RESUMO
The Hunchback (Hb) transcription factor is crucial for anterior-posterior patterning of the Drosophila embryo. The maternal hb mRNA acts as a paradigm for translational regulation due to its repression in the posterior of the embryo. However, little is known about the translatability of zygotically transcribed hb mRNAs. Here, we adapt the SunTag system, developed for imaging translation at single-mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. Whereas the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Biossíntese de Proteínas/genética , RNA Mensageiro Estocado/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , Zigoto/fisiologiaRESUMO
MOTIVATION: The MS2-MCP (MS2 coat protein) live imaging system allows for visualization of transcription dynamics through the introduction of hairpin stem-loop sequences into a gene. A fluorescent signal at the site of nascent transcription in the nucleus quantifies mRNA production. Computational modelling can be used to infer the promoter states along with the kinetic parameters governing transcription, such as promoter switching frequency and polymerase loading rate. However, modelling of the fluorescent trace presents a challenge due its persistence; the observed fluorescence at a given time point depends on both current and previous promoter states. A compound state Hidden Markov Model (cpHMM) was recently introduced to allow inference of promoter activity from MS2-MCP data. However, the computational time for inference scales exponentially with gene length and the cpHMM is therefore not currently practical for application to many eukaryotic genes. RESULTS: We present a scalable implementation of the cpHMM for fast inference of promoter activity and transcriptional kinetic parameters. This new method can model genes of arbitrary length through the use of a time-adaptive truncated compound state space. The truncated state space provides a good approximation to the full state space by retaining the most likely set of states at each time during the forward pass of the algorithm. Testing on MS2-MCP fluorescent data collected from early Drosophila melanogaster embryos indicates that the method provides accurate inference of kinetic parameters within a computationally feasible timeframe. The inferred promoter traces generated by the model can also be used to infer single-cell transcriptional parameters. AVAILABILITY AND IMPLEMENTATION: Python implementation is available at https://github.com/ManchesterBioinference/burstInfer, along with code to reproduce the examples presented here. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Drosophila melanogaster , Animais , Drosophila melanogaster/genética , Fatores de Tempo , Simulação por ComputadorRESUMO
The capacity for embryonic cells to differentiate relies on a large-scale reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this reprogramming step is achieved by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Yet, it remains unclear whether cells lacking this reprogramming step are directed towards wild type states or towards novel developmental canals in the Waddington landscape of embryonic development. Here we investigate the developmental fate of embryonic cells mutant for NPS by analyzing their single-cell gene expression profiles. We find that cells lacking the first developmental reprogramming steps can acquire distinct cell states. These states are manifested by gene expression modules that result from a failure of nuclear reprogramming, the persistence of the maternal program, and the activation of somatic compensatory programs. As a result, most mutant cells follow new developmental canals and acquire new mixed cell states in development. In contrast, a group of mutant cells acquire primordial germ cell-like states, suggesting that NPS-dependent reprogramming is dispensable for these cell states. Together, these results demonstrate that developmental reprogramming after fertilization is required to differentiate most canonical developmental programs, and loss of the transient totipotent state canalizes embryonic cells into new developmental states in vivo.
RESUMO
CRISPR-Cas9 genome editing has transformed biology by enabling site-specific genome modifications to be simply engineered. Here, we describe two CRISPR-Cas9 approaches to introduce MS2 stem-loop sequences into endogenous gene loci in Drosophila. This can facilitate live imaging of nascent transcription in Drosophila. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).
Assuntos
Drosophila/embriologia , Edição de Genes/métodos , Perfilação da Expressão Gênica/métodos , Animais , Sistemas CRISPR-Cas , Genoma , Sequências Repetidas Invertidas/genética , RNA Mensageiro/genéticaRESUMO
Visualizing transcription live in Drosophila is providing important new insights into the spatiotemporal regulation of transcription. Here, we describe a protocol to visualize and quantitate transcription from gene loci that are tagged with MS2 stem-loop sequences in the Drosophila embryo. MS2 stem-loop sequences are recognized by a coat protein fused to a fluorescent protein and visualized with microscopy. We also describe an analysis pipeline to extract and subsequently quantify transcription dynamics. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).
Assuntos
Drosophila/embriologia , Perfilação da Expressão Gênica/métodos , Microscopia de Fluorescência/métodos , Animais , Proteínas do Capsídeo/genética , Sequências Repetidas Invertidas/genética , RNA Mensageiro/genética , Tomografia Computadorizada por Raios X/métodosRESUMO
Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021).
Assuntos
Embrião não Mamífero/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/análise , Imagem Individual de Molécula/métodos , Animais , Drosophila/genética , Drosophila/metabolismo , Embrião não Mamífero/química , Feminino , Hibridização in Situ Fluorescente , Masculino , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Anticorpos de Cadeia ÚnicaRESUMO
Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient's conserved role in embryonic dorsal-ventral axis patterning. Here, we elucidate how the BMP gradient is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, we show that BMP signaling controls burst frequency by regulating the promoter activation rate. We provide evidence that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. We suggest that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/patologia , Animais , Humanos , Splicing de RNA , RNA não Traduzido , Sequências Reguladoras de Ácido Nucleico , Mutação Silenciosa , Regiões não TraduzidasRESUMO
Hormone-gated nuclear receptors (NRs) are conserved transcriptional regulators of metabolism, reproduction, and homeostasis. Here we show that C. elegans NHR-8 NR, a homolog of vertebrate liver X and vitamin D receptors, regulates nematode cholesterol balance, fatty acid desaturation, apolipoprotein production, and bile acid metabolism. Loss of nhr-8 results in a deficiency in bile acid-like steroids, called the dafachronic acids, which regulate the related DAF-12/NR, thus controlling entry into the long-lived dauer stage through cholesterol availability. Cholesterol supplementation rescues various nhr-8 phenotypes, including developmental arrest, unsaturated fatty acid deficiency, reduced fertility, and shortened life span. Notably, nhr-8 also interacts with daf-16/FOXO to regulate steady-state cholesterol levels and is synthetically lethal in combination with insulin signaling mutants that promote unregulated growth. Our studies provide important insights into nuclear receptor control of cholesterol balance and metabolism and their impact on development, reproduction, and aging in the context of larger endocrine networks.