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1.
Nat Genet ; 53(10): 1480-1492, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34611363

RESUMO

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.


Assuntos
Diferenciação Celular , Genoma , Crista Neural/citologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Integrases/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/metabolismo , Células Musculares/citologia , Crista Neural/metabolismo , Ligação Proteica , Domínios Proteicos , Proteólise , Fatores de Transcrição/química , Transcrição Genética
2.
Int J Mol Sci ; 22(19)2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34639080

RESUMO

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.


Assuntos
Proteínas Fúngicas/genética , Edição de Genes , Vetores Genéticos , Integrases/metabolismo , Plasmídeos/genética , Yarrowia/genética , Engenharia Genética , Integrases/genética , Recombinação Genética , Yarrowia/crescimento & desenvolvimento
3.
Nat Commun ; 12(1): 5520, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535684

RESUMO

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.


Assuntos
Fator de Transcrição PAX7/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Animais , Cruzamento , Diferenciação Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Integrases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout , Desenvolvimento Muscular , PTEN Fosfo-Hidrolase/deficiência , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rabdomiossarcoma/genética
4.
Nat Commun ; 12(1): 5080, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426574

RESUMO

Bed nucleus of the stria terminalis (BNST) neurons that synthesize corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety. Here, we found that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2 + synaptic input from the paraventricular thalamus (PVT) that releases glutamate directly onto BNSTCRF neurons but also engages a large BNST interneuron population to ultimately inhibit BNSTCRF neurons, and this polysynaptic PVTVGLUT2-BNSTCRF circuit is more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking only in female mice, while activation reduced avoidance behavior in both sexes. Lastly, repeated binge drinking produced a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex, feedforward inhibitory PVTVGLUT2-BNSTCRF circuit that is sex-dependent in its function, behavioral roles, and alcohol-induced plasticity.


Assuntos
Consumo de Bebidas Alcoólicas/patologia , Aprendizagem da Esquiva , Hormônio Liberador da Corticotropina/metabolismo , Sistema Límbico/patologia , Neurônios/patologia , Sinapses/patologia , Tálamo/patologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Animais , Ansiedade/fisiopatologia , Comportamento Animal , Potenciais Pós-Sinápticos Excitadores , Feminino , Ácido Glutâmico/metabolismo , Potenciais Pós-Sinápticos Inibidores , Integrases/metabolismo , Sistema Límbico/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Núcleos Septais/patologia , Núcleos Septais/fisiopatologia , Caracteres Sexuais , Tálamo/fisiopatologia
5.
Cells ; 10(7)2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34359992

RESUMO

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/-ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/-ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.


Assuntos
Arginase/metabolismo , Citocinas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Salmonelose Animal/enzimologia , Salmonella typhimurium/fisiologia , Animais , Células da Medula Óssea/microbiologia , Proteínas de Transporte de Cátions , Integrases/metabolismo , Interleucina-4/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirrolidinas/farmacologia , Regulação para Cima
6.
J Biol Chem ; 297(4): 101093, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34416236

RESUMO

Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.


Assuntos
Integrases/química , Proteínas Intrinsicamente Desordenadas/química , Retroelementos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Integrases/genética , Integrases/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
J Biol Chem ; 297(4): 101119, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34450162

RESUMO

The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.


Assuntos
Escherichia coli , Expressão Gênica , Proteínas de Fluorescência Verde , Integrases , Microrganismos Geneticamente Modificados , Proteínas Recombinantes de Fusão , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Integrases/genética , Integrases/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Multimerização Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
8.
J Immunol ; 207(3): 799-808, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34301844

RESUMO

Protein kinase CK2 (also known as Casein Kinase 2) is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory CK2ß subunits. CK2 is overexpressed and overactive in B cell acute lymphoblastic leukemia and diffuse large B cell lymphomas, leading to inappropriate activation of the NF-κB, JAK/STAT, and PI3K/AKT/mTOR signaling pathways and tumor growth. However, whether CK2 regulates normal B cell development and differentiation is not known. We generated mice lacking CK2α specifically in B cells (using CD19-driven Cre recombinase). These mice exhibited cell-intrinsic expansion of marginal zone B cells at the expense of transitional B cells, without changes in follicular B cells. Transitional B cells required CK2α to maintain adequate BCR signaling. In the absence of CK2α, reduced BCR signaling and elevated Notch2 signaling activation increased marginal zone B cell differentiation. Our results identify a previously unrecognized function for CK2α in B cell development and differentiation.


Assuntos
Linfócitos B/imunologia , Caseína Quinase II/metabolismo , Células Precursoras de Linfócitos B/imunologia , Animais , Antígenos CD19/metabolismo , Caseína Quinase II/genética , Diferenciação Celular , Células Cultivadas , Integrases/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais
9.
eNeuro ; 8(4)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34326065

RESUMO

Bombesin receptor subtype-3 (BRS3) is an orphan receptor that regulates energy homeostasis. We compared Brs3 driver mice with constitutive or inducible Cre recombinase activity. The constitutive BRS3-Cre mice show a reporter signal (Cre-dependent tdTomato) in the adult brain because of lineage tracing in the dentate gyrus, striatal patches, and indusium griseum, in addition to sites previously identified in the inducible BRS3-Cre mice (including hypothalamic and amygdala subregions, and parabrachial nucleus). We detected Brs3 reporter expression in the dentate gyrus at day 23 but not at postnatal day 1 or 5 months of age. Hypothalamic sites expressed reporter at all three time points, and striatal patches expressed Brs3 reporter at 1 day but not 5 months. Parabrachial nucleus Brs3 neurons project to the preoptic area, hypothalamus, amygdala, and thalamus. Both Cre recombinase insertions reduced Brs3 mRNA levels and BRS3 function, causing obesity phenotypes of different severity. These results demonstrate that driver mice should be characterized phenotypically and illustrate the need for knock-in strategies with less effect on the endogenous gene.


Assuntos
Integrases , Receptores da Bombesina , Animais , Encéfalo/metabolismo , Hipotálamo/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Receptores da Bombesina/metabolismo
10.
PLoS One ; 16(7): e0254426, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34292968

RESUMO

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.


Assuntos
Regulação Neoplásica da Expressão Gênica , Integrases , Proteínas de Neoplasias , Proteínas Serina-Treonina Quinases , Proteína A4 de Ligação a Cálcio da Família S100 , Sarcoma Experimental , Fator de Transcrição Sp7 , Animais , Indução Enzimática , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologia , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo
11.
Sci Rep ; 11(1): 15325, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321513

RESUMO

We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.


Assuntos
Animais Geneticamente Modificados , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Técnicas de Genotipagem , Integrases/genética , Tribolium/genética , Animais , Embrião não Mamífero , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Expressão Gênica , Engenharia Genética/economia , Marcadores Genéticos , Vetores Genéticos/química , Heterozigoto , Histonas/genética , Histonas/metabolismo , Homozigoto , Integrases/metabolismo , Masculino , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
12.
Aging (Albany NY) ; 13(11): 15479-15490, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099590

RESUMO

Sirtuin 1 (SIRT1) is a class III histone deacetylase that exerts an anti-inflammatory effect in airway diseases. Activated macrophages play an important role in asthma. However, the roles of SIRT1 on allergic airway inflammation in macrophages remain largely unexplored. In this study, we aimed to determine the roles of SIRT1 on allergic airway inflammation in macrophages. The effect of myeloid-specific SIRT1 deletion (Sirt1fl/fl-LysMcre) on airway inflammation was assessed by using in vivo models of asthma following allergen exposure and in vitro culture of primary bone marrow-derived macrophages (BMDMs) exposed to house dust mite (HDM). We observed that Sirt1fl/fl-LysMcre mice substantially enhanced airway inflammation and mucus production in response to allergen exposure. Expression of chemokine ligand (CXCL) 2, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α were reduced in BMDMs with myeloid-specific deletion of Sirt1 after stimulation of HDM. Moreover, SIRT1 suppressed the inflammatory cytokines expression in BMDMs partially via the ERK/p38 MAPK pathways. Our study demonstrated that SIRT1 suppresses the allergic airway inflammation in macrophages, and suggested that activation of SIRT1 in macrophages may represent therapeutic strategy for asthma.


Assuntos
Asma/patologia , Deleção de Genes , Hipersensibilidade/patologia , Inflamação/patologia , Pulmão/patologia , Células Mieloides/metabolismo , Sirtuína 1/metabolismo , Alérgenos/efeitos adversos , Animais , Asma/complicações , Citocinas/metabolismo , Modelos Animais de Doenças , Hipersensibilidade/complicações , Inflamação/complicações , Integrases/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Transgênicos , Pyroglyphidae
13.
J Bacteriol ; 203(16): e0070320, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34060907

RESUMO

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.


Assuntos
Bacteriófagos/enzimologia , Pareamento Cromossômico , Integrases/química , Integrases/metabolismo , Listeria monocytogenes/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Integração Viral , Motivos de Aminoácidos , Sítios de Ligação Microbiológicos , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/fisiologia , Integrases/genética , Listeria monocytogenes/genética , Prófagos/química , Prófagos/enzimologia , Prófagos/genética , Prófagos/fisiologia , Domínios Proteicos , Recombinação Genética , Proteínas Virais/genética
14.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064885

RESUMO

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Deleção de Genes , Integrases/metabolismo , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Transgenes , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Vetores Genéticos , Integrases/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Recombinação Genética , Reprodução
15.
Cell Stem Cell ; 28(6): 989-990, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34087159

RESUMO

Specific cell targeting with one site-specific recombinase is challenging. In this issue of Cell Stem Cell, Han et al. (2021) released a collection of Dre drivers and demonstrate how two recombinases can be combined to improve the cell specificity of lineage tracing and gene inactivation in mice.


Assuntos
Integrases , Recombinases , Animais , Sequência de Bases , Movimento Celular , Integrases/metabolismo , Camundongos , Recombinases/genética , Recombinases/metabolismo
16.
Cells ; 10(6)2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072453

RESUMO

The catalytic domain of most 'cut and paste' DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp-DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.


Assuntos
Domínio Catalítico/fisiologia , Integrases/metabolismo , Transposases/metabolismo , Elementos de DNA Transponíveis/fisiologia , Galium/genética , Galium/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Integrases/genética , Mutagênese Sítio-Dirigida/métodos , Transposases/genética
17.
Sci Rep ; 11(1): 11603, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079011

RESUMO

Blood vessels in the adult mammal exist in a highly organized and stable state. In the ischemic heart, limited expansion capacity of the myocardial vascular bed cannot satisfy demands for oxygen supply and the myocardium eventually undergoes irreversible damage. The predominant contribution of endogenous c-Kit+ cells is understood to be in the development and homeostasis of cardiac endothelial cells, which suggests potential for their targeting in treatments for cardiac ischemic injury. Quiescent cells in other tissues are known to contribute to the long-term maintenance of a cell pool, preserve proliferation capacity and, upon activation, facilitate tissue homeostasis and regeneration in response to tissue injury. Here, we present evidence of a Setd4-expressing quiescent c-Kit+ cell population in the adult mouse heart originating from embryonic stages. Conditional knock-out of Setd4 in c-Kit-CreERT2;Setd4f/f;Rosa26TdTomato mice induced an increase in vascular endothelial cells of capillaries in both neonatal and adult mice. We show that Setd4 regulates quiescence of c-Kit+ cells by the PI3K-Akt-mTOR signaling pathway via H4K20me3 catalysis. In myocardial infarction injured mice, Setd4 knock-out resulted in attenuated cardiomyocyte apoptosis, decreased infarction size and improved cardiac function. Lineage tracing in Setd4-Cre;Rosa26mT/mG mice showed that Setd4+ cells contribute to each cardiac lineage. Overall, Setd4 epigenetically controls c-Kit+ cell quiescence in the adult heart by facilitating heterochromatin formation via H4K20me3. Beyond activation, endogenous quiescent c-Kit+ cells were able to improve cardiac function in myocardial infarction injured mice via the neovascularization of capillaries.


Assuntos
Células Endoteliais/metabolismo , Epigênese Genética , Metiltransferases/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Apoptose , Capilares/crescimento & desenvolvimento , Divisão Celular , Proliferação de Células , Modelos Animais de Doenças , Ecocardiografia , Células Endoteliais/citologia , Feminino , Histonas/genética , Histonas/metabolismo , Integrases/genética , Integrases/metabolismo , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Neovascularização Fisiológica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
18.
Methods Mol Biol ; 2317: 177-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028769

RESUMO

Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the barau gene flanked by loxP sites in the plastid genome. For marker excision Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the barau gene, confers a visual aurea leaf phenotype, thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The success of in planta plastid marker excision proves that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA and the availability of visual marker genes.


Assuntos
Agrobacterium/metabolismo , Marcadores Genéticos , Integrases/metabolismo , Brotos de Planta/genética , Plastídeos/genética , Tabaco/genética , Transformação Genética , Agrobacterium/genética , Genes de Plantas , Engenharia Genética , Genomas de Plastídeos , Integrases/genética , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Recombinação Genética , Tabaco/crescimento & desenvolvimento
19.
Nucleic Acids Res ; 49(10): 5654-5670, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34048565

RESUMO

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.


Assuntos
Cromossomos/metabolismo , Transferência Genética Horizontal/genética , Integrases/metabolismo , Integrons/genética , Vibrio cholerae/metabolismo , Cromossomos/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Integrases/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Recombinação Genética/genética , Vibrio cholerae/genética
20.
Science ; 372(6538)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833095

RESUMO

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.


Assuntos
Linhagem da Célula , Expressão Gênica , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Análise de Célula Única , Animais , Encéfalo/citologia , Linhagem Celular , Células Clonais/citologia , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Perfilação da Expressão Gênica , Resposta ao Choque Térmico , Hibridização in Situ Fluorescente , Integrases/metabolismo , Camundongos , Mutagênese , Análise Espacial , Imagem com Lapso de Tempo , Transcrição Genética
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