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1.
Mol Cell ; 81(16): 3356-3367.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34297910

RESUMO

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.


Assuntos
Proteínas de Drosophila/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RNA Polimerase II/genética , Transcrição Genética , Animais , Drosophila melanogaster/genética , Metiltransferases/genética , Regiões Promotoras Genéticas/genética
2.
Sci Rep ; 11(1): 13197, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162956

RESUMO

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.

3.
Elife ; 102021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33594977

RESUMO

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using in situ expression mapping and developmental and transcriptome analysis, we reveal PP heterogeneity and identify molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, we show that PP activate STAT92E and remain undifferentiated, promoting survival. Stat92E knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. We discuss how heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable.

4.
Br J Cancer ; 124(4): 805-816, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33214683

RESUMO

BACKGROUND: Understanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment. METHODS: We used transgenic mouse models to determine the role of Hoxb9 in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options. RESULTS: Our human ACC dataset analyses showed high expression of HOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression of Hoxb9 in the adrenal cortex of mice with activated Ctnnb1 led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, including Ccne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor. CONCLUSIONS: These studies show Hoxb9 can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

5.
PLoS Biol ; 18(12): e3000948, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33284790

RESUMO

Chronic inflammation is now a well-known precursor for cancer development. Infectious prostatitis are the most common causes of prostate inflammation, but emerging evidence points the role of metabolic disorders as a potential source of cancer-related inflammation. Although the widely used treatment for prostate cancer based on androgen deprivation therapy (ADT) effectively decreases tumor size, it also causes profound alterations in immune tumor microenvironment within the prostate. Here, we demonstrate that prostates of a mouse model invalidated for nuclear receptors liver X receptors (LXRs), crucial lipid metabolism and inflammation integrators, respond in an unexpected way to androgen deprivation. Indeed, we observed profound alterations in immune cells composition, which was associated with chronic inflammation of the prostate. This was explained by the recruitment of phagocytosis-deficient macrophages leading to aberrant hyporesponse to castration. This phenotypic alteration was sufficient to allow prostatic neoplasia. Altogether, these data suggest that ADT and inflammation resulting from metabolic alterations interact to promote aberrant proliferation of epithelial prostate cells and development of neoplasia. This raises the question of the benefit of ADT for patients with metabolic disorders.


Assuntos
Imunidade/fisiologia , Receptores X do Fígado/metabolismo , Próstata/metabolismo , Antagonistas de Androgênios/imunologia , Androgênios/metabolismo , Animais , Modelos Animais de Doenças , Imunidade/imunologia , Receptores X do Fígado/genética , Receptores X do Fígado/imunologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Neoplasias/etiologia , Neoplasias/imunologia , Neoplasias/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Receptores Citoplasmáticos e Nucleares/metabolismo , Microambiente Tumoral
6.
Nat Commun ; 11(1): 2300, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385236

RESUMO

One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. However, due to lack of adequate in vivo model, implication of emblematic signalling pathways such as Ras/Mitogen-Activated Protein Kinase (MAPK) and phosphoinositide 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathways, is scarcely described in this phenomenon. We have developed a unique model of basal extrusion in the Drosophila accessory gland. There, we demonstrate that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, we show that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Drosophila , Glândulas Exócrinas/metabolismo , Masculino , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia
7.
Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829940

RESUMO

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.


Assuntos
Canais de Cálcio/biossíntese , Doença do Sistema de Condução Cardíaco/genética , Doença do Sistema de Condução Cardíaco/fisiopatologia , Regulação da Expressão Gênica , Distrofia Miotônica/complicações , Animais , Canais de Cálcio/genética , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Humanos , Camundongos
8.
Dis Model Mech ; 11(5)2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29716962

RESUMO

Steinert disease, or myotonic dystrophy type 1 (DM1), is a multisystemic disorder caused by toxic noncoding CUG repeat transcripts, leading to altered levels of two RNA binding factors, MBNL1 and CELF1. The contribution of CELF1 to DM1 phenotypes is controversial. Here, we show that the Drosophila CELF1 family member, Bru-3, contributes to pathogenic muscle defects observed in a Drosophila model of DM1. Bru-3 displays predominantly cytoplasmic expression in muscles and its muscle-specific overexpression causes a range of phenotypes also observed in the fly DM1 model, including affected motility, fiber splitting, reduced myofiber length and altered myoblast fusion. Interestingly, comparative genome-wide transcriptomic analyses revealed that Bru-3 negatively regulates levels of mRNAs encoding a set of sarcomere components, including Actn transcripts. Conversely, it acts as a positive regulator of Actn translation. As CELF1 displays predominantly cytoplasmic expression in differentiating C2C12 myotubes and binds to Actn mRNA, we hypothesize that it might exert analogous functions in vertebrate muscles. Altogether, we propose that cytoplasmic Bru-3 contributes to DM1 pathogenesis in a Drosophila model by regulating sarcomeric transcripts and protein levels.


Assuntos
Proteínas de Drosophila/metabolismo , Músculos/metabolismo , Distrofia Miotônica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sarcômeros/metabolismo , Animais , Fusão Celular , Linhagem Celular , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Larva/metabolismo , Camundongos , Modelos Biológicos , Movimento , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Músculos/patologia , Músculos/fisiopatologia , Mioblastos/metabolismo , Distrofia Miotônica/patologia , Distrofia Miotônica/fisiopatologia , Fenótipo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
J Vis Exp ; (103)2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26381166

RESUMO

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.


Assuntos
RNA Mensageiro/isolamento & purificação , Animais , Animais Geneticamente Modificados , Cromatografia de Afinidade/métodos , Drosophila , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Magnetismo/métodos , Músculos/química , Músculos/citologia , Músculos/fisiologia , Polirribossomos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribossomos/química , Ribossomos/genética
10.
Development ; 142(5): 994-1005, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25715399

RESUMO

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.


Assuntos
Proteínas de Drosophila/metabolismo , Coração/embriologia , Proteínas de Choque Térmico Pequenas/metabolismo , Músculos/embriologia , Músculos/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Filaminas/genética , Filaminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Choque Térmico Pequenas/genética , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia
11.
EMBO Rep ; 15(4): 411-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24562610

RESUMO

In Drosophila, the piRNA cluster, flamenco, produces most of the piRNAs (PIWI-interacting RNAs) that silence transposable elements in the somatic follicle cells during oogenesis. These piRNAs are thought to be processed from a long single-stranded precursor transcript. Here, we demonstrate that flamenco transcription is initiated from an RNA polymerase II promoter containing an initiator motif (Inr) and downstream promoter element (DPE) and requires the transcription factor, Cubitus interruptus. We show that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed to piRNAs. Our data reveal dynamic processing steps giving rise to piRNA cluster precursors.


Assuntos
Drosophila melanogaster/genética , RNA Interferente Pequeno/genética , Transcrição Genética , Processamento Alternativo , Animais , Caderinas/genética , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Feminino , Família Multigênica , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , RNA Polimerase II/fisiologia , Splicing de RNA , Fatores de Transcrição/fisiologia
12.
Hum Mol Genet ; 22(14): 2795-810, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23525904

RESUMO

Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant disorder characterized by myotonia and muscle degeneration. In DM1 patients, the mutant DMPK transcripts containing expanded CUG repeats form nuclear foci and sequester the Muscleblind-like 1 splicing factor, resulting in mis-splicing of its targets. However, several pathological defects observed in DM1 and their link with disease progression remain poorly understood. In an attempt to fill this gap, we generated inducible transgenic Drosophila lines with increasing number of CTG repeats. Targeting the expression of these repeats to the larval muscles recapitulated in a repeat-size-dependent manner the major DM1 symptoms such as muscle hypercontraction, splitting of muscle fibers, reduced fiber size or myoblast fusion defects. Comparative transcriptional profiling performed on the generated DM1 lines and on the muscleblind (mbl)-RNAi line revealed that nuclear accumulation of toxic CUG repeats can affect gene expression independently of splicing or Mbl sequestration. Also, in mblRNAi contexts, the largest portion of deregulated genes corresponded to single-transcript genes, revealing an unexpected impact of the indirect influence of mbl on gene expression. Among the single-transcript Mbl targets is Muscle protein 20 involved in myoblast fusion and causing the reduced number of nuclei in muscles of mblRNAi larvae. Finally, by combining in silico prediction of Mbl targets with mblRNAi microarray data, we found the calcium pump dSERCA as a Mbl splice target and show that the membrane dSERCA isoform is sufficient to rescue a DM1-induced hypercontraction phenotype in a Drosophila model.


Assuntos
Modelos Animais de Doenças , Drosophila melanogaster/genética , Distrofia Miotônica/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Músculos/metabolismo , Músculos/fisiopatologia , Distrofia Miotônica/metabolismo , Distrofia Miotônica/fisiopatologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Expansão das Repetições de Trinucleotídeos
13.
Nucleic Acids Res ; 40(Web Server issue): W134-9, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22641854

RESUMO

DroPNet (Drosophila Protein Network) is a Drosophila-dedicated web portal for generating and analyzing protein-protein interaction (PPI) networks. This platform integrates users' experimental data provided as one or two lists of genes with PPI data from Drosophila and other species. These experimental data can, for example, come from RNAi screens, for which this approach is known to be valuable. DroPNet, therefore, provides an essential basis for further biological analysis by linking functional and physical interactions and reinforcing the relevance of each. DroPNet focuses on the search of PPIs between genes of the entry list, and includes the possibility of searching for intermediate genes for which the corresponding protein indirectly links two entry data. It also offers multiple functions for editing the networks obtained, providing users with interactive possibilities to progressively improve and refine the results. This approach gives a global view of the studied process and makes it possible to highlight specific interactions that have so far been understudied. DroPNet is freely available at http://dropnet.isima.fr.


Assuntos
Proteínas de Drosophila/metabolismo , Mapeamento de Interação de Proteínas/métodos , Software , Animais , Drosophila/metabolismo , Internet , Integração de Sistemas
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