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Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.
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Proteínas de Drosophila , Animales , Proteínas Cromosómicas no Histona/genética , Drosophila/genética , Proteínas de Drosophila/genética , Heterocromatina , Homeostasis , Humanos , Receptores Notch/genéticaRESUMEN
Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.
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Drosophila melanogaster/genética , Ingeniería Genética/métodos , Animales , Sistemas CRISPR-Cas/genética , Expresión Génica , Interferencia de ARN , Activación TranscripcionalRESUMEN
In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173â¯EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled.
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Diferenciación Celular/genética , Redes Reguladoras de Genes , Interferencia de ARN , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Genes Esenciales/genética , Genómica , Mutación , Ovario/citología , Ovario/metabolismo , Fenotipo , Empalme del ARN , Transducción de Señal/genéticaRESUMEN
In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the mutant progeny can be generated by only injecting a DNA plasmid encoding synthetic guide RNA (sgRNA) under the control of the U6b promoter into transgenic fly embryos in which Cas9 is specifically expressed in the progenitor cells, thus the gene of interest can be edited by the CRISPR in germ cells, with high rate of heritable mutations and few side effects.
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Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.
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Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority choice to study gene functions due to its simple and practical characteristics in Drosophila. We established a novel system that works well in both soma and germ cells which is efficient and specific. With this system, we can precisely and efficiently modulate highly expressed genes, and simultaneously knock down multiple genes in one step. In this study, we provide a detailed protocol of the pNP system, which replaces other transgenic systems, and expect it can provide some help to researchers who are using this system.
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Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, we develop a next generation transgenic RNAi system. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, we increase versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, we have generated a collection of transgenic RNAi lines for those genes that are highly homologous to human disease genes.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Dysregulation of the homeostatic balance of histone H3 di- and tri-methyl lysine 27 (H3K27me2/3) levels caused by the mis-sense mutation of histone H3 (H3K27M) is reported to be associated with various types of cancers. In this study, we found that reduction in H3K27me2/3 caused by H3.1K27M, a mutation of H3 variants found in patients with diffuse intrinsic pontine glioma (DIPG), dramatically attenuated the presence of 53BP1 (also known as TP53BP1) foci and the capability of non-homologous end joining (NHEJ) in human dermal fibroblasts. H3.1K27M mutant cells showed increased rates of genomic insertions/deletions and copy number variations, as well as an increase in p53-dependent apoptosis. We further showed that both hypo-H3K27me2/3 and H3.1K27M interacted with FANCD2, a central player in the choice of DNA repair pathway. H3.1K27M triggered the accumulation of FANCD2 on chromatin, suggesting an interaction between H3.1K27M and FANCD2. Interestingly, knockdown of FANCD2 in H3.1K27M cells recovered the number of 53BP1-positive foci, NHEJ efficiency and apoptosis rate. Although these findings in HDF cells may differ from the endogenous regulation of the H3.1K27M mutant in the specific tumor context of DIPG, our results suggest a new model by which H3K27me2/3 facilitates NHEJ and the maintenance of genome stability.This article has an associated First Person interview with the first author of the paper.
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Cromatina/metabolismo , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Histonas/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Línea Celular , Cromatina/genética , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Fibroblastos , Inestabilidad Genómica , Glioma/genética , Glioma/metabolismo , Células HEK293 , Histonas/genética , Humanos , Metilación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.
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Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Proteínas de Drosophila , Regulación de la Expresión Génica/genética , Factores de Transcripción , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Drosophila ovary is recognized as one of the best model systems to study stem cell biology in vivo. We had previously identified an autonomous role of the histone H1 in germline stem cell (GSC) maintenance. Here, we found that histone H1 depletion in escort cells (ECs) resulted in an increase of spectrosome-containing cells (SCCs), an ovary tumor-like phenotype. Further analysis showed that the Dpp pathway is excessively activated in these SCC cells, while the expression of bam is attenuated. In the H1-depleted ECs, both transposon activity and DNA damage had increased dramatically, followed by EC apoptosis, which is consistent with the role of H1 in other somatic cells. Surprisingly, H1-depleted ECs acquired cap cell characteristics including dpp expression, and the resulting abnormal Dpp level inhibits SCC further differentiation. Most interestingly, double knockdown of H1 and dpp in ECs can reduce the number of SCCs to the normal level, indicating that the additional Dpp secreted by ECs contributes to the germline tumor. Taken together, our findings indicate that histone H1 is an important epigenetic factor in controlling EC characteristics and a key suppressor of germline tumor.
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Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Células Germinativas/patología , Histonas/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Animales , Apoptosis , Recuento de Células , Daño del ADN , Elementos Transponibles de ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Modelos Biológicos , Fenotipo , Transducción de Señal , Transcripción Genética , Regulación hacia ArribaRESUMEN
Response surface methodology (RSM) based on Box-Behnken design (BBD) was employed to investigate the effect of ultrasonic treatment on beta-carotene production by Blakeslea trispora. The optimized strategy involved exposing three-day-old mycelial cultures to ultrasonic treatment at a fixed frequency of 20 kHz, power of 491 W, treatment time of 3 min, working time of 3 s, and rest time of 5.8 s, repeated four times at a 24-h interval. Mycelium growth was not significantly promoted under ultrasonic stimulation; however, the glucose metabolism increased by about 10%, the average size of the aggregates significantly decreased, and the uptake rate of imidazole into cells was increased about 2.5-fold. After a 6-d culture, the technique produced 173 mg/L of beta-carotene and 82 mg/L of lycopene, which represented an increase of nearly 40.7% and 52.7%, respectively, over the yields obtained in cultures without ultrasonic treatment.