RESUMO
Mouse postnatal neural stem cells (pNSCs) can be expanded in vitro in the presence of epidermal growth factor and fibroblast growth factor 2 and upon removal of these factors cease proliferation and generate neurons, astrocytes, and oligodendrocytes. The genetic requirements for self-renewal and lineage-commitment of pNSCs are incompletely understood. In this study, we show that the transcription factors NFIA and NFIB, previously shown individually, to be essential for the normal commitment of pNSCs to the astrocytic lineage in vivo, are jointly required for normal self-renewal of pNSCs in vitro and in vivo. Using conditional knockout alleles of Nfia and Nfib, we show that the simultaneous loss of these two genes under self-renewal conditions in vitro reduces the expression of the proliferation markers PCNA and Ki67, eliminates clonogenicity of the cells, reduces the number of cells in S phase, and induces aberrant differentiation primarily into the neuroblast lineage. This phenotype requires the loss of both genes and is not seen upon loss of Nfia or Nfib alone, nor with combined loss of Nfia and Nfix or Nfib and Nfix. These data demonstrate a unique combined requirement for both Nfia and Nfib for pNSC self-renewal.
Assuntos
Fatores de Transcrição NFI , Células-Tronco Neurais , Animais , Camundongos , Diferenciação Celular/fisiologia , Autorrenovação Celular , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismoRESUMO
Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this 'privileged' repertoire. Our experiments identify early transcription as a potential 'epigenetic' contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.
Assuntos
Neurônios Receptores Olfatórios , Receptores Odorantes , Animais , Camundongos , Receptores Odorantes/genética , Epigenômica , Alelos , Epigênese GenéticaRESUMO
Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this "privileged" repertoire. Our experiments identify early transcription as a potential "epigenetic" contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.
RESUMO
BACKGROUND AND OBJECTIVES: Non-communicable diseases may reflect an evolutionary mismatch between our human ancestry and modern environments. To explore the mismatch hypothesis for Acne vulgaris, we studied the prevalence and severity of acne in Dogon adolescents in Mali, West Africa. METHODOLOGY: We graded the prevalence and severity of acne in 1182 Dogon adolescents aged 11-18 years from nine villages using facial photos taken as part of a prospective cohort study. Eighty-nine (89%) of the individuals in the cohort migrated to the city during adolescence, enabling us to assess the effect of urban migration. Using multivariable logistic regression, we estimated the effect of predictor variables on the presence of acne. RESULTS: The prevalence of acne in the cohort was 28%, with 90% of cases being mild or very mild. Thus, the prevalence and severity of acne was much lower than for adolescents in high-income countries. Controlling for age, puberty, and body mass index (BMI), the odds of boys developing acne was 85% lower in the city than in the villages (P = 0.002). CONCLUSION AND IMPLICATIONS: Acne is similar to the 'diseases of civilization' in being promoted by the pro-inflammatory properties of modern diets. The low prevalence and severity of acne in the Dogon supports the mismatch hypothesis and suggests that acne should join the list of diseases of modern lifestyles. However, we also observed an unexpected decrease in acne in urban boys. Future research is needed for a deeper mechanistic understanding of the interplay between diet, inflammation, immune function and other environmental exposures that differ between urban and rural environments.
RESUMO
Murine postnatal neural stem cells (NSCs) give rise to neurons, astrocytes, or oligodendrocytes (OLs); however, our knowledge of the genes that control this lineage specification is incomplete. In this study, we show that nuclear factor I X (NFIX), a transcription factor known to regulate NSC quiescence, also suppresses oligodendrogenesis (ODG) from NSCs. Immunostaining reveals little or no expression of NFIX in OL lineage cells both in vivo and in vitro. Loss of NFIX from subventricular zone (SVZ) NSCs results in enhanced ODG both in vivo and in vitro, while forced expression of NFIX blocks NSC differentiation into OLs in vitro. RNA-seq analysis shows that genes previously shown to be differentially expressed in OL progenitors are significantly enriched in RNA from Nfix(-/-) versus wild-type NSCs. These data indicate that NFIX influences the lineage specification of postnatal SVZ NSCs, specifically suppressing ODG.
Assuntos
Ventrículos Laterais/embriologia , Fatores de Transcrição NFI/genética , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Oligodendroglia/citologia , Animais , Astrócitos/citologia , Linhagem da Célula , Células Cultivadas , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Fatores de Transcrição SOXE/metabolismoRESUMO
We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.
Assuntos
Membrana Celular/metabolismo , Modelos Biológicos , Sinais Direcionadores de Proteínas , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Sequência de Aminoácidos , Animais , Polaridade Celular , Sequência Conservada , Cães , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Madin Darby de Rim Canino , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/química , Transportadores de Sódio Acoplados à Vitamina C/genéticaRESUMO
The Nuclear factor I (NFI) transcription factor family consists of four genes (Nfia, Nfib, Nfic and Nfix) that regulate the development of multiple organ systems in mice and humans. Nfib is expressed in both lung mesenchyme and epithelium and mice lacking Nfib have severe lung maturation defects and die at birth. Here we continue our analysis of the phenotype of Nfibâ»/â» lungs and show that Nfib specifically in lung mesenchyme controls late epithelial and mesenchymal cell proliferation and differentiation. There are more PCNA, BrdU, PHH3 and Ki67 positive cells in Nfibâ»/â» lungs than in wild type lungs at E18.5 and this increase in proliferation marker expression is seen in both epithelial and mesenchymal cells. The loss of Nfib in all lung cells decreases the expression of markers for alveolar epithelial cells (Aqp5 and Sftpc), Clara cells (Scgb1a1) and ciliated cells (Foxj1) in E18.5 lungs. To test for a specific role of Nfib in lung mesenchyme we generated and analyzed Nfib(flox/flox), Dermo1-Cre mice. Loss of Nfib only in mesenchyme results in decreased Aqp5, Sftpc and Foxj1 expression, increased cell proliferation, and a defect in sacculation similar to that seen in Nfibâ»/â» mice. In contrast, mesenchyme specific loss of Nfib had no effect on the expression of Scgb1a1 in the airway. Microarray and QPCR analyses indicate that the loss of Nfib in lung mesenchyme affects the expression of genes associated with extracellular matrix, cell adhesion and FGF signaling which could affect distal lung maturation. Our data indicate that mesenchymal Nfib regulates both mesenchymal and epithelial cell proliferation through multiple pathways and that mesenchymal NFI-B-mediated signals are essential for the maturation of distal lung epithelium.
Assuntos
Diferenciação Celular , Proliferação de Células , Pulmão/crescimento & desenvolvimento , Fatores de Transcrição NFI/metabolismo , Mucosa Respiratória/citologia , Animais , Aquaporina 5/metabolismo , Bromodesoxiuridina/metabolismo , Epitélio/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular , Antígeno Ki-67/metabolismo , Pulmão/citologia , Pulmão/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína C Associada a Surfactante Pulmonar , Transdução de Sinais , Uteroglobina/metabolismoRESUMO
Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/embriologia , Fatores de Transcrição NFI/metabolismo , Transcrição Gênica , Animais , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoenzimas/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Fatores de Regulação Miogênica/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fator de Transcrição PAX7/metabolismo , Fosfopiruvato Hidratase , Proteína Quinase C/metabolismo , Proteína Quinase C-thetaRESUMO
Human sodium-dependent vitamin C transporters, SVCT1 and SVCT2, share 66% sequence identity yet localize in the apical and basolateral membranes of epithelial cells, respectively. This pair thus serves as a model for studying multipass membrane protein targeting. Domain swaps, deletions, insertions, and point mutations were performed on EGFP-tagged hSVCT1 and hSVCT2 plasmids. Mutant proteins stably expressed in MDCK cells were analyzed by confocal microscopy and Transwell ascorbate transport assays. These studies identified an SVCT2 basolateral targeting sequence (BTS) in the N-terminus, which is conserved among mammalian SVCT2 forms. The less conserved N-terminus of SVCT1 is not required for apical localization. The destruction of SVCT2 BTS led to apical localization of the protein in a manner independent of the C-terminal sequence. A C-terminal sequence present in both SVCTs appears to be required for plasma membrane incorporation and retention as its deletion led to an increased level of intracellular appearance of both apically and basolaterally targeted SVCTs in the absence or presence of BTS. Nevertheless, all C-terminal deletion mutants showed preferential apical transport activity, suggesting a greater importance of this sequence for basolateral targeting. Our results collectively suggested a default apical targeting of SVCT, which is consistent with the evolution-based prediction. The SVCT sorting model with a hierarchal contribution of N- and C-terminal sequences was compared to the observations made for other multipass membrane proteins. The involvement of both intracellularly localized termini of multipass membrane proteins in the sorting pathway suggests a more complex sorting mechanism compared to that for single-pass proteins.
Assuntos
Células Epiteliais/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Homologia de Sequência de Aminoácidos , Simportadores/química , Simportadores/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Deleção de Sequência , Transportadores de Sódio Acoplados à Vitamina C , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice. RESULTS: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals. CONCLUSION: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.
Assuntos
Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Genes Controladores do Desenvolvimento , Fatores de Transcrição NFI/genética , Animais , Western Blotting , Córtex Cerebral/crescimento & desenvolvimento , Ventrículos Cerebrais/crescimento & desenvolvimento , Embrião de Mamíferos , Células-Tronco Embrionárias , Feminino , Marcação de Genes , Hipocampo/crescimento & desenvolvimento , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
Three members of the Nuclear Factor I (Nfi) gene family of transcription factors; Nfia, Nfib, and Nfix are highly expressed in the developing mouse brain. Nfia and Nfib knockout mice display profound defects in the development of midline glial populations and the development of forebrain commissures (das Neves et al. [1999] Proc Natl Acad Sci U S A 96:11946-11951; Shu et al. [2003] J Neurosci 23:203-212; Steele-Perkins et al. [2005] Mol Cell Biol 25:685-698). These findings suggest that Nfi genes may regulate the substrate over which the commissural axons grow to reach targets in the contralateral hemisphere. However, these genes are also expressed in the cerebral cortex and, thus, it is important to assess whether this expression correlates with a cell-autonomous role in cortical development. Here we detail the protein expression of NFIA and NFIB during embryonic and postnatal mouse forebrain development. We find that both NFIA and NFIB are expressed in the deep cortical layers and subplate prenatally and display dynamic expression patterns postnatally. Both genes are also highly expressed in the developing hippocampus and in the diencephalon. We also find that principally neither NFIA nor NFIB are expressed in callosally projecting neurons postnatally, emphasizing the role for midline glial cell populations in commissure formation. However, a large proportion of laterally projecting neurons express both NFIA and NFIB, indicating a possible cell-autonomous role for these transcription factors in corticospinal neuron development. Collectively, these data suggest that, in addition to regulating the formation of axon guidance substrates, these genes also have cell-autonomous roles in cortical development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Transcrição NFI/metabolismo , Prosencéfalo , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFI/genética , Vias Neurais/citologia , Vias Neurais/embriologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismoRESUMO
Sodium-dependent vitamin C transporters, SVCT1 and SVCT2, are the only two known proteins for the uptake of ascorbate, the active form of vitamin C. Little structural information is available for SVCTs, although a transport activity increase from pH 5.5 to 7.5 suggests a functional role of one or more conserved histidines (p K a approximately 6.5). Confocal fluorescence microscopy and uptake kinetic analyses were used here to characterize cells transfected with mutants of EGFP-tagged hSVCTs. Mutating any of the four conserved histidine residues (His51, 147, 210, or 354) in hSVCT1 to alanine did not affect the apical membrane localization in polarized MDCK cells. His51Ala (in putative transmembrane segment 1, TM1) was the only mutation that resulted in a significant loss of ascorbate transport and an increase in apparent Km with no significant effect on Vmax. The corresponding mutation in hSVCT2, His109Ala, also led to a loss of transport activity. Among eight other mutations of His51 in hSVCT1, significant sodium-dependent ascorbate transport activity was only observed with asparagine or tyrosine replacement. Thus, our results suggest that uncharged His51, directly or indirectly, contributes to substrate binding through the hydrogen bond. His51 cannot account for the observed pH dependence as neutral amino acid substitutions failed to abolish the pH-dependent activity increase. The importance of TM1 is further strengthened by the comparable loss of sodium-dependent ascorbate transport activity upon the mutation of adjacent conserved Gln50 and the apparent change in substrate specificity in the hSVCT1-His51Gln mutation, which showed a specific increase in sodium-independent dehydroascorbate transport.
Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Ácido Ascórbico/metabolismo , Transporte Biológico , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Estrutura Terciária de Proteína , Transportadores de Sódio Acoplados à Vitamina C , Relação Estrutura-Atividade , Simportadores/química , Simportadores/genéticaRESUMO
Deletion 22q11.2 syndrome is the most frequent known microdeletion syndrome and is associated with a highly variable phenotype, including DiGeorge and Shprintzen (velocardiofacial) syndromes. Although haploinsufficiency of the T-box transcription factor gene TBX1 is thought to cause the phenotype, to date, only four different point mutations in TBX1 have been reported in association with six of the major features of 22q11.2 deletion syndrome. Although, for the two truncating mutations, loss of function was previously shown, the pathomechanism of the missense mutations remains unknown. We report a novel heterozygous missense mutation, H194Q, in a familial case of Shprintzen syndrome and show that this and the two previously reported missense mutations result in gain of function, possibly through stabilization of the protein dimer DNA complex. We therefore conclude that TBX1 gain-of-function mutations can result in the same phenotypic spectrum as haploinsufficiency caused by loss-of-function mutations or deletions.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Síndrome de DiGeorge/genética , Dosagem de Genes/genética , Mutação de Sentido Incorreto/genética , Proteínas com Domínio T/genética , Pré-Escolar , Cristalografia por Raios X , Feminino , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fragmentos de Peptídeos/genética , Fenótipo , Conformação ProteicaRESUMO
The T-box transcription factor T-bet is expressed in a number of hematopoetic cell types and plays an essential role in the lineage determination of Th1 T-helper cells. In the absence of T-bet, CD4(+) T-cells fail to induce IFNgamma, the cytokine whose expression characterizes Th1 cells. Here we show that, surprisingly, T-bet induces the expression of endogenous IFNgamma in non-immune human cells, including 293 and other cell lines. Thus T-bet can induce IFNgamma expression independently of its role in T-cell lineage determination. In addition, mutations in T-bet, and chimeras of T-bet with other transcription factors including the T-box transcription factor, TBX2, differentially affect the ability of T-bet to activate expression of endogenous IFNgamma versus a T-site regulated reporter gene. A truncated T-betVp16 fusion protein strongly activates the T-site reporter but fails to activate endogenous IFNgamma. Conversely, native T-bet strongly activates endogenous IFNgamma expression but only weakly activates the reporter gene. Fusion of the Vp16 activation domain to full-length T-bet greatly increases its activation of both endogenous IFNgamma and transfected T-site reporter gene expression. In contrast, TBX2Vp16 potently activates the T-site reporter but has a negligible effect on endogenous IFNgamma expression. Butyrate treatment of T-bet expressing cells potentiates the expression of endogenous IFNgamma but weakly represses expression of the T-site reporter gene. These data indicate that induction of endogenous IFNgamma can be uncoupled from differentiation into the Th1 lineage and that the expression of endogenous IFNgamma versus a T-site reporter gene is differentially regulated by T-bet and other T-box proteins.
Assuntos
Interferon gama/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Linhagem Celular , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Luciferases/genética , Modelos Biológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas com Domínio T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.
Assuntos
Ácido Ascórbico/farmacocinética , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Células CHO , Células CACO-2 , Linhagem Celular , Cricetinae , Cricetulus , Cães , Humanos , Camundongos , Transportadores de Sódio Acoplados à Vitamina C , Distribuição TecidualRESUMO
The determinants of in vivo target site selectivity by transcription factors are poorly understood. To find targets for the developmentally regulated transcription factor TBX2, we generated stable transfectants of human embryonic kidney cells (293) that express a TBX2-ecdysone receptor (EcR) chimeric protein. While constitutive expression of TBX2 is toxic to 293 cells, clones expressing TBX2EcR are viable in the absence of an EcR ligand. Using cDNA arrays and quantitative PCR, we discovered nine genes whose expression was increased, but no genes whose expression was reduced, following 24 h of induction with Ponasterone A (PonA), a ligand for EcR. Since TBX2 was reported previously to be a transcriptional repressor, we also generated cell lines expressing a TBX2VP16EcR protein which we showed was a potent conditional transcriptional activator in transient transfection assays. Treatment of these cells with PonA induced the expression of five genes, none of which were affected in TBX2EcR-expressing cells. This discordance between TBX2- and TBX2VP16-regulated genes strongly suggests that specific transactivation domains can be a major determinant of gene target site selectivity by transcription factors that possess the same DNA-binding domain.