RESUMO
The rearrangement and expression of the immunoglobulin µ heavy chain (Igh) gene require communication of the intragenic Eµ and 3' regulatory region (RR) enhancers with the variable (VH) gene promoter. Eµ binding of the transcription factor YY1 has been implicated in enhancer-promoter communication, but the YY1 protein network remains obscure. By analyzing the comprehensive proteome of the 1-kb Eµ wild-type enhancer and that of Eµ lacking the YY1 binding site, we identified the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network. We found that MSL2 recruitment depends on YY1 and that gene knockout of Msl2 in primary pre-B cells reduces µ gene expression and chromatin looping of Eµ to the 3' RR enhancer and VH promoter. Moreover, Mof heterozygosity in mice impaired µ expression and early B cell differentiation. Together, these data suggest that the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication.
Assuntos
Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fator de Transcrição YY1 , Animais , Masculino , Camundongos , Diferenciação Celular , Elementos Facilitadores Genéticos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genética , FemininoRESUMO
Heterozygous beta-actin (ACTB) indel and nonsense mutations are linked to developmental disorders. We generated two CRISPR/Cas9 human induced pluripotent stem cell (iPSC) lines, WTSIi018-B-19 and WTSIi018-B-20, carrying heterozygous and homozygous indel mutations in ACTB exon 4. Both iPSCs exhibited normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. While iPSCs with a heterozygous ACTB mutation maintain genome integrity, homozygous mutants showed a loss of heterozygosity in chromosome three. These mutants provide a powerful model to study the onset, progression, and complex interplay of genetic compensation and phenotypic variation of ACTB-related diseases.
Assuntos
Actinas , Sistemas CRISPR-Cas , Edição de Genes , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Actinas/metabolismo , Actinas/genética , Mutação com Perda de Função , Diferenciação CelularRESUMO
Beta-actin (ACTB) heterozygous loss-of-function mutations are associated with pleiotropic developmental disorders entailing intellectual disability and frequent organ malformations in affected individuals. We generated two CRISPR/Cas9 prime-edited human induced pluripotent stem cell (iPSC) lines, IUFi004-A-1 and IUFi004-A-2, carrying a heterozygous missense mutation in exon 4 of ACTB. Mutant iPSCs exhibited normal cell morphology and genomic integrity, maintained expression of pluripotency markers, and differentiated into the three primary germ layers. The mutants offer a valuable platform for examining the molecular and functional consequences of ACTB haploinsufficiency, developing effective treatments, and exploring mechanisms underlying phenotypic variability and genetic compensation observed in monogenic diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Actinas/genética , Actinas/metabolismo , Mutação , Diferenciação Celular , Deficiência Intelectual/genética , Sistemas CRISPR-Cas/genéticaRESUMO
The attribution of seizure freedom is yet to be achieved for patients suffering from refractory epilepsy, e.g. Dravet Syndrome (DS). The confined ability of mono-chemical entity-based antiseizure drugs (ASDs) to act directly at genomic level is one of the factors, combined with undetermined seizure triggers lead to recurrent seizure (RS) in DS, abominably affecting the sub-genomic architecture of neural cells. Thus, the RS and ASD appear to be responsible for the spectrum of exorbitant clinical pathology. The RS distresses the 5-HT-serotonin pathway, hypomethylates genes of CNS, and modulates the microRNA (miRNA)/long non-coding RNA (lncRNA), eventually leading to frozen molecular alterations. These changes shall be reverted by compatible epigenetic regulators (EGR) like, miRNA and lncRNA from Breast milk (BML) and Bacopa monnieri (BMI). The absence of studious seizure in SCN1A mutation-positive babies for the first 6 months raises the possibility that the consequences of mutation in SCN1A are subsidized by EGRs from BML. EGR-dependent-modifier gene effect is likely imposed by the other members of the SCN family. Therefore, we advocate that miRNA/lncRNA from BML and bacosides/miRNA from BMI buffer the effect of SCN1A mutation by sustainably maintaining modifier gene effect in the aberrant neurons. The presence of miRNA-155-5p, -30b-5p, and -30c-5p family in BML and miR857, miR168, miR156, and miR158 in BMI target at regulating SCN family and CLCN5 as visualized by Cystoscope. Thus, we envisage that the possible effects of EGR might include (a) upregulating the haploinsufficient SCN1A strand, (b) down-regulating seizure-elevated miRNA, (c) suppressing the seizure-induced methyltransferases, and (d) enhancing the GluN2A subunit of NMDA receptor to improve cognition. The potential of these EGRs from BML and BML is to further experimentally strengthen, long-haul step forward in molecular therapeutics.
Assuntos
Epilepsia Resistente a Medicamentos , Epilepsias Mioclônicas , MicroRNAs , RNA Longo não Codificante , Lactente , Feminino , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Epilepsia Resistente a Medicamentos/genética , RNA Longo não Codificante/genética , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Convulsões , Mutação , MicroRNAs/genética , Epigênese GenéticaRESUMO
Human dermal fibroblasts from a Leigh Syndrome (LS) patient harboring the heterozygous NDUFS1 R557X/D618N compound mutation were reprogrammed to generate integration-free induced pluripotent stem cells (iPSCs). The full characterization of IUFi002-A-iPSCs demonstrated that the line is free of exogenous reprogramming genes and maintains the genomic integrity. IUFi002-A-iPSCs' pluripotency was confirmed by the expression of pluripotency markers and embryoid body-based differentiation into cell types representative of each of the three germ layers. The generated iPSC line provides a powerful tool to investigate LS and analyze the molecular mechanisms underlying NDUFS1 mutations-induced pathology.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Leigh , NADH Desidrogenase , Humanos , Genômica , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Leigh/genética , Doença de Leigh/patologia , Mutação , NADH Desidrogenase/genética , Linhagem CelularRESUMO
Due to the ubiquity of environmental micro- and nanoplastics (MNPs), inhalation and ingestion by humans is very likely, but human health effects remain largely unknown. The NLRP3 inflammasome is a key player of the innate immune system and is involved in responses towards foreign particulate matter and the development of chronic intestinal and respiratory inflammatory diseases. We established NLRP3-proficient and -deficient THP-1 cells as an alternative in vitro screening tool to assess the potential of MNPs to activate the NLRP3 inflammasome. By investigating cytokine release (IL-1ß and IL-8) and cytotoxicity after treatment with engineered nanomaterials, this in vitro approach was compared to earlier published ex vivo murine bone marrow-derived macrophages and in vivo data. This approach showed a strong correlation with previously published data, verifying that THP-1 cells are a suitable model to investigate NLRP3 inflammasome activation. We then investigated the proinflammatory potential of eight MNPs of different size, shape, and chemical composition. Only amine-modified polystyrene (PS-NH2) acted as a direct NLRP3 activator. However, polyethylene terephthalate (PET), polyacrylonitrile (PAN), and nylon (PA6) induced a significant increase in IL-8 release in NLRP3-/- cells. Our results suggest that most MNPs are not direct activators of the NLRP3 inflammasome, but specific MNP types might still possess pro-inflammatory potential via other pathways.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Humanos , Inflamassomos/metabolismo , Interleucina-8 , Camundongos , Microplásticos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1RESUMO
Genomic mutations are the driving force of biological diversity but they are also the cause of a plethora of human diseases ranging from heritable disorders to neurological pathologies and cancer. For most genetic disorders, there is no curative treatment available to date. The demand for precise, preferably patient-specific, treatment regimen offering cure is naturally high. Genome editing by Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas enables targeted manipulation of genomes, thereby offering the opportunity to treat such diseases. While ethical and regulatory guidelines need to be developed and considered, the prospect of genome editing for curative treatment is certainly exciting. Here, we review the current state of therapeutics based on genome editing techniques. We highlight recent breakthroughs, describe clinical trials employing genome editing-based medicine, discuss the benefits and pitfalls, and take a look into the future of genome editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma , Humanos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Ciência Translacional BiomédicaRESUMO
The NLRP3 inflammasome plays an important role in intestinal homeostasis as well as inflammation. However, in vivo studies investigating the role of the NLRP3 inflammasome in inflammatory bowel disease (IBD) report contrasting results, leaving it unclear if the NLRP3 inflammasome augments or attenuates intestinal inflammation. To investigate the role of the NLRP3/caspase-1 pathway in a model of acute intestinal inflammation, we modified a previously established in vitro triple culture model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1). Using THP-1 knockout cell lines, we analyzed how the NLRP3 inflammasome and its downstream enzyme caspase-1 (CASP1) affect inflammatory parameters including barrier integrity and cytotoxicity, as well as gene expression and secretion of pro-inflammatory cytokines and mucus. Furthermore, we investigated differences in inflammation-mediated cytotoxicity towards enterocyte-like (Caco-2) or goblet-like (HT29-MTX-E12) epithelial cells. As a complementary approach, inflammation-related cytotoxicity and gene expression of cytokines was analyzed in intestinal tissue explants from wildtype (WT) and Nlrp3-/- mice. Induction of intestinal inflammation impaired the barrier, caused cytotoxicity, and altered gene expression of pro-inflammatory cytokines and mucins in vitro, while the knockout of NLRP3 and CASP1 in THP 1 cells led to attenuation of these inflammatory parameters. The knockout of CASP1 tended to show a slightly stronger attenuating effect compared to the NLRP3 knockout model. We also found that the inflammation-mediated death of goblet-like cells is NLRP3/caspase-1 dependent. Furthermore, inflammation-related cytotoxicity and upregulation of pro-inflammatory cytokines was present in ileal tissue explants from WT, but not Nlrp3-/- mice. The here presented observations indicate a pro-inflammatory and adverse role of the NLRP3 inflammasome in macrophages during acute intestinal inflammation.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Células CACO-2 , Caspase 1/genética , Caspase 1/metabolismo , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Intestinos/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1RESUMO
Genome engineering-induced cleavage sites can be resolved by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Identifying genetically modified clones at the target locus remains an intensive and laborious task. Different workflows and software that rely on deep sequencing data have been developed to detect and quantify targeted mutagenesis. Nevertheless, these pipelines require high-quality reads generated by Next Generation Sequencing (NGS) platforms. Here, we have developed a robust, versatile, and easy-to-use computational webserver named CRISPRnano (www.CRISPRnano.de) that enables the analysis of low-quality reads generated by affordable and portable sequencers including Oxford Nanopore Technologies (ONT) devices. CRISPRnano allows fast and accurate identification, quantification, and visualization of genetically modified cell lines, it is compatible with NGS and ONT sequencing reads, and it can be used without an internet connection.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Nanoporos , Análise de Sequência de DNA , Software , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma , Edição de Genes/métodos , Fluxo de Trabalho , Mutagênese Sítio-Dirigida , Internet , Computadores , Visualização de Dados , Fatores de Tempo , Linhagem CelularRESUMO
The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K âR ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K âR or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K âR ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K âR cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.
Assuntos
Células-Tronco Embrionárias , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Diferenciação Celular/genética , Cromatina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods.
Assuntos
Separação Celular/métodos , Edição de Genes/métodos , Animais , Biomarcadores/análise , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma/genética , Células HEK293 , Humanos , Magnetismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.
Assuntos
Ácido Fólico/metabolismo , Interleucina-7/fisiologia , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética , Transativadores/metabolismo , Animais , Carbono/metabolismo , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Camundongos , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfócitos B/patologia , Ligação Proteica , Análise de Célula Única , Transativadores/genéticaRESUMO
Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children. Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment. The zinc-finger protein GLIS2/NPHP7 represents an exception as it has been implicated in transcriptional regulation; only two families with GLIS2/NPHP7 mutations and typical NPH manifestations have been identified so far. We describe here that the recently identified GLIS2/NPHP7(C175R) point mutation abolished the nuclear localization of GLIS2/NPHP7. Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein. We further observed that wild type, but not GLIS2/NPHP7(C175R), prevented the cyst formation caused by depletion of nphp7 in zebrafish embryos. Taken together, our findings indicate that the C175R mutation affects both localization and function of GLIS2/NPHP7, supporting a role of this mutation in NPH, but questioning the direct involvement of GLIS2/NPHP7 in ciliary functions.
Assuntos
Núcleo Celular/metabolismo , Doenças Renais Císticas/genética , Fatores de Transcrição Kruppel-Like/genética , Mutação de Sentido Incorreto , Transporte Ativo do Núcleo Celular , Animais , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Ligação Proteica , Ativação Transcricional , Peixe-ZebraRESUMO
Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Repetição de Anquirina , Proteínas de Transporte/genética , Citoplasma/metabolismo , Células HEK293 , Humanos , Doenças Renais Císticas/metabolismo , Camundongos , Peso Molecular , Complexos Multiproteicos/metabolismo , Mutação , Quinases Relacionadas a NIMA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ratos , Serina/metabolismo , Treonina/metabolismo , TransfecçãoRESUMO
Glis2/NPHP7 is a transcriptional regulator mutated in type 7 nephronophthisis, an autosomal recessive ciliopathy associated with cystic and fibrotic kidney disease as well as characteristic extrarenal manifestations. While most ciliopathy-associated molecules are found in the cilium, Glis2/NPHP7 presumably localizes to the nucleus. However, the detection of endogenous Glis2/NPHP7 has remained unsuccessful, potentially due to its ubiquitylation-dependent rapid degradation. We report now that Glis2/NPHP7 is also SUMOylated, preferentially by PIAS4, which conjugates Glis2/NPHP7 to SUMO3. SUMOylation interferes with ubiquitylation and degradation of Glis2/NPHP7, suggesting that Glis2/NPHP7 protein levels are regulated by competing ubiquitylation/ SUMOylation. SUMOylation also alters the transcriptional activity of Glis2/NPHP7. While Glis2/NPHP7 activates the mouse insulin-2-promotor (mIns2), SUMOylated Glis2/NPHP7 lacks this property, and seems to act as a repressor. Taken together, our data reveal that Glis2/NPHP7 is extensively modified by post-translational modifications, suggesting that a tight control of this transcriptional regulator is required for normal development and tissue homeostasis.
Assuntos
Doenças Renais Císticas/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteólise , Sumoilação , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/química , Camundongos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/metabolismo , Estabilidade Proteica , Ubiquitinas/metabolismoRESUMO
Nephronophthisis (NPH) is a heterogenetic autosomal recessive disorder associated with kidney cysts and multiple extrarenal manifestations. The disease-associated gene products (NPHPs) typically contain domains involved in protein-protein interactions, and appear to exert their tissue-specific functions in large protein complexes. Most NPHPs localize to the cilium and/or basal body; however, their precise molecular functions remain largely unknown. We have recently identified the SAM-domain containing protein Anks3 as a potential ANKS6/NPHP16-interacting protein, and report now that Anks3 interacts with several NPHPs as well as with Bicc1 and the oxygen-sensitive asparaginyl hydroxylase HIF1AN. Knockdown of anks3 in zebrafish embryos was associated with NPH-typical manifestations, including ciliary abnormalities, cyst formation, and laterality defects. In multi-ciliated epidermal cells, GFP-tagged Anks3 localizes to the cilium, but forms large aggregates in the absence of NPHP1, indicating that the negatively charged NPHP1 curtails the polymerization of Anks3. Collectively, these findings suggest that Anks3 is a cilia-associated molecule that partners with the ANKS6- and via NPHP1 to the NPHP1-4-8 module. Thus, developmental defects associated with Anks3 depletion in zebrafish suggest that ANKS3 mutations may cause NPH or NPH-like disease in humans.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Rim/embriologia , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cílios/metabolismo , Proteínas do Citoesqueleto , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Rim/anormalidades , Doenças Renais Císticas/metabolismo , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Quinases Relacionadas a NIMA , Proteínas Nucleares/metabolismo , Polimerização , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Situs Inversus/genética , Xenopus , Proteínas de Xenopus/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Although the two ciliopathies Bardet-Biedl syndrome and nephronophthisis share multiple clinical manifestations, the molecular basis for this overlap remains largely unknown. Both BBS11 and NPHP7 are unusual members of their respective gene families. Although BBS11/TRIM32 represents a RING finger E3 ubiquitin ligase also involved in hereditary forms of muscular dystrophy, NPHP7/Glis2 is a Gli-like transcriptional repressor that localizes to the nucleus, deviating from the ciliary localization of most other ciliopathy-associated gene products. We found that BBS11/TRIM32 and NPHP7/Glis2 can physically interact with each other, suggesting that both proteins form a functionally relevant protein complex in vivo. This hypothesis was further supported by the genetic interaction and synergist cyst formation in the zebrafish pronephros model. However, contrary to our expectation, the E3 ubiquitin ligase BBS11/TRIM32 was not responsible for the short half-life of NPHP7/Glis2 but instead promoted the accumulation of mixed Lys(48)/Lys(63)-polyubiquitylated NPHP7/Glis2 species. This modification not only prolonged the half-life of NPHP7/Glis2, but also altered the subnuclear localization and the transcriptional activity of NPHP7/Glis2. Thus, physical and functional interactions between NPHP and Bardet-Biedl syndrome gene products, demonstrated for Glis2 and TRIM32, may help to explain the phenotypic similarities between these two syndromes.
Assuntos
Síndrome de Bardet-Biedl/metabolismo , Doenças Renais Císticas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Animais , Síndrome de Bardet-Biedl/genética , Células HEK293 , Humanos , Doenças Renais Císticas/genética , Fatores de Transcrição Kruppel-Like/análise , Fatores de Transcrição Kruppel-Like/genética , Mapas de Interação de Proteínas , Transporte Proteico , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Peixe-ZebraRESUMO
Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1-4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.
Assuntos
Rim/embriologia , Rim/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Serina-Treonina Quinase 3 , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.