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Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos.

ABSTRACT: The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. We explored the possibility that interallelic interactions between two homologous alleles might affect gene regulation. Using an MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcripts of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the corresponding hemizygous allele, suggesting the possibility of allelic competition in homozygotes. However, the competition was not observed when the enhancer-promoter interaction was weakened by placing the reporter construct in a different chromosome location or by moving the enhancer further away from the promoter. Moreover, the reporter gene showed reduced transcriptional activity when a partial transcription unit (either an enhancer or reporter gene only) was in the homologous position. We propose that the transcriptional machinery that binds both the enhancer and promoter regions, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. We showed that the degree of allelic interference increased over developmental time as more Pol II was needed to activate zygotic genes. Such allelic competition was observed for an endogenous gene as well. Our study provides new insights into the role of 3D interallelic interactions in gene regulation.

SUBMITTER: Deng H

PROVIDER: S-EPMC9311490 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMorphogen molecules form concentration gradients to provide spatial information to cells in a developing embryo. Precisely how cells decode such information to form patterns with sharp boundaries remains an open question. For example, it remains controversial whether the Drosophila morphogenetic protein Bicoid (Bcd) plays a transient or sustained role in activating its target genes to establish sharp expression boundaries during development.Methodology/principal findingsIn this study, we describe a method to simultaneously detect Bcd and the nascent transcripts of its target genes in developing embryos. This method allows us to investigate the relationship between Bcd and the transcriptional status of individual copies of its target genes on distinct scales. We show that, on three scales analyzed concurrently--embryonic, nuclear and local, the actively-transcribing gene copies are associated with high Bcd concentrations. These results underscore the importance of Bcd as a sustained input for transcriptional decisions of individual copies of its target genes during development. We also show that the Bcd-dependent transcriptional decisions have a significantly higher noise than Bcd-dependent gene products, suggesting that, consistent with theoretical studies, time and/or space averaging reduces the noise of Bcd-activated transcriptional output. Finally, our analysis of an X-linked Bcd target gene reveals that Bcd-dependent transcription bursts at twice the frequency in males as in females, providing a mechanism for dosage compensation in early Drosophila embryos.Conclusion/significanceOur study represents a first experimental uncovering of the actions of Bcd in controlling the actual transcriptional events while its positional information is decoded during development. It establishes a sustained role of Bcd in transcriptional decisions of individual copies of its target genes to generate sharp expression boundaries. It also provides an experimental evaluation of the effect of time and/or space averaging on Bcd-dependent transcriptional output, and establishes a dosage compensation mechanism in early Drosophila embryos.

Project description:Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the "parts list" of cell types that comprise each organ. Here we perform single cell RNA sequencing (scRNA-seq) of the Drosophila embryo to identify the genes that characterize different cell and tissue types during development. We assay three different timepoints, revealing a coordinated change in gene expression within each tissue. Interestingly, we find that the elav and Mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, show broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. We next focus on the central nervous system (CNS), where we identify genes whose expression is enriched at each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, we ask whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, we find that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, we were able to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, neurons which share a temporal identity (e.g. Hunchback, Kruppel, Pdm, and Castor temporal transcription factors in the NB7-1 lineage) have shared transcriptional features, allowing for the identification of candidate novel temporal factors or targets of the temporal transcription factors. In conclusion, we have characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor.

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Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos.

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