Biology subjects

Eisen, M.

Publications and source records attributed to Eisen, M..

10 recordsLinked to original sources

bioRxiv2026

The Zelda interactome reveals diverse co-factors essential for the Drosophila zygotic genome activation

Zelda (Zld) is the master pioneer transcription factor essential for zygotic genome activation (ZGA) during the Drosophila maternal-to-zygotic transition (MZT). While Zld is known to promote chromatin accessibility at early enhancers and possess multiple conserved functional domains, the specific protein complexes it recruits to execute its functions remain poorly defined. Using an optimized immunoprecipitation and mass spectrometry (IP-MS) pipeline in Stage 4-5 embryos, we identified a diverse Zld interactome. This repertoire includes the coactivators dCBP and Fsh (the Drosophila Brd4 ortholog), subunits of major nucleosome remodeling complexes(e.g. PBAP/BAP, NURF, FACT), RNA polymerase II (RNAPII), the corepressor Smrter (Smr), and the Tousled-like kinase (Tlk). We confirmed the enrichment of ten key factors at Zld-bound regions using CUT&RUN. Notably, our results demonstrate that RNAPII associates not only with Zld-bound promoters but also with distal Zld-binding sites. This finding, which contrasts with previous ChIP-based studies, suggests that Zld facilitates transcription by actively scaffolding or pre-recruiting the transcriptional machinery at enhancers. Functional analyses revealed that dCBP and Fsh are required for Zld-mediated activation, while RNAi knockdown of smr led to the broad derepression of Zld-target genes, suggesting that Smr modulates Zld activity to prevent premature expression. Finally, we show that Tlk, a kinase typically associated with DNA replication, directly interacts with a specific Zld domain in vitro and co-localizes with Zld in vivo. This suggests a novel mechanism by which Zld may coordinate transcriptional activation with the rapid mitotic cycles of early embryogenesis. Collectively, our findings provide a comprehensive map of the Zld interactome and reveal how a pioneer factor integrates diverse chromatin and transcriptional regulators to orchestrate zygotic genome activation.

genomics↗

bioRxiv2023

Evolutionary analyses of IDRs reveal widespread signals of conservation

Intrinsically disordered regions (IDRs) are segments of proteins without stable three-dimensional structures. As this flexibility allows them to interact with diverse binding partners, IDRs play key roles in cell signaling and gene expression. Despite the prevalence and importance of IDRs in eukaryotic proteomes and various biological processes, associating them with specific molecular functions remains a significant challenge due to their high rates of sequence evolution. However, by comparing the observed values of various IDR-associated properties against those generated under a simulated model of evolution, a recent study found most IDRs across the entire yeast proteome contain conserved features. Furthermore, it showed clusters of IDRs with common "evolutionary signatures," i.e. patterns of conserved features, were associated with specific biological functions. To determine if similar patterns of conservation are found in the IDRs of other systems, in this work we applied a series of phylogenetic models to over 8,500 orthologous IDRs identified in the Drosophila genome to dissect the forces driving their evolution. By comparing models of constrained and unconstrained continuous trait evolution using the Brownian motion and Ornstein-Uhlenbeck models, respectively, we identified signals of widespread constraint, indicating conservation of distributed features is mechanism of IDR evolution common to multiple biological systems. In contrast to the previous study in yeast, however, we observed limited evidence of IDR clusters with specific biological functions, which suggests a more complex relationship between evolutionary constraints and function in the IDRs of multicellular organisms.

bioinformatics↗

bioRxiv2023

Leveraging genomic redundancy to improve inference and alignment of orthologous proteins

Identifying protein sequences with common ancestry is a core task in bioinformatics and evolutionary biology. However, methods for inferring and aligning such sequences in annotated genomes have not kept pace with the increasing scale and complexity of the available data. Thus, in this work we implemented several improvements to the traditional methodology that more fully leverage the redundancy of closely related genomes and the organization of their annotations. Two highlights include the application of the more flexible k-clique percolation algorithm for identifying clusters of orthologous proteins and the development of a novel technique for removing poorly supported regions of alignments with a phylogenetic HMM. In making the latter, we also wrote a fully documented Python package Homomorph that implements standard HMM algorithms and created a set of tutorials to promote its use by a wide audience. We applied the resulting pipeline to a set of 33 annotated Drosophila genomes, generating 22,813 orthologous groups and 8,566 high-quality alignments.

bioinformatics↗

bioRxiv2022

Early dosage compensation of zygotically-expressed genes in Drosophila melanogaster is mediated through a post-transcriptional regulatory mechanism

Many key regulators of early embryogenesis in Drosophila melanogaster are X-linked. However, the canonical, MSL-mediated dosage compensation, which involves hyper-transcription of the genes on the single X chromosome in males, is not active until the post-syncytial stage of development. A separate MSL-independent dosage compensation system active earlier in development has been described, though the mechanism through which the process functions remain unclear. In this study, we quantified transcription in living embryos at single-locus resolution to determine if early dosage compensation of the X-linked genes buttonhead and giant is sensitive to X chromosome dose. We found no evidence for a transcriptionally regulated mechanism of early dosage compensation, suggesting that the previously observed compensation of mRNA levels for these genes is achieved via a post-transcriptional regulatory mechanism.

molecular biology↗

bioRxiv2021

Single-nucleus RNA-sequencing in pre-cellularization Drosophila melanogaster embryos

Our current understanding of the regulation of gene expression in the early Drosophila melanogaster embryo comes from observations of a few genes at a time, as with in situ hybridizations, or observation of gene expression levels without regards to patterning, as with RNA-sequencing. Single-nucleus RNA-sequencing however, has the potential to provide new insights into the regulation of gene expression for many genes at once while simultaneously retaining information regarding the position of each nucleus prior to dissociation based on patterned gene expression. In order to establish the practicality of single-nucleus RNA sequencing in the context of a real biological question, here we look at the difference in gene expression between control and an insulator protein, dCTCF, maternal null embryos during zygotic genome activation at nuclear cycle 14. We find that early embryonic nuclei can be grouped into distinct clusters according to gene expression. From both virtual and published in situ hybridizations, we also find that these clusters correspond to spatial regions of the embryo. Lastly, we present multiple examples of differential gene expression between control and maternal CTCF null nuclei in one or more clusters, but not in bulk when grouping expression across all nuclei. These results highlight the potential for single-nucleus RNA-sequencing to reveal new insights into the regulation of gene expression in the early Drosophila melanogaster embryo.

genomics↗

bioRxiv2021

Embryo-to-embryo variability in RNAi knockdown efficiency of dKDM5/lid in Drosophila melanogaster

We used the maternal-Gal4 shRNA system to knock down expression of dKDM5/lid in Drosophila melanogaster embryos, and analyzed the efficacy of the knockdown by qRT-PCR. Although average relative expression of lid was significantly lower in knockdown conditions compared to the driver-only control, we observed a wide and overlapping range of relative gene expression between individual control and knockdown embryos.

genetics↗

bioRxiv2021

Eliminating Animal Agriculture Would Negate 56 Percent of Anthropogenic Greenhouse Gas Emissions Through 2100

Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change. Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy. Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture.

ecology↗

bioRxiv2018

Mutation of sequences flanking and separating transcription factor binding sites in a Drosophila enhancer significantly alter its output

Here we explore how mutating different sequences in an enhancer that regulates patterned gene expression in Drosophila melanogaster embryos can affect its output. We used quantitative imaging to analyze the effects of a wide variety of mutations in the hunchback distal anterior enhancer. This enhancer has been shown to respond to the anterior morphogen Bicoid, but we found that mutations in only one of the five strong Bicoid sites in the enhancer has a significant effect on its binding. The pioneer factor Zelda, which binds to this enhancer and is the only other factor implicated in its activity besides Bicoid. However, we found that mutations of all its sites only has modest effect that is limited to reduction of its output in more posterior regions of the embryo, where Bicoid levels are low. In contrast to the modest effects of mutating known transcription factor binding sites, randomizing the sequences between Zelda and Bicoid sites significantly compromised enhancer activity. Finer mapping suggested that the sequences that determine activity are broadly distributed in the enhancer. Mutations in short sequences flanking Bicoid binding sites have stronger effects than mutations to Bicoid sites themselves, highlighting the complex and counterintuitive nature of the relationship between enhancer sequence and activity.

developmental biology↗

bioRxiv2017

The ancestral animal genetic toolkit revealed by diverse choanoflagellate transcriptomes

The changes in gene content that preceded the origin of animals can be reconstructed by comparison with their sister group, the choanoflagellates. However, only two choanoflagellate genomes are currently available, providing poor coverage of their diversity. We sequenced transcriptomes of 19 additional choanoflagellate species to produce a comprehensive reconstruction of the gains and losses that shaped the ancestral animal gene repertoire. We find roughly 1,700 gene families with origins on the animal stem lineage, of which only a core set of 36 are conserved across animals. We find more than 350 gene families that were previously thought to be animal-specific actually evolved before the animal-choanoflagellate divergence, including Notch and Delta, Toll-like receptors, and glycosaminoglycan hydrolases that regulate animal extracellular matrix (ECM). In the choanoflagellate Salpingoeca helianthica, we show that a glycosaminoglycan hydrolase modulates rosette colony size, suggesting a link between ECM regulation and morphogenesis in choanoflagellates and animals.\n\nData AvailabilityRaw sequencing reads: NCBI BioProject PRJNA419411 (19 choanoflagellate transcriptomes), PRJNA420352 (S. rosetta polyA selection test)\n\nTranscriptome assemblies, annotations, and gene families: https://dx.doi.org/10.6084/m9.figshare.5686984\n\nProtocols: https://dx.doi.org/10.17504/protocols.io.kwscxee

evolutionary biology↗

bioRxiv2017

Or22 allelic variation alone does not explain differences in discrimination of yeast-produced volatiles by D. melanogaster

Different lines of the fruit fly Drosophila melanogaster show variation in the ability to discriminate between volatiles produced by the yeast Saccharomyces cerevisiae under natural (nitrogen-limiting, YVN) or laboratory (sugar-limiting, YVL) conditions. Previous work in our laboratory uncovered a strong correlation between heightened sensitivity to YVN wild D. melanogaster lines that harbored a chimeric variant of the highly variable odorant receptor 22 (Or22) locus of D. melanogaster. We sought to determine if this trend held for an extended set of D. melanogaster lines, if observed variation within chimeric and non-chimeric lines could be explained by nucleotide polymorphisms and if replacing Or22 with a chimeric allele in a non-chimeric background could confer the enhanced ability to detect YVN. In parallel, we performed crosses of chimeric and non-chimeric fly lines and assayed the behavior of their progeny for enhanced sensitivity to YVN to assess the heritability of the Or22 locus. Ultimately, we found that, while the overall trend of increased sensitivity to YVN in chimeric lines persists, there are exceptions and variation that cannot be explained by sequence variation at the Or22 locus. In addition, we did not observe increased sensitivity for YVN upon replacing the Or22 allele in a non-chimeric line (OreR) with that from our most YVN-sensitive, chimeric line (ME). Though our results do not support our hypothesis that Or22 is the primary driver of sensitivity to YVN, Or22 remains an interesting locus in the context of fly-yeast ecology.

genetics↗