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1.
Front Cell Infect Microbiol ; 14: 1408451, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38828264

RESUMO

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.


Assuntos
Vesículas Extracelulares , Malária Vivax , Plasmodium vivax , Proteínas de Protozoários , Baço , Vesículas Extracelulares/metabolismo , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Humanos , Baço/metabolismo , Baço/parasitologia , Malária Vivax/parasitologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Eritrócitos/parasitologia , Eritrócitos/metabolismo , Fibroblastos/parasitologia , Fibroblastos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Adesão Celular , Interações Hospedeiro-Parasita
2.
STAR Protoc ; 3(4): 101788, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36345375

RESUMO

NanoDam is a technique for genome-wide profiling of the binding targets of any endogenously tagged chromatin-binding protein in vivo, without the need for antibodies, crosslinking, or immunoprecipitation. Here, we explain the procedure for NanoDam experiments in Drosophila, starting from a genetic cross, to the generation of sequencing libraries and, finally, bioinformatic analysis. This protocol can be readily adapted for use in other model systems after simple modifications. For complete details on the use and execution of this protocol, please refer to Tang et al. (2022).


Assuntos
Cromatina , Drosophila , Animais , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Drosophila/genética , Proteínas de Transporte/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos
3.
EMBO J ; 41(21): e111338, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36121125

RESUMO

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.


Assuntos
Pulmão , Fatores de Transcrição SOX9 , Células-Tronco , Humanos , Diferenciação Celular/fisiologia , Pulmão/embriologia , Transdução de Sinais , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo
4.
Nat Commun ; 13(1): 2210, 2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468895

RESUMO

The Notch signalling pathway is a master regulator of cell fate transitions in development and disease. In the brain, Notch promotes neural stem cell (NSC) proliferation, regulates neuronal migration and maturation and can act as an oncogene or tumour suppressor. How NOTCH and its transcription factor RBPJ activate distinct gene regulatory networks in closely related cell types in vivo remains to be determined. Here we use Targeted DamID (TaDa), requiring only thousands of cells, to identify NOTCH and RBPJ binding in NSCs and their progeny in the mouse embryonic cerebral cortex in vivo. We find that NOTCH and RBPJ associate with a broad network of NSC genes. Repression of NSC-specific Notch target genes in intermediate progenitors and neurons correlates with decreased chromatin accessibility, suggesting that chromatin compaction may contribute to restricting NOTCH-mediated transactivation.


Assuntos
Cromatina , Células-Tronco Neurais , Animais , Diferenciação Celular/fisiologia , Camundongos , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais
5.
Sci Adv ; 6(24): eaaz5057, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32577509

RESUMO

Malaria transmission requires that some asexual parasites convert into sexual forms termed gametocytes. The initial stages of sexual development, including sexually committed schizonts and sexual rings, remain poorly characterized, mainly because they are morphologically identical to their asexual counterparts and only a small subset of parasites undergo sexual development. Here, we describe a system for controlled sexual conversion in the human malaria parasite Plasmodium falciparum, based on conditional expression of the PfAP2-G transcription factor. Using this system, ~90 percent of the parasites converted into sexual forms upon induction, enabling the characterization of committed and early sexual stages without further purification. We characterized sexually committed schizonts and sexual rings at the transcriptomic and phenotypic levels, which revealed down-regulation of genes involved in solute transport upon sexual commitment, among other findings. The new inducible lines will facilitate the study of early sexual stages at additional levels, including multiomic characterization and drug susceptibility assays.


Assuntos
Malária , Parasitos , Animais , Regulação da Expressão Gênica , Humanos , Malária/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Fatores de Transcrição/metabolismo
6.
Sci Rep ; 9(1): 14595, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601834

RESUMO

Transmission of malaria parasites from humans to mosquito vectors requires that some asexual parasites differentiate into sexual forms termed gametocytes. The balance between proliferation in the same host and conversion into transmission forms can be altered by the conditions of the environment. The ability to accurately measure the rate of sexual conversion under different conditions is essential for research addressing the mechanisms underlying sexual conversion, and to assess the impact of environmental factors. Here we describe new Plasmodium falciparum transgenic lines with genome-integrated constructs in which a fluorescent reporter is expressed under the control of the promoter of the gexp02 gene. Using these parasite lines, we developed a sexual conversion assay that shortens considerably the time needed for an accurate determination of sexual conversion rates, and dispenses the need to add chemicals to inhibit parasite replication. Furthermore, we demonstrate that gexp02 is expressed specifically in sexual parasites, with expression starting as early as the sexual ring stage, which makes it a candidate marker for circulating sexual rings in epidemiological studies.


Assuntos
Técnicas Genéticas , Plasmodium falciparum/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Eritrócitos/parasitologia , Corantes Fluorescentes , Genes Reporter , Humanos , Proteínas de Protozoários/genética , Transgenes
7.
Brief Funct Genomics ; 18(5): 329-341, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31114839

RESUMO

Transcriptional differences enable the generation of alternative phenotypes from the same genome. In malaria parasites, transcriptional plasticity plays a major role in the process of adaptation to fluctuations in the environment. Multiple studies with culture-adapted parasites and field isolates are starting to unravel the different transcriptional alternatives available to Plasmodium falciparum and the underlying molecular mechanisms. Here we discuss how epigenetic variation, directed transcriptional responses and also genetic changes that affect transcript levels can all contribute to transcriptional variation and, ultimately, parasite survival. Some transcriptional changes are driven by stochastic events. These changes can occur spontaneously, resulting in heterogeneity within parasite populations that provides the grounds for adaptation by dynamic natural selection. However, transcriptional changes can also occur in response to external cues. A better understanding of the mechanisms that the parasite has evolved to alter its transcriptome may ultimately contribute to the design of strategies to combat malaria to which the parasite cannot adapt.


Assuntos
Cromatina/metabolismo , Plasmodium falciparum/genética , Transcrição Gênica , Adaptação Fisiológica , Cromatina/química , Cromatina/enzimologia , Cromatina/genética , Epigênese Genética , Variação Genética , Genoma de Protozoário , Mutação , Fenótipo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/metabolismo , Seleção Genética , Análise de Célula Única , Transcriptoma/genética
8.
Nat Microbiol ; 4(1): 144-154, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30478286

RESUMO

Human to vector transmission of malaria requires that some blood-stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here, we study this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually committed parasite stages that precede the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route in which PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon re-invasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Reanalysis of published single-cell RNA-sequencing (RNA-seq) data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle.


Assuntos
Estágios do Ciclo de Vida/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Desenvolvimento Sexual/fisiologia , Sequência de Bases , Eritrócitos/parasitologia , Humanos , Malária Falciparum/patologia , Esquizontes/metabolismo , Análise de Sequência de RNA
9.
J Mol Cell Cardiol ; 102: 74-82, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27894866

RESUMO

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.


Assuntos
Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Transcrição Gênica , Animais , Sítios de Ligação , Linhagem Celular , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Humanos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos
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