RESUMO
Wnt signaling is essential for many aspects of embryonic development including the formation of the primary embryonic axis. In addition, excessive Wnt signaling drives multiple diseases including cancer, highlighting its importance for disease pathogenesis. ß-catenin is a key effector in this pathway that translocates into the nucleus and activates Wnt responsive genes. However, due to our lack of understanding of ß-catenin nuclear transport, therapeutic modulation of Wnt signaling has been challenging. Here, we took an unconventional approach to address this long-standing question by exploiting a heterologous model system, the budding yeast Saccharomyces cerevisiae, which contains a conserved nuclear transport machinery. In contrast to prior work, we demonstrate that ß-catenin accumulates in the nucleus in a Ran-dependent manner, suggesting the use of a nuclear transport receptor (NTR). Indeed, a systematic and conditional inhibition of NTRs revealed that only Kap104, the ortholog of Kap-ß2/Transportin-1 (TNPO1), was required for ß-catenin nuclear import. We further demonstrate direct binding between TNPO1 and ß-catenin that is mediated by a conserved PY-NLS. Finally, using Xenopus secondary axis and TCF/LEF (T Cell factor/lymphoid enhancer factor family) reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the nuclear localization signal in ß-catenin and its cognate NTR, our study suggests new therapeutic targets for a host of human diseases caused by excessive Wnt signaling. Indeed, we demonstrate that a small chimeric peptide designed to target TNPO1 can reduce Wnt signaling as a first step toward therapeutics.
Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , Humanos , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Carioferinas/metabolismo , Fatores de Transcrição/metabolismo , Sinais de Localização Nuclear/metabolismoRESUMO
Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with ß-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Complexos Multiproteicos/metabolismo , Crista Neural/embriologia , Transtornos do Neurodesenvolvimento/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Membranas Intracelulares/patologia , Complexos Multiproteicos/genética , Crista Neural/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Frog model organisms have been appreciated for their utility in exploring physiological phenomena for nearly a century. Now, a vibrant community of biologists that utilize this model organism has poised Xenopus to serve as a high throughput vertebrate organism to model patient-driven genetic diseases. This has facilitated the investigation of effects of patient mutations on specific organs and signaling pathways. This approach promises a rapid investigation into novel mechanisms that disrupt normal organ morphology and function. Considering that many disease states are still interrogated in vitro to determine relevant biological processes for further study, the prospect of interrogating genetic disease in Xenopus in vivo is an attractive alternative. This model may more closely capture important aspects of the pathology under investigation such as cellular micro environments and local forces relevant to a specific organ's development and homeostasis. This review aims to highlight recent methodological advances that allow investigation of genetic disease in organ-specific contexts in Xenopus as well as provide examples of how these methods have led to the identification of novel mechanisms and pathways important for understanding human disease.
RESUMO
Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.
Assuntos
Regiões 3' não Traduzidas/genética , Células Germinativas/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Animais , Linhagem Celular Tumoral , Hidrazinas/farmacologia , Camundongos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Peixe-ZebraRESUMO
In recent years, the zebrafish has become a critical contributor to various areas of biomedical research, advancing our fundamental understanding of biomedicine and helping discover candidate therapeutics for human diseases. Nevertheless, to further extend the power of this important model organism requires a robust and simple-to-use genome editing platform that will enable targeted gene knockouts and introduction of specific mutations identified in human diseases into the zebrafish genome. We describe here protocols for creating insertion or deletion (indel) mutations or precise sequence modifications in zebrafish genes using customizable CRISPR-Cas9 RNA-guided nucleases (RGNs). These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Desoxirribonucleases/metabolismo , Engenharia Genética/métodos , Mutagênese , RNA/genética , Peixe-Zebra/genética , Animais , Embrião não Mamífero/metabolismo , Vetores Genéticos/genética , Mutação , Peixe-Zebra/embriologiaRESUMO
The transcription activator-like effector (TALE) nucleases, or TALENs, are customizable restriction enzymes that may be used to induce mutations at nearly any investigator-specified DNA sequence in zebrafish. The DNA-binding specificities of TALENs are determined by a protein array comprised of four types of TALE repeats, where each repeat recognizes a different DNA base. Here, we describe methods for constructing TALEN vectors that have been shown to achieve high success rates and mutation efficiencies in zebrafish. In addition, we discuss simple techniques and protocols that can be used to detect TALEN-induced mutations at almost any genomic locus. These methods should enable zebrafish researchers to quickly generate targeted mutations at their genes-of-interest.
Assuntos
Mutagênese Sítio-Dirigida/métodos , Peixe-Zebra/genética , Animais , Desoxirribonucleases/química , Desoxirribonucleases/genética , Engenharia de ProteínasRESUMO
We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.
Assuntos
Sistemas CRISPR-Cas , Genoma , Peixe-Zebra/genética , Animais , Sequência de Bases , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Engenharia Genética , Mutação em Linhagem Germinativa , Mutação INDEL , Dados de Sequência Molecular , Mutagênese Insercional , Taxa de Mutação , Oligonucleotídeos/química , Oligonucleotídeos/genética , Alinhamento de SequênciaRESUMO
In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.
Assuntos
Genoma , Sequências Repetidas Invertidas , Peixe-Zebra/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , DNA/genética , Clivagem do DNA , Embrião não Mamífero , Endonucleases/genética , Engenharia Genética , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Edição de RNA , Pequeno RNA não TraduzidoRESUMO
Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Técnicas de Inativação de Genes , Mutação , Peixe-Zebra/genética , Alelos , Animais , Sequência de Bases , Proteínas de Ligação a DNA/química , Dimerização , Dados de Sequência Molecular , Mutagênese , Transativadores/químicaRESUMO
Impairments in mitochondria and transcription are important factors in the pathogenesis of Huntington disease (HD), a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. This study investigated the effect of different metabolic states and peroxisome proliferator-activated receptor γ (PPARγ) activation on sensitivity to cellular stressors such as H(2)O(2) or thapsigargin in HD. Striatal precursor cells expressing wild type (STHdh(Q7)) or mutant huntingtin (STHdh(Q111)) were prepared in different metabolic conditions (glucose vs. pyruvate). Due to the fact that STHdh(Q111) cells exhibit mitochondrial deficits, we expected that in the pyruvate condition, where ATP is generated primarily by the mitochondria, there would be greater differences in cell death between the two cell types compared to the glucose condition. Intriguingly, it was the glucose condition that gave rise to greater differences in cell death. In the glucose condition, thapsigargin treatment resulted in a more rapid loss of mitochondrial membrane potential (ΔΨm), a greater activation of caspases (3, 8, and 9), and a significant increase in superoxide/reactive oxygen species (ROS) in STHdh(Q111) compared to STHdh(Q7), while both cell types showed similar kinetics of ΔΨm-loss and similar levels of superoxide/ROS in the pyruvate condition. This suggests that bioenergetic deficiencies are not the primary contributor to the enhanced sensitivity of STHdh(Q111) cells to stressors compared to the STHdh(Q7) cells. PPARγ activation significantly attenuated thapsigargin-induced cell death, concomitant with an inhibition of caspase activation, a delay in ΔΨm loss, and a reduction of superoxide/ROS generation in STHdh(Q111) cells. Expression of mutant huntingtin in primary neurons induced superoxide/ROS, an effect that was significantly reduced by constitutively active PPARγ. These results provide significant insight into the bioenergetic disturbances in HD with PPARγ being a potential therapeutic target for HD.