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1.
Nat Commun ; 12(1): 714, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514705

RESUMO

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase critical for maintaining gene silencing during eukaryotic development. In mammals, PRC2 activity is regulated in part by the selective incorporation of one of two paralogs of the catalytic subunit, EZH1 or EZH2. Each of these enzymes has specialized biological functions that may be partially explained by differences in the multivalent interactions they mediate with chromatin. Here, we present two cryo-EM structures of PRC2:EZH1, one as a monomer and a second one as a dimer bound to a nucleosome. When bound to nucleosome substrate, the PRC2:EZH1 dimer undergoes a dramatic conformational change. We demonstrate that mutation of a divergent EZH1/2 loop abrogates the nucleosome-binding and methyltransferase activities of PRC2:EZH1. Finally, we show that PRC2:EZH1 dimers are more effective than monomers at promoting chromatin compaction, and the divergent EZH1/2 loop is essential for this function, thereby tying together the methyltransferase, nucleosome-binding, and chromatin-compaction activities of PRC2:EZH1. We speculate that the conformational flexibility and the ability to dimerize enable PRC2 to act on the varied chromatin substrates it encounters in the cell.


Assuntos
Cromatina/metabolismo , Inativação Gênica , Complexo Repressor Polycomb 2/ultraestrutura , Animais , Linhagem Celular , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Mutação , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Multimerização Proteica , Células Sf9 , Spodoptera , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
2.
Nat Commun ; 12(1): 568, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495454

RESUMO

The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon's exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Eletrônica , Mutação , Multimerização Proteica , Schizosaccharomyces , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
3.
Nucleic Acids Res ; 48(17): 9538-9549, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32766790

RESUMO

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.


Assuntos
DNA/metabolismo , Histonas/síntese química , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatina/química , Cromatina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histonas/genética , Lisina/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
4.
Nucleic Acids Res ; 48(15): 8782-8795, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633758

RESUMO

The stability and processing of cellular RNA transcripts are efficiently controlled via non-templated addition of single or multiple nucleotides, which is catalyzed by various nucleotidyltransferases including poly(A) polymerases (PAPs). Germline development defective 2 (GLD-2) is among the first reported cytoplasmic non-canonical PAPs that promotes the translation of germline-specific mRNAs by extending their short poly(A) tails in metazoan, such as Caenorhabditis elegans and Xenopus. On the other hand, the function of mammalian GLD-2 seems more diverse, which includes monoadenylation of certain microRNAs. To understand the structural basis that underlies the difference between mammalian and non-mammalian GLD-2 proteins, we determine crystal structures of two rodent GLD-2s. Different from C. elegans GLD-2, mammalian GLD-2 is an intrinsically robust PAP with an extensively positively charged surface. Rodent and C. elegans GLD-2s have a topological difference in the ß-sheet region of the central domain. Whereas C. elegans GLD-2 prefers adenosine-rich RNA substrates, mammalian GLD-2 can work on RNA oligos with various sequences. Coincident with its activity on microRNAs, mammalian GLD-2 structurally resembles the mRNA and miRNA processor terminal uridylyltransferase 7 (TUT7). Our study reveals how GLD-2 structurally evolves to a more versatile nucleotidyltransferase, and provides important clues in understanding its biological function in mammals.


Assuntos
Proteínas de Caenorhabditis elegans/genética , MicroRNAs/genética , Nucleotidiltransferases/genética , Polinucleotídeo Adenililtransferase/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Xenopus/genética , Animais , Caenorhabditis elegans/genética , Citoplasma/genética , Células Germinativas/crescimento & desenvolvimento , Mamíferos , Poli A/genética , Interferência de RNA
5.
PLoS Biol ; 18(7): e3000750, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667916

RESUMO

Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation-of the order of 10 pN-applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods' phototransduction.


Assuntos
Transdução de Sinal Luminoso , Mecanotransdução Celular , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Xenopus laevis/metabolismo , Animais , Cálcio/metabolismo , Fluorescência , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Mecanotransdução Celular/efeitos da radiação , Família Multigênica , Estimulação Luminosa , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Canais de Cátion TRPC/genética , Proteínas de 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.
BMC Mol Cell Biol ; 21(1): 39, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32466750

RESUMO

BACKGROUND: Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFß ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFß signaling network. RESULTS: Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression. CONCLUSION: Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA-binding activity is required for repression of TGFß signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling.


Assuntos
Ativinas/farmacologia , Ectoderma/metabolismo , Camadas Germinativas/metabolismo , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Fosforilação , Domínios Proteicos/genética , Transdução de Sinais/genética , Proteína Smad1/genética , Proteína Smad2/genética , Proteínas com Domínio T/química , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis
9.
J Mol Biol ; 432(10): 3149-3158, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32277988

RESUMO

Ligand-independent activation of receptor tyrosine kinases (RTKs) allows for dissecting out the receptor-specific signaling outcomes from the pleiotropic effects of the ligands. In this regard, RTK intracellular domains (ICD) are of interest due to their ability to recapitulate signaling activity in a ligand-independent manner when fused to chemical or optical dimerizing domains. A common strategy for synthetic activation of RTKs involves membrane tethering of dimerizer-RTK ICD fusions. Depending on the intrinsic signaling capacity, however, this approach could entail undesirable baseline signaling activity in the absence of stimulus, thereby diminishing the system's sensitivity. Here, we observed toxicity in early Xenopus laevis embryos when using such a conventional optogenetic design for the fibroblast growth factor receptor (FGFR). To surpass this challenge, we developed a cytoplasm-to-membrane translocation approach, where FGFR ICD is recruited from the cytoplasm to the plasma membrane by light, followed by its subsequent activation via homo-association. This strategy results in the optical activation of FGFR with low background activity and high sensitivity, which allows for the light-mediated formation of ectopic tail-like structures in developing X. laevis embryos. We further generalized this strategy by developing optogenetic platforms to control three neurotrophic tropomyosin receptor kinases, TrkA, TrkB, and TrkC. We envision that these ligand-independent optogenetic RTKs will provide useful toolsets for the delineation of signaling sub-circuits in developing vertebrate embryos.


Assuntos
Optogenética/métodos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Animais , Membrana Celular/metabolismo , Citoplasma/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Humanos , Transporte Proteico , Receptores Proteína Tirosina Quinases/química , Transdução de Sinais , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética
10.
Nat Commun ; 11(1): 1345, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165637

RESUMO

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Blástula/embriologia , Blástula/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade , Histonas/química , Histonas/genética , Domínios Proteicos , Origem de Replicação , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
11.
PLoS Genet ; 16(2): e1008590, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32053595

RESUMO

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development.


Assuntos
Encéfalo/embriologia , Cromossomos Humanos Par 3/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Deficiência Intelectual/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Xenopus/genética , Animais , Apoptose/genética , Encéfalo/patologia , Ciclo Celular/genética , Deleção Cromossômica , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Deficiência Intelectual/patologia , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis
12.
J Clin Invest ; 130(2): 813-826, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31904590

RESUMO

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/genética
13.
Genesis ; 58(3-4): e23354, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31909537

RESUMO

To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.


Assuntos
Olho/embriologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Interferência de RNA , Xenopus laevis/embriologia , Xenopus laevis/genética , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Estudos de Associação Genética , Mutação com Perda de Função , Organogênese/genética , Fenótipo , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
14.
Methods Mol Biol ; 2041: 87-106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646482

RESUMO

Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.


Assuntos
5'-Nucleotidase/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , Receptores Purinérgicos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , 5'-Nucleotidase/genética , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Feminino , Sondas RNA , Receptores Purinérgicos/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
15.
Biochem Biophys Res Commun ; 521(4): 907-913, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31711643

RESUMO

ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I motifs) enzymes play an important role in various morphogenesis processes. To determine the functions of Adamts18 in the early stages of organogenesis, we created Adamts18 deficient zebrafish using morpholino antisense oligonucleotides (MO) to generate exon 3 skipped adamts18 mRNA transcripts. Results showed that Adamts18 deficiency in zebrafish embryos caused developmental defects, including expanded brain ventricle and hindbrain edema, eye defects, and accumulation of blood in the caudal vein. Adamts18 deficiency also led to impaired trunk angiogenesis and formation of the caudal vein plexus (CVP). Consequently, Adamts18 deficient zebrafish embryos exhibited incomplete formation of intersegment vessels (ISVs), disruption of the honeycomb structure of CVP, and reduced CVP area and loop number. Furthermore, Adamts18 deficiency resulted in impaired blood circulation in major trunk, caudal vein (CV), and common cardinal vein (CCV). These aberrant vascular phenotypes in mutant zebrafish embryos were shown to be associated with a decreased expression of multiple angiogenesis-related signaling genes, including slit/robo, dll4/Notch, cox2, and fgfr. These findings indicate the critical role of Adamts18 in the early stages of vascular network development.


Assuntos
Metaloendopeptidases/genética , Neovascularização Fisiológica/genética , Veias/embriologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Proteínas ADAMTS , Animais , Animais Geneticamente Modificados , Circulação Sanguínea/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Metaloendopeptidases/metabolismo , Oligonucleotídeos Antissenso/genética , Proteínas de Xenopus/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
16.
Mol Cell ; 77(5): 1080-1091.e8, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31862156

RESUMO

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Instabilidade Genômica , Animais , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Modelos Genéticos , Complexos Multiproteicos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Imagem Individual de Molécula , Fatores de Tempo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
17.
Dev Biol ; 459(2): 138-148, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31846624

RESUMO

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Sistema Cardiovascular/embriologia , Colágeno/metabolismo , Citocinas/metabolismo , Proteínas Musculares/metabolismo , Hormônios Peptídicos/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Colágeno/genética , Citocinas/genética , Ectoderma/metabolismo , Desenvolvimento Embrionário/genética , Eritrócitos/metabolismo , Eritropoese/genética , Feminino , Técnicas de Silenciamento de Genes , Masculino , Proteínas Musculares/genética , Hormônios Peptídicos/genética , RNA-Seq , Transdução de Sinais/genética , Proteínas de Xenopus/genética , Xenopus laevis
18.
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
19.
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
20.
Dev Biol ; 459(2): 109-125, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31884020

RESUMO

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.


Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Células Epidérmicas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Hidrocefalia/genética , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , Cauda do Espermatozoide/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Traqueia/citologia , Proteínas de Xenopus/genética , Xenopus laevis
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