatives of the virus had been previously described as insect viruses based on the presence of viral genomes in transcriptomes assembled from RNA extracted from wild dipterans. By analyzing sequencing data from these earlier reports, we show that both dipteran samples were co-infected with E. muscae. We also find the virus in RNA sequencing data from samples of two other species of dipterans, Musca domestica and Delia radicum, known to be infected with E. muscae. These data establish that Entomophthovirus is widely, and seemingly obligately, associated with E. muscae. As other members of the iflaviridae cause behavioral changes in insects, we speculate on the possibility that Entomophthovirus plays a role in E. muscae involved host manipulation.

microbiology↗

bioRxiv2018

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shape complex developmental patterns.

developmental biology↗

bioRxiv2018

The ecology of the Drosophila-yeast mutualism in wineries

The fruit fly, Drosophila melanogaster, is preferentially found on fermenting fruits. The yeasts that dominate the microbial communities of these substrates are the primary food source for developing D. melanogaster larvae, and adult flies manifest a strong olfactory system-mediated attraction for the volatile compounds produced by these yeasts during fermentation. Although most work on this interaction has focused on the standard laboratory yeast Saccharomyces cerevisiae, a wide variety of other yeasts naturally ferment fallen fruit. Here we address the open question of whether D. melanogaster preferentially associates with distinct yeasts in different, closely-related environments. We characterized the spatial and temporal dynamics of Drosophila-associated fungi in Northern California wineries that use organic grapes and natural fermentation using high-throughput, short-amplicon sequencing. We found that there is nonrandom structure in the fungal communities that are vectored by flies both between and within vineyards. Within wineries, the fungal communities associated with flies in cellars, fermentation tanks, and pomace piles are distinguished by varying abundances of a small number of yeast species. To investigate the origins of this structure, we assayed Drosophila attraction to, oviposition on, larval development in, and longevity when consuming the yeasts that distinguish vineyard microhabitats from each other. We found that wild fly lines did not respond differentially to the yeast species that distinguish winery habitats in habitat specific manner. Instead, this subset of yeast shares traits that make them attractive to and ensure their close association with Drosophila.

ecology↗

bioRxiv2018

Convergent evolution of gene expression in two high-toothed stickleback populations

Changes in developmental gene regulatory networks enable evolved changes in morphology. These changes can be in cis regulatory elements that act in an allele-specific manner, or changes to the overall trans regulatory environment that interacts with cis regulatory sequences. Here we address several questions about the evolution of gene expression accompanying a convergently evolved constructive morphological trait, increases in tooth number in two independently derived freshwater populations of threespine stickleback fish (Gasterosteus aculeatus). Are convergently evolved cis and/or trans changes in gene expression associated with convergently evolved morphological evolution? Do cis or trans regulatory changes contribute more to the evolutionary gain of a morphological trait? Transcriptome data from dental tissue of ancestral low-toothed and two independently derived high-toothed stickleback populations revealed significantly shared gene expression changes that have convergently evolved in the two high-toothed populations. Comparing cis and trans regulatory changes using phased gene expression data from F1 hybrids, we found that trans regulatory changes were predominant and more likely to be shared among both high-toothed populations. In contrast, while cis regulatory changes have evolved in both high-toothed populations, overall these changes were distinct and not shared among high-toothed populations. Together these data suggest that a convergently evolved trait can occur through genetically distinct regulatory changes that converge on similar trans regulatory environments.\n\nAuthor SummaryConvergent evolution, where a similar trait evolves in different lineages, provides an opportunity to study the repeatability of evolution. Convergent morphological evolution has been well studied at multiple evolutionary time scales ranging from ancient, to recent, such as the gain in tooth number in freshwater stickleback fish. However, much less is known about the accompanying evolved changes in gene regulation during convergent evolution. Here we compared evolved changes in gene expression in dental tissue of ancestral low-toothed marine fish to fish from two independently derived high-toothed freshwater populations. We also partitioned gene expression changes into those affecting a genes regulatory elements (cis), and those affecting the overall regulatory environment (trans). Both freshwater populations have evolved similar gene expression changes, including a gain of expression of putative dental genes. These similar gene expression changes are due mainly to shared changes to the trans regulatory environment, while the cis changes are largely population specific. Thus, during convergent evolution, overall similar and perhaps predictable transcriptome changes can evolve despite largely different underlying genetic bases.

genomics↗

bioRxiv2017

A fungal pathogen that robustly manipulates the behavior of Drosophila melanogaster in the laboratory

Many microbes induce striking behavioral changes in their animal hosts, but how they achieve this is poorly understood, especially at the molecular level. Mechanistic understanding has been largely constrained by the lack of a model system with advanced tools for molecular manipulation. We recently discovered a strain of the behavior-manipulating fungal pathogen Entomophthora muscae infecting wild Drosophila, and established methods to infect D. melanogaster in the lab. Lab-infected flies manifest the moribund behaviors characteristic of E. muscae infection: hours before death, they climb upward, extend their proboscides and affix in place, then raise their wings, clearing a path for infectious spores to launch from their abdomens. We found that E. muscae invades the fly nervous system, suggesting a direct means by which the fungus could induce behavioral changes. Given the vast molecular toolkit available for D. melanogaster, we believe this new system will enable rapid progress in understanding the mechanistic basis of E. muscaes behavioral manipulation in the fly.

animal behavior and cognition↗

bioRxiv2017

Microbiome-by-ethanol interactions impact Drosophila melanogaster fitness and physiology

Ethanol is one of the worlds most abused drugs yet the impacts of chronic ethanol consumption are debated. Ethanol is a prevalent component in the diets of diverse animals and can act as a nutritional source, behavior modulator, and a toxin. The source of ethanol is microbes, which can both produce and degrade ethanol, and the gut microbiome has been associated with differential health outcomes in chronic alcoholism. To disentangle the various and potentially interacting roles of bacteria and ethanol on host health, we developed a model for chronic ethanol ingestion in the adult fruit fly, Drosophila melanogaster, which naturally consumes a diet between 0 and 5% ethanol. We took advantage of the tractability of the fly microbiome, which can be experimentally removed to separate the direct and indirect effects of commensal microbes. We found that moderate to heavy ethanol ingestion decreased lifespan and reproduction, without causing inebriation. These effects were more pronounced in flies lacking a microbiome, but could not be explained by simple bacterial degradation of ethanol. However, moderate ethanol ingestion increased reproduction in bacterially-colonized flies, relative to bacteria-free flies. Ethanol decreased intestinal stem cell turnover in bacterially-colonized flies and decreased intestinal barrier failure and increased fat content in all flies, regardless of microbiome status. Analysis of host gene expression finds that ethanol triggers the innate immune response, but only in flies colonized with bacteria. Taken together we show that, chronic ethanol ingestion negatively impacts fly health in a microbiome-dependent manner.

microbiology↗

bioRxiv2017

Spatially uniform establishment of chromatin accessibility in the early Drosophila embryo

As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However, it remains unclear whether open chromatin is a product of activity - transcription at promoters and patterning transcription factor binding at enhancers - or whether it is established by independent mechanisms. Recent work has implicated the ubiquitously expressed, maternal factor Zelda in this process. To assess the relative contribution of activity in the establishment of chromatin accessibility, we have probed chromatin accessibility across the anterior-posterior axis of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is remarkably similar between the two halves. Promoters and enhancers that are active in exclusively one half of the embryo have open chromatin in the other half, demonstrating that chromatin accessibility is not a direct result of activity. However, there is a small skew at enhancers that drive transcription exclusively in either the anterior or posterior half of the embryo, with greater accessibility in the region of activity. Taken together these data support a model in which regions of chromatin accessibility are defined and established by ubiquitous factors, and fine-tuned subsequently by activity.

developmental biology↗

bioRxiv2017

Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo

Evidence has emerged in recent years linking insulators and the proteins that bind them to the higher order structure of animal chromatin, but the precise nature of this relationship and the manner by which insulators influence chromatin structure have remained elusive. Here we present high-resolution genome-wide chromatin conformation capture (Hi-C) data from early Drosophila melanogaster embryos that allow us to map three-dimensional interactions to 500 base pairs. We observe a complex, nested pattern of regions of chromatin self-association, and use a combination of computational and manual annotation to identify boundaries between these topological associated domains (TADs). We demonstrate that, when mapped at high resolution, boundaries resemble classical insulators: short (500 - 1000 bp) genomic regions that are sensitive to DNase digestion and strongly bound by known insulator proteins. Strikingly, we show that for regions where the banding pattern of polytene chromosomes has been mapped to genomic position at comparably high resolution, there is a perfect correspondence between polytene banding and our chromatin conformation maps, with boundary insulators forming the interband regions that separate compacted bands that correspond to TADs. We propose that this precise, high-resolution relationship between insulators and TADs on the one hand and polytene bands and interbands on the other extends across the genome, and suggest a model in which the decompaction of insulator regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.

genomics↗

bioRxiv2017

Association Mapping From Sequencing Reads Using K-mers

Genome wide association studies (GWAS) rely on microarrays, or more recently mapping of whole-genome sequencing reads, to genotype individuals. The reliance on prior sequencing of a reference genome for the organism on which the association study is to be performed limits the scope of association studies, and also precludes the identification of differences between cases and controls outside of the reference. We present an alignment free method for association studies that is based on counting k-mers in sequencing reads, testing for associations directly between k-mers and the trait of interest, and local assembly of the statistically significant k-mers to identify sequence differences. Results with simulated data and an analysis of the 1000 genomes data provide a proof of principle for the approach. In a pairwise comparison of the Toscani in Italia (TSI) and the Yoruba in Ibadan, Nigeria (YRI) populations we find that sequences identified by our method largely agree with results obtained using standard GWAS based on variant calling from mapped reads. However unlike standard GWAS, we find that our method identifies associations with structural variations and sites not present in the reference genome revealing sequences absent from the human reference genome. We also analyze data from the Bengali from Bangladesh (BEB) population to explore possible genetic basis of high rate of mortality due to cardiovascular diseases (CVD) among South Asians and find significant differences in frequencies of a number of non-synonymous variants in genes linked to CVDs between BEB and TSI samples, including the site rs1042034, which has been associated with higher risk of CVDs previously, and the nearby rs676210 in the Apolipoprotein B (ApoB) gene.\n\nAuthor SummaryWe present a method for associating regions in genomes to traits or diseases. The method is based on finding differences in frequencies of short strings of letters in sequencing reads and do not require reads to be aligned to a reference genome. This makes it applicable to study of organisms with no or incomplete reference genomes. We test our method with simulated data and sequencing data from the 1000 genomes project and find agreement with the conventional approach based on alignment to a reference genome. In addition, our method finds associations with sequences not in reference genomes and reveals sequences missing from the human reference genome. We also explore high rates of mortality due to cardiovascular diseases among South Asians and find prevalence of variations in genes associated with heart diseases in samples from the Bengali from Bangladesh population including one that has been reported to be associated with early onset of cardiovascular diseases.

bioinformatics↗

bioRxiv2017

A chromatin extension model for insulator function based on comparison of high-resolution chromatin conformation capture and polytene banding maps

Insulator proteins bind to specific genomic loci and have been shown to play a role in partitioning genomes into independent domains of gene expression and chromatin structure. Despite decades of study, the mechanism by which insulators establish these domains remains elusive. Here, we use genome-wide chromatin conformation capture (Hi-C) to generate a high-resolution map of spatial interactions of chromatin from Drosophila melanogaster embryos. We show that from the earliest stages of development the genome is divided into distinct topologically associated domains (TADs), that we can map the boundaries between TADs to sub-kilobase resolution, and that these boundaries correspond to 500-2000 bp insulator elements. Comparing this map with a detailed assessment of the banding pattern of a region of a polytene chromosome, we show that these insulator elements correspond to low density polytene interbands that divide compacted bands, which correspond to TADs. It has been previously shown that polytene interbands have low packing ratios allowing the conversion of small genomic distances (in base pairs) into a large physical distances. We therefore suggest a simple mechanism for insulator function whereby insulators increase the physical space between adjacent domains via the unpacking and extension of intervening chromatin. This model provides an intuitive explanation for known features of insulators, including the ability to block enhancer-promoter interactions, limit the spread of heterochromatin, and organize the structural features of interphase chromosomes.

genomics↗