RESUMO
The gene regulatory network governing anterior-posterior axis formation in Drosophila is a well-established paradigm to study transcription in developmental biology. The rapid temporal dynamics of gene expression during early stages of development, however, are difficult to track with standard techniques. We optimized the bright and fast-maturing fluorescent protein mNeonGreen as a real-time, quantitative reporter of enhancer expression. We derive enhancer activity from the reporter fluorescence dynamics with high spatial and temporal resolution, using a robust reconstruction algorithm. By comparing our results with data obtained with the established MS2-MCP system, we demonstrate the higher detection sensitivity of our reporter. We used the reporter to quantify the activity of variants of a simple synthetic enhancer, and observe increased activity upon reduction of enhancer-promoter distance or addition of binding sites for the pioneer transcription factor Zelda. Our reporter system constitutes a powerful tool to study spatio-temporal gene expression dynamics in live embryos.
Assuntos
Drosophila , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcriptoma/genética , Animais , Sítios de Ligação/genética , Padronização Corporal/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Feminino , Redes Reguladoras de Genes/genética , Genes Reporter/genética , Masculino , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Measurements of open chromatin in specific cell types are widely used to infer the spatiotemporal activity of transcriptional enhancers. How reliable are these predictions? In this review, it is argued that the relationship between the accessibility and activity of an enhancer is insufficiently described by simply considering open versus closed chromatin, or active versus inactive enhancers. Instead, recent studies focusing on the quantitative nature of accessibility signal reveal subtle differences between active enhancers and their different inactive counterparts: the closed silenced state and the accessible primed and repressed states. While the open structure as such is not a specific indicator of enhancer activity, active enhancers display a higher degree of accessibility than the primed and repressed states. Molecular mechanisms that may account for these quantitative differences are discussed. A model that relates molecular events at an enhancer to changes in its activity and accessibility in a developing tissue is also proposed.
Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário , Histonas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
Establishment of spatial coordinates during Drosophila embryogenesis relies on differential regulatory activity of axis patterning enhancers. Concentration gradients of activator and repressor transcription factors (TFs) provide positional information to each enhancer, which in turn promotes transcription of a target gene in a specific spatial pattern. However, the interplay between an enhancer regulatory activity and its accessibility as determined by local chromatin organization is not well understood. We profiled chromatin accessibility with ATAC-seq in narrow, genetically tagged domains along the antero-posterior axis in the Drosophila blastoderm. We demonstrate that one-quarter of the accessible genome displays significant regional variation in its ATAC-seq signal immediately after zygotic genome activation. Axis patterning enhancers are enriched among the most variable intervals, and their accessibility changes correlate with their regulatory activity. In an embryonic domain where an enhancer receives a net activating TF input and promotes transcription, it displays elevated accessibility in comparison to a domain where it receives a net repressive input. We propose that differential accessibility is a signature of patterning cis-regulatory elements in the Drosophila blastoderm and discuss potential mechanisms by which accessibility of enhancers may be modulated by activator and repressor TFs.