RESUMO
Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.
Assuntos
Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Regiões Promotoras Genéticas/genética , TATA Box/genética , Imagem com Lapso de Tempo/métodos , Transcrição Gênica/genética , Algoritmos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Teóricos , Proteína Vermelha FluorescenteRESUMO
Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.
Assuntos
Linhagem da Célula/genética , Cromatina/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Genômica , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Análise de Célula Única , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Much is known about the factors involved in the translation of messenger RNA (mRNA) into protein; however, this multistep process has not been imaged in living multicellular organisms. Here, we deploy the SunTag method to visualize and quantify the timing, location, and kinetics of the translation of single mRNAs in living Drosophila embryos. By focusing on the translation of the conserved major epithelial-mesenchymal transition-inducing transcription factor Twist, we identify spatial heterogeneity in mRNA translation efficiency and reveal the existence of translation factories, where clustered mRNAs are cotranslated preferentially at basal perinuclear regions. Observing the location and dynamics of mRNA translation in a living multicellular organism opens avenues for understanding gene regulation during development.
Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Cinética , RNA Mensageiro/genéticaRESUMO
The original version of this Article contained an error in Fig. 4a, in which the "=" sign of the equation was inadvertently replaced with a "-" sign. This has been corrected in the PDF and HTML versions of the Article.
RESUMO
Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and to the establishment of transcriptional competence. However, the impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains unclear. Here we employ quantitative live imaging methods and mathematical modeling to test the effect of the pioneer factor Zelda on transcriptional dynamics and memory in Drosophila embryos. We demonstrate that increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation regardless of their transcriptional past. Despite its known pioneering activities, we show that Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory. We further reveal that Zelda forms sub-nuclear dynamic hubs where Zelda binding events are transient. We propose that Zelda facilitates transcriptional activation by accumulating in microenvironments where it could accelerate the duration of multiple pre-initiation steps.