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1.
Nature ; 586(7827): 151-155, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968275

RESUMO

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.


Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/metabolismo , Animais , Biocatálise , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Metilação de DNA , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética
2.
Nature ; 585(7826): 563-568, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939088

RESUMO

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.


Assuntos
Endotelinas/metabolismo , Evolução Molecular , Crista Neural/citologia , Petromyzon/metabolismo , Transdução de Sinais , Xenopus/metabolismo , Animais , Desenvolvimento Ósseo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Linhagem da Célula , Endotelinas/genética , Feminino , Cabeça/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Ligantes , Masculino , Petromyzon/genética , Petromyzon/crescimento & desenvolvimento , Receptores de Endotelina/deficiência , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismo , Xenopus/genética , Xenopus/crescimento & desenvolvimento
3.
PLoS Genet ; 16(8): e1008967, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813698

RESUMO

Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.


Assuntos
Deficiências do Desenvolvimento/genética , Desenvolvimento Embrionário/genética , Fator de Transcrição PAX9/genética , Ribossomos/genética , Animais , Nucléolo Celular/genética , Deficiências do Desenvolvimento/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Crista Neural/patologia , Biossíntese de Proteínas/genética , RNA Polimerase II/genética , Ribossomos/patologia , Xenopus/genética , Xenopus/crescimento & desenvolvimento
4.
Mol Pharmacol ; 98(4): 328-342, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690626

RESUMO

Epibatidine is a potent analgetic agent with very high affinity for brain nicotinic acetylcholine receptors (nAChR). We determined the activity profiles of three epibatidine derivatives, RTI-36, RTI-76, and RTI-102, which have affinity for brain nAChR equivalent to that of epibatidine but reduced analgetic activity. RNAs coding for nAChR monomeric subunits and/or concatamers were injected into Xenopus oocytes to obtain receptors of defined subunit composition and stoichiometry. The epibatidine analogs produced protracted activation of high sensitivity (HS) α4- and α2-containing receptors with the stoichiometry of 2alpha:3beta subunits but not low sensitivity (LS) receptors with the reverse ratio of alpha and beta subunits. Although not strongly activated by the epibatidine analogs, LS α4- and α2-containing receptors were potently desensitized by the epibatidine analogs. In general, the responses of α4(2)ß2(2)α5 and ß3α4ß2α6ß2 receptors were similar to those of the HS α4ß2 receptors. RTI-36, the analog closest in structure to epibatidine, was the most efficacious of the three compounds, also effectively activating α7 and α3ß4 receptors, albeit with lower potency and less desensitizing effect. Although not the most efficacious agonist, RTI-76 was the most potent desensitizer of α4- and α2-containing receptors. RTI-102, a strong partial agonist for HS α4ß2 receptors, was effectively an antagonist for LS α4ß2 receptors. Our results highlight the importance of subunit stoichiometry and the presence or absence of specific accessory subunits for determining the activity of these drugs on brain nAChR, affecting the interpretation of in vivo studies since in most cases these structural details are not known. SIGNIFICANCE STATEMENT: Epibatidine and related compounds are potent ligands for the high-affinity nicotine receptors of the brain, which are therapeutic targets and mediators of nicotine addiction. Far from being a homogeneous population, these receptors are diverse in subunit composition and vary in subunit stoichiometry. We show the importance of these structural details for drug activity profiles, which present a challenge for the interpretation of in vivo experiments since conventional methods, such as in situ hybridization and immunohistochemistry, cannot illuminate these details.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Agonistas Nicotínicos/farmacologia , Subunidades Proteicas/metabolismo , Piridinas/química , Receptores Nicotínicos/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Humanos , Estrutura Molecular , Complexos Multiproteicos/metabolismo , Agonistas Nicotínicos/química , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Tropanos/química , Tropanos/farmacologia , Xenopus/genética
5.
Am J Physiol Cell Physiol ; 319(2): C359-C370, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520610

RESUMO

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.


Assuntos
Proteínas de Transporte de Ânions/genética , Transporte Biológico/genética , Distrofias Hereditárias da Córnea/genética , Lisina/genética , Simportadores/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bicarbonatos/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Modelos Animais de Doenças , Epitélio Posterior/metabolismo , Epitélio Posterior/patologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Lisina/metabolismo , Camundongos , Mutação/genética , Oócitos/metabolismo , Oócitos/patologia , Sódio , Xenopus/genética
6.
Arch Biochem Biophys ; 689: 108436, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32492375

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium (Kv) and cyclic nucleotide-gated (CNG) channels. HCN channels contain the glycine-tyrosine-glycine (GYG) sequence that forms part of the selectivity filter, a similar structure than some potassium channels; however, they permeate both sodium and potassium, giving rise to an inward current. Yet a second amino acid sequence, leucine-cysteine-isoleucine (LCI), next to GYG, is well-preserved in all HCNs but not in the selective potassium channels. In this study we used site-directed mutagenesis and electrophysiology in frog oocytes to determine whether the LCI sequence affects the kinetics of HCN2 currents. Permeability and voltage dependence were evaluated, and we found a role of LCI in the gating mechanism combined with changes in ion permeability. The I residue resulted critical to this function.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Ativação do Canal Iônico , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Permeabilidade , Potássio/metabolismo , Sódio/metabolismo , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
7.
Am J Physiol Cell Physiol ; 319(2): C371-C380, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579473

RESUMO

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.


Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
8.
Genes Cells ; 25(7): 498-509, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32323394

RESUMO

Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Xenopus/genética , Animais , Feminino , Mutação da Fase de Leitura , Biblioteca Gênica , Estudos de Associação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação INDEL , Masculino , Fenótipo , Software , Fluxo de Trabalho
9.
Nucleic Acids Res ; 48(6): 3014-3028, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31980815

RESUMO

The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.


Assuntos
Replicação do DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , N-Glicosil Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Fibroblastos/metabolismo , Ficusina/farmacologia , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Xenopus/genética
10.
Mol Biol Evol ; 37(1): 149-166, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553476

RESUMO

Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.


Assuntos
RNA Nucleolar Pequeno/genética , Xenopus/genética , Animais , Genoma , Humanos , Anotação de Sequência Molecular , Mutação Puntual
11.
Biochim Biophys Acta Gen Subj ; 1864(3): 129482, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31734465

RESUMO

BACKGROUND: Asymmetric arginine dimethylation of histone H4R3 to H4R3me2a by protein arginine methyltransferase 1 (PRMT1) has been implicated to play a key role in gene activation throughout vertebrates. PRMT1 knockout in mouse leads to embryonic lethality. This and the uterus-enclosed nature of the mouse embryo make it difficult to determine the development role of PRMT1 in mammals. METHODS: We took advantage of the external development of the diploid anuran Xenopus tropicalis and adapted the TALEN genome editing technology to knock out PRMT1 in order to investigate how PRMT1 participates in vertebrate development. RESULTS: We observed that PRMT1 knockout had no apparent effect on embryogenesis because normally feeding tadpoles were formed, despite the reduced asymmetric H4R3 di-methylation (H4R3me2a) due to the knockout. However, PRMT1 knockout tadpoles had severely reduced growth even with normal growth hormone gene expression. These tadpoles were also stalled in development shortly after feeding began at stages 44/45 and died within 2 weeks, well before the onset of metamorphosis. In situ analyses revealed broad cessation or drastic reduction in cell proliferation in diverse organs including the eye, brain, spinal cord, liver, and intestine. CONCLUSIONS: Our findings suggest that PRMT1 is not required for embryogenesis but is a key regulator for normal progression of vertebrate development and growth. GENERAL SIGNIFICANCE: The similarities and differences between PRMT1 knockout Xenopus tropicalis and mouse suggest that two distinct phases of vertebrate development: early embryogenesis and subsequent growth/organ maturation, have different but evolutionally conserved requirement for epigenetic modifications.


Assuntos
Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Proliferação de Células , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes/métodos , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Larva/metabolismo , Masculino , Metamorfose Biológica/genética , Metamorfose Biológica/fisiologia , Metilação , Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Ativação Transcricional , Xenopus/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
12.
Nucleic Acids Res ; 48(D1): D776-D782, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31733057

RESUMO

Xenbase (www.xenbase.org) is a knowledge base for researchers and biomedical scientists that employ the amphibian Xenopus as a model organism in biomedical research to gain a deeper understanding of developmental and disease processes. Through expert curation and automated data provisioning from various sources Xenbase strives to integrate the body of knowledge on Xenopus genomics and biology together with the visualization of biologically significant interactions. Most current studies utilize next generation sequencing (NGS) but until now the results of different experiments were difficult to compare and not integrated with other Xenbase content. Xenbase has developed a suite of tools, interfaces and data processing pipelines that transforms NCBI Gene Expression Omnibus (GEO) NGS content into deeply integrated gene expression and chromatin data, mapping all aligned reads to the most recent genome builds. This content can be queried and visualized via multiple tools and also provides the basis for future automated 'gene expression as a phenotype' and gene regulatory network analyses.


Assuntos
Bases de Dados Genéticas , Redes Reguladoras de Genes/genética , Genômica , Software , Xenopus/genética , Animais , Sequenciamento de Cromatina por Imunoprecipitação , Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA-Seq , Interface Usuário-Computador
13.
Nucleic Acids Res ; 48(4): 1925-1940, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31828326

RESUMO

DNA single-strand breaks (SSBs) represent the most abundant type of DNA damage. Unrepaired SSBs impair DNA replication and transcription, leading to cancer and neurodegenerative disorders. Although PARP1 and XRCC1 are implicated in the SSB repair pathway, it remains unclear how SSB repair and SSB signaling pathways are coordinated and regulated. Using Xenopus egg extract and in vitro reconstitution systems, here we show that SSBs are first sensed by APE1 to initiate 3'-5' SSB end resection, followed by APE2 recruitment to continue SSB end resection. Notably, APE1's exonuclease activity is critical for SSB repair and SSB signaling pathways. An APE1 exonuclease-deficient mutant identified in somatic tissue from a cancer patient highlighted the significance of APE1 exonuclease activity in cancer etiology. In addition, APE1 interacts with APE2 and PCNA, although PCNA is dispensable for APE1's exonuclease activity. Taken together, we propose a two-step APE1/APE2-mediated mechanism for SSB end resection that couples DNA damage response with SSB repair in a eukaryotic system.


Assuntos
Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Enzimas Multifuncionais/genética , Proteínas de Xenopus/genética , Animais , Quebras de DNA de Cadeia Simples , Dano ao DNA/genética , Replicação do DNA/genética , Humanos , Transdução de Sinais/genética , Xenopus/genética , Xenopus/crescimento & desenvolvimento
14.
Hum Mol Genet ; 29(2): 305-319, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31813957

RESUMO

Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.


Assuntos
Anormalidades Múltiplas/enzimologia , Face/anormalidades , Doenças Hematológicas/enzimologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Crista Neural/metabolismo , Doenças Vestibulares/enzimologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Acetilação , Animais , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Face/patologia , Doenças Hematológicas/genética , Doenças Hematológicas/metabolismo , Doenças Hematológicas/patologia , Histonas/metabolismo , Mutação com Perda de Função , Metilação , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Crista Neural/enzimologia , Crista Neural/patologia , Placa Neural/crescimento & desenvolvimento , Placa Neural/metabolismo , Placa Neural/patologia , Semaforinas/genética , Semaforinas/metabolismo , Doenças Vestibulares/genética , Doenças Vestibulares/metabolismo , Doenças Vestibulares/patologia , Xenopus/embriologia , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/fisiologia
15.
Methods Mol Biol ; 2047: 377-395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31552666

RESUMO

Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function. MOs are widely used to alter gene expression in development of Xenopus and Zebrafish, where they are typically injected into the fertilized egg or blastomeres. Here we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells of the brain with current passing between 2 platinum plate electrodes, positioned on either side of the target brain area. The method is relatively straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell type-specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.


Assuntos
Morfolinos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Xenopus/crescimento & desenvolvimento , Animais , Encéfalo/crescimento & desenvolvimento , Fenômenos Eletrofisiológicos , Eletroporação/instrumentação , Técnicas de Silenciamento de Genes , Morfolinos/farmacologia , Fenótipo , Imagem com Lapso de Tempo , Xenopus/genética
16.
Gen Comp Endocrinol ; 287: 113349, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31794731

RESUMO

Anuran metamorphosis is perhaps the most dramatic developmental process regulated by thyroid hormone (TH). One of the unique processes that occur during metamorphosis is the complete resorption of the tail, including the notochord. Interestingly, recent gene knockout studies have shown that of the two known vertebrate TH receptors, TRα and TRß, TRß appears to be critical for notochord regression during tail resorption in Xenopus tropicalis. To determine the mechanisms underlying notochord regression, we carried out a comprehensive gene expression analysis in the notochord during metamorphosis by using RNA-Seq analyses of whole tail at stage 60 before any noticeable tail length reduction, whole tail at stage 63 when the tail length is reduced by about one half, and the rest of the tail at stage 63 after removing the notochord. This allowed us to identify many notochord-enriched, metamorphosis-induced genes at stage 63. Future studies on these genes should help to determine if they are regulated by TRß and play any roles in notochord regression.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Notocorda/crescimento & desenvolvimento , RNA-Seq/métodos , Cauda/crescimento & desenvolvimento , Xenopus laevis/crescimento & desenvolvimento , Xenopus/genética , Animais
17.
Elife ; 82019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31818368

RESUMO

Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication.


Assuntos
Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Receptores de Feromonas/metabolismo , Atrativos Sexuais/metabolismo , Animais , Comportamento Animal , Sistemas CRISPR-Cas , Drosophila/genética , Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Lepidópteros/genética , Lepidópteros/metabolismo , Masculino , Mariposas/genética , Receptores Odorantes , Receptores de Feromonas/classificação , Receptores de Feromonas/genética , Spodoptera/genética , Spodoptera/metabolismo , Transcriptoma , Xenopus/genética , Xenopus/metabolismo
18.
Biochem J ; 476(24): 3791-3804, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31808793

RESUMO

Oxidative DNA damage represents one of the most abundant DNA lesions. It remains unclear how DNA repair and DNA damage response (DDR) pathways are co-ordinated and regulated following oxidative stress. While XRCC1 has been implicated in DNA repair, it remains unknown how exactly oxidative DNA damage is repaired and sensed by XRCC1. In this communication, we have demonstrated evidence that XRCC1 is dispensable for ATR-Chk1 DDR pathway following oxidative stress in Xenopus egg extracts. Whereas APE2 is essential for SSB repair, XRCC1 is not required for the repair of defined SSB and gapped plasmids with a 5'-OH or 5'-P terminus, suggesting that XRCC1 and APE2 may contribute to SSB repair via different mechanisms. Neither Polymerase beta nor Polymerase alpha is important for the repair of defined SSB structure. Nonetheless, XRCC1 is important for the repair of DNA damage following oxidative stress. Our observations suggest distinct roles of XRCC1 for genome integrity in oxidative stress in Xenopus egg extracts.


Assuntos
Extratos Celulares/química , Genoma , Óvulo/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Xenopus/metabolismo , Animais , Cromatina , Dano ao DNA , Reparo do DNA , Regulação da Expressão Gênica , Masculino , Óvulo/química , Estresse Oxidativo , Plasmídeos , Espermatozoides , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Xenopus/genética
19.
Nat Commun ; 10(1): 4269, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537794

RESUMO

Embryonic development yields many different cell types in response to just a few families of inductive signals. The property of signal-receiving cells that determines how they respond to inductive signals is known as competence, and it differs in different cell types. Here, we explore the ways in which maternal factors modify chromatin to specify initial competence in the frog Xenopus tropicalis. We identify early-engaged regulatory DNA sequences, and infer from them critical activators of the zygotic genome. Of these, we show that the pioneering activity of the maternal pluripotency factors Pou5f3 and Sox3 determines competence for germ layer formation by extensively remodelling compacted chromatin before the onset of inductive signalling. This remodelling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work identifies significant developmental principles that inform our understanding of how pluripotent stem cells interpret inductive signals.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/crescimento & desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Xenopus/genética
20.
Sci Rep ; 9(1): 13543, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537823

RESUMO

Here, we show that human Connexin 26 (hCx26 or Cx26WT) hemichannel opening rapidly enables the transport of small molecules when triggered by temperature and by compensation of the Ca2+ blockade with EDTA. Point mutations within Cx26 were analysed by a novel optical microarray-based Lucifer Yellow uptake assay or by two electrode voltage clamp (TEVC) on frog oocytes to monitor simultaneous activities of channel proteins. Point mutations L90P, F161S, R184P or K188N influenced the temperature-dependent activity drastically. Since several mutations blocked trafficking, the temperature-dependent activity of the recombinant synthesized and purified wild-type Cx26WT and Cx26K188N hemichannel was tested by liposome flux assay (LFA) and on a microarray-based Lucifer Yellow uptake assay under warm conditions (>30 °C). The data from TEVC measurements and dye flux experiments showed that the mutations gave no or only a weak activity at increased temperature (>30 °C). We conclude that the position K188 in the Cx26WT forms a temperature-sensitive salt bridge with E47 whereas the exchange to K188N destabilizes the network loop- gating filter, which was recently identified as a part of the flexible Ca2+ binding site. We assume that the temperature sensitivity of Cx26 is required to protect cells from uncontrolled release or uptake activities through Cx26 hemichannels.


Assuntos
Conexina 26/genética , Conexina 26/fisiologia , Animais , Cálcio/metabolismo , Junções Comunicantes/metabolismo , Humanos , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oócitos/metabolismo , Porinas/genética , Porinas/metabolismo , Transporte Proteico/fisiologia , Temperatura , Xenopus/genética
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