RESUMO
Members of the fibroblast growth factor (FGF) family play important roles during various developmental processes including eye development. FRS (FGF receptor substrate) proteins bind to FGFR and serve as adapters for coordinated assembly of multi-protein complexes involved in Ras/MAPK and PI3 kinase/Akt pathways. Here, we identified Xenopus laevis Frs3 (XFrs3), a homolog of vertebrate Frs3, and investigated its roles during embryogenesis. XFrs3 is expressed maternally and zygotically with specific expression patterns throughout the early development. Knockdown of XFrs3 using a specific antisense morpholino oligonucleotide (MO) caused reduction of Pax6 expression in the lens placode, and defects in the eye ranging from microphthalmia to anophthalmia. XFrs3 MO-induced defects were alleviated by wild type XFrs3 or a mutant XFrs3 (XFrs3-4YF), in which the putative tyrosine phosphorylation sites served as Grb2-binding sites are mutated. However, another XFrs3 mutant (XFrs3-2YF), in which the putative Shp2-binding sites are mutated, could not rescue the defects of XFrs3 morphants. In addition, we found that XFrs3 is important for FGF or IGF-induced ERK activation in ectodermal tissue. Taken together, our results suggest that signaling through Shp2-binding sites of XFrs3 is necessary for the eye development in Xenopus laevis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Cristalino/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Sítios de Ligação , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Mutação , Oligonucleotídeos/química , Fator de Transcrição PAX6 , Fosforilação , Ligação Proteica , Transdução de Sinais , Tirosina/químicaRESUMO
The inflammatory response contributes substantially to secondary injury cascades after spinal cord injury, with both neurotoxic and protective effects. However, epigenetic regulations of inflammatory genes following spinal cord injury have yet to be characterized thoroughly. In this study, we found that histone H3K27me3 demethylase Jmjd3 expression is acutely up-regulated in blood vessels of the injured spinal cord. We also observed up-regulation of Jmjd3 gene expression in bEnd.3 endothelial cells that were subjected to oxygen-glucose deprivation/reperfusion injury. When Jmjd3 was depleted by siRNA, oxygen-glucose deprivation/reperfusion injury-induced up-regulation of IL-6 was significantly inhibited. In addition, Jmjd3 associated with NF-κB (p65/p50) and CCAAT-enhancer-binding protein ß at the IL-6 gene promoter. The recruitment of Jmjd3 coincided with decreased levels of tri-methylated H3K27 as well as increased levels of mono-methylated H3K27 at the IL-6 gene promoter. Furthermore, Jmjd3 depletion did not result in significant changes of methylation level of H3K27 at the IL-6 gene promoter. Collectively, our findings imply that Jmjd3-mediated H3K27me3 demethylation is crucial for IL-6 gene activation in endothelial cells, and this molecular event may regulate acute inflammatory response and integrity of the blood-spinal cord barrier following spinal cord injury.
Assuntos
Células Endoteliais/metabolismo , Interleucina-6/genética , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/fisiologia , Traumatismos da Medula Espinal/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Hipóxia Celular/fisiologia , Células Cultivadas , Metilação de DNA , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/fisiologia , Glucose/deficiência , Imuno-Histoquímica , Imunoprecipitação , Inflamação/metabolismo , Interleucina-6/biossíntese , Masculino , Reação em Cadeia da Polimerase , RNA/biossíntese , RNA/isolamento & purificação , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Regulação para CimaRESUMO
Fibroblast growth factors (FGFs) function as mitogens and morphogens during vertebrate development. In the present study, to characterise the regulatory mechanism of FGF8 gene expression in developing Xenopus embryos the upstream region of the Xenopus FGF8 (XFGF8) gene was isolated. The upstream region of the XFGF8 gene contains two putative binding sites for the SRY (sex-determining region Y)-box 2 (SOX2) transcription factor. A reporter assay with serially deleted constructs revealed that the putative SOX2-binding motif may be a critical cis-element for XFGF8 gene activation in developing Xenopus embryos. Furthermore, Xenopus SOX2 (XSOX2) physically interacted with the SOX2-binding motif within the upstream region of the XFGF8 gene in vitro and in vivo. Depletion of endogenous XSOX2 resulted in loss of XFGF8 gene expression in midbrain-hindbrain junction, auditory placode, lens placode and forebrain in developing Xenopus embryos. Collectively, our results suggest that XSOX2 directly upregulates XFGF8 gene expression in the early embryonic development of Xenopus.
Assuntos
Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Técnicas de Cultura Embrionária , Embrião não Mamífero/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Microinjeções , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologiaRESUMO
It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3ß RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3ß RNA exon 1 and 2, which resulted in generation of N'-terminal truncated GSK3ß protein. This event further caused decreased phosphorylation of ß-catenin and subsequently increased ß-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3ß RNA splicing and canonical Wnt/ß-catenin signaling.
Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Olho/crescimento & desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Splicing de RNA , Proteínas de Xenopus/genética , Animais , Diferenciação Celular , Olho/citologia , Olho/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Organogênese , Fosforilação , Estabilidade Proteica , Transdução de Sinais , Fator de Processamento U2AF/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Catenina/química , beta Catenina/metabolismoRESUMO
It has been shown that epigenetic regulation plays an important role in skin wound healing. We previously found that histone H3K27me3 demethylase JMJD3 regulates inflammation and cell migration in keratinocyte wound healing. In this study, we identified Notch1 as a direct target of JMJD3 and NF-κB in wounded keratinocytes using in vitro cell and in vivo animal models. We found that Notch1 is up-regulated in the wound edge and its expression is dependent on JMJD3 and NF-κB in wounded keratinocytes. We also found that Notch1 activates the expression of RhoU and PLAU gene, which are critical regulators of cell migration. Consistently, depletion or inactivation of Notch1 resulted in decreased filopodia formation, increased focal adhesion and actin stress fiber, leading to reduced keratinocyte migration and skin wound healing. Thus, our findings provide the molecular mechanism involving JMJD3/NF-κB-Notch pathway in keratinocyte wound healing.
Assuntos
Movimento Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Queratinócitos/fisiologia , NF-kappa B/metabolismo , Receptor Notch1/metabolismo , Cicatrização , Ferimentos e Lesões/patologia , Animais , Linhagem Celular , Epigênese Genética , Humanos , Camundongos , Transdução de SinaisRESUMO
The blood-brain barrier (BBB) exhibits a highly selective permeability to support the homeostasis of the central nervous system (CNS). The tight junctions in the BBB microvascular endothelial cells seal the paracellular space to prevent diffusion. Thus, disruption of tight junctions results in harmful effects in CNS diseases and injuries. It has recently been demonstrated that glucocorticoids have beneficial effects on maintaining tight junctions in both in vitro cell and in vivo animal models. In the present study, we found that dexamethasone suppresses the expression of JMJD3, a histone H3K27 demethylase, via the recruitment of glucocorticoid receptor α (GRα) and nuclear receptor co-repressor (N-CoR) to the negative glucocorticoid response element (nGRE) in the upstream region of JMJD3 gene in brain microvascular endothelial cells subjected to TNFα treatment. The decreased JMJD3 gene expression resulted in the suppression of MMP-2, MMP-3, and MMP-9 gene activation. Dexamethasone also activated the expression of the claudin 5 and occludin genes. Collectively, dexamethasone attenuated the disruption of the tight junctions in the brain microvascular endothelial cells subjected to TNFα treatment. Therefore, glucocorticoids may help to preserve the integrity of the tight junctions in the BBB via transcriptional and post-translational regulation following CNS diseases and injuries.
Assuntos
Encéfalo/irrigação sanguínea , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glucocorticoides/farmacologia , Histona Desmetilases com o Domínio Jumonji/genética , Junções Íntimas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Camundongos , Microvasos/citologia , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Receptores de Glucocorticoides/metabolismo , Junções Íntimas/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An organic-inorganic photopolymers have been studied for their potential in of reducing the volume shrinkage during photopolymerization and enhancing the dimensional stability of photopolymers. We demonstrate the diffraction efficiency of photopolymers could be significantly enhanced by the interfacial interactions induced at the surface of inorganic nanoparticles.
RESUMO
Histone H3K27me3 demethylase JMJD3 has been shown to be involved in keratinocyte differentiation and wound healing. However, the exact molecular mechanism underlying JMJD3-mediated keratinocyte wound healing has not been fully elucidated. In this study, we report on the biological function of JMJD3 in keratinocyte wound healing using in vitro cell and in vivo animal models. Our results indicate that JMJD3 up-regulation and NF-κB activation occur in the region of the wound edge during keratinocyte wound healing. We also found that JMJD3 interacts with NF-κB, resulting in increased expression of the inflammatory, matrix metalloproteinase, and growth factor genes via demethylation of H3K27me3 at the gene promoters. Consistently, inactivation of JMJD3 or NF-κB resulted in aberrant keratinocyte wound healing. Our study suggests that regulation of JMJD3 may provide a new therapeutic intervention for treating the chronic skin wound.
Assuntos
Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Queratinócitos/enzimologia , Subunidade p50 de NF-kappa B/metabolismo , Cicatrização , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Histonas/química , Humanos , Inflamação , Histona Desmetilases com o Domínio Jumonji/genética , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Pele/metabolismoRESUMO
Metastatic prostate cancer is the leading cause of morbidity and mortality in men. In this study, we found that expression level of SFMBT2 is altered during prostate cancer progression and has been associated with the migration and invasion of prostate cancer cells. The expression level of SFMBT2 is high in poorly metastatic prostate cancer cells compared to highly metastatic prostate cancer cells. We also found that SFMBT2 knockdown elevates MMP-2, MMP-3, MMP-9, and MMP-26 expression, leading to increased cell migration and invasion in LNCaP and VCaP cells. SFMBT2 interacts with YY1, RNF2, N-CoR and HDAC1/3, as well as repressive histone marks such as H3K9me2, H4K20me2, and H2AK119Ub which are associated with transcriptional repression. In addition, SFMBT2 knockdown decreased KAI1 gene expression through up-regulation of N-CoR gene expression. Expression of SFMBT2 in prostate cancer was strongly associated with clinicopathological features. Patients having higher Gleason score (≥ 8) had substantially lower SFMBT2 expression than patients with lower Gleason score. Moreover, tail vein or intraprostatic injection of SFMBT2 knockdown LNCaP cells induced metastasis. Taken together, our findings suggest that regulation of SFMBT2 may provide a new therapeutic strategy to control prostate cancer metastasis as well as being a potential biomarker of metastatic prostate cancer.
Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , TransfecçãoRESUMO
The Xenopus FGF-8a and FGF-8b isoforms have been reported to be neural crest and neuronal inducers, respectively. However, cloning of Xenopus FGF-8b (XFGF-8b) has not been reported previously and the two isoforms do not seem to have been clearly distinguished in Xenopus experiments. Here, we describe the cloning and expression of XFGF-8b and compare the effects of the two isoforms. XFGF-8b has an 11 amino acid insert in its N-terminal region compared with XFGF-8a. Both isoforms are expressed in the anterior neural regions of the early embryo, and in the apical ectodermal ridge of limb buds and tips of growing digits in the larval stages. However, XFGF-8b is more abundant than XFGF-8a throughout early development. The two isoforms are also regulated in similar fashion by retinoic acid in early development. However, although both XFGF-8a and XFGF-8b induce ectopic neurogenesis, only XFGF-8a appears to be involved in neural crest induction.
Assuntos
Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/isolamento & purificação , Isoformas de Proteínas/biossíntese , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/química , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Alinhamento de Sequência , Tretinoína/farmacologia , Tubulina (Proteína)/biossíntese , Xenopus laevis/embriologiaRESUMO
We demonstrate that colloidal lithography with self-assembled monolayers of crosslinkable polymeric particles can be used to create hierarchical arrays of nanopores on substrates.
Assuntos
Coloides/química , Reagentes de Ligações Cruzadas/química , Nanoestruturas , Nanotecnologia/métodos , Cristalização , Tamanho da Partícula , Fotoquímica , PorosidadeRESUMO
The blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) formed by capillary endothelial cells provides a physical wall between the central nervous system (CNS) and circulating blood with highly selective permeability. BBB/BSCB disruption by activation of matrix metalloproteinases (MMPs) has been shown to result in further neurological damage after CNS injury. Recently it has been discovered that estrogen attenuates BBB/BSCB disruption in in vitro and in vivo models. However, the molecular mechanism underlying the estrogen-mediated attenuation of BBB/BSCB disruption has not been elucidated fully. In the present study, we found that 17ß-estradiol (E2) suppresses nuclear factor-κB-dependent MMP-1b, MMP-2, MMP-3, MMP-9, MMP-10, and MMP-13 gene activation in microvessel endothelial bEnd.3 cells subjected to oxygen and glucose deprivation/reperfusion injury. E2 induced the recruitment of ERα and nuclear receptor corepressor to the nuclear factor-κB binding site on the MMPs' gene promoters. Consistently, ER antagonist ICI 182.780 showed opposite effects of E2. We further found that E2 attenuates tight junction disruption through the decreased degradation of tight junction proteins in bEnd.3 cells subjected to oxygen and glucose deprivation-reperfusion injury. In addition, E2 suppressed the up-regulation of MMP expression, leading to a decreased BSCB disruption in the injured spinal cord. In conclusion, we discovered the molecular mechanism underlying the protective role of estrogenin BBB/BSCB disruption using an in vitro and in vivo model. Our study suggests that estrogens may provide a potential therapeutic intervention for preserving BBB/BSCB integrity after CNS injury.
Assuntos
Barreira Hematoencefálica/patologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Metaloproteinases da Matriz/genética , NF-kappa B/antagonistas & inibidores , Animais , Lesões Encefálicas/patologia , Linhagem Celular , Impedância Elétrica , Células Endoteliais/citologia , Ativação Enzimática , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Fulvestranto , Masculino , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Junções Íntimas/metabolismoRESUMO
A range of tetrahydropyrans and piperidines were produced by Fe(III)-catalyzed intramolecular hydroalkoxylation and hydroamination reactions of allenes. Various Fe catalysts with different counterions were tested. Their activities toward allene and alkene activation depended sensitively on their counterion and reaction conditions. Mechanistic study of the reaction intermediates found a new reaction pattern involving the Fe catalysts and diene substrates.
RESUMO
Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5) as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.
Assuntos
Criptocromos/biossíntese , Regulação Enzimológica da Expressão Gênica , Proteínas Circadianas Period/biossíntese , Proteínas Metiltransferases/fisiologia , Proteína-Arginina N-Metiltransferases/fisiologia , Animais , Especificidade de Anticorpos , Arginina/química , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Metilação , Camundongos , Modelos Genéticos , Células NIH 3T3 , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
We propose an algorithm that converts a full-parallax hologram to a horizontal-parallax-only (HPO) hologram for 3D display. We first record a full-parallax hologram of an object. Subsequently, we filter the hologram with a Gaussian low-pass filter and a fringe-matched filter along the vertical direction. The final filtered output becomes an HPO hologram. To the best of our knowledge, this is the first algorithm proposed for converting full-parallax holographic information to HPO-holographic information.
RESUMO
Retinoic acid and the synthetic retinoid, arotinoid, were compared for their efficacy in inducing proximodistal (PD) pattern duplication in regenerating axolotl limbs, after amputation through either the distal zeugopodium (lower arm or leg) or distal stylopodium (upper arm or leg). At each level of amputation, the morphology of the duplications produced was the same for both retinoids, and the mean level of proximalization was dose-dependent. Blastema formation was delayed by both retinoids and the delay was associated with regression of the limb stump. Blastemas which produced PD duplication to the stylopodial or girdle level grew out from the stump in a posterior direction. In several zeugopodial regenerates, a partially duplicated, PD-reversed zeugopodium regenerated between the stump cartilages and a completely duplicated zeugopodium distally. Arotinoid was 50 times more effective than retinoic acid in evoking duplication. The dose of arotinoid required to duplicate a stylopodium in a stylopodial regenerate was several times higher than the dose required to duplicate a zeugopodium in a zeugopodial regenerate, suggesting differences either in the sensitivity of zeugopodial and stylopodial cells to retinoid, or in the numbers of positional value specifying these segments.
RESUMO
Retinoids induce proximodistal (PD) pattern duplication in zeugopodial (lower arm or leg) regenerates of normal limbs and PD pattern duplication plus anteroposterior (AP) pattern completion in double anterior half zeugopodial regenerates. In contrast, retinoids inhibit the regeneration of double posterior half zeugopodia (Kim and Stocum, 1986). Here we describe the developmental histology of regenerating normal, double anterior half and double posterior half zeugopodia in axolotls after intraperitoneal injection of retinoic acid (RA) at the stage of initial blastema cell accumulation. In all three classes of RA-treated limbs, the accumulation of blastema cells disappeared within 3 days after injection, and dedifferentiation continued to a much more proximal extent than in controls. Subsequently, however, the developmental histology of the three limb classes was different. RA-treated double posterior limbs exhibited the histological features typical of non-regenerating limbs: the premature appearance of a thick basement membrane under the wound epidermis, formation of a thick connective tissue mat between the basement membrane and the cut ends of the stump cartilages, and failure of blastema formation. In contrast, RA-treated normal zeugopodia reformed single blastemas which grew out in a posterior or posterodorsal direction. RA-treated double anterior zeugopodia formed twin blastemas that were spatially separated to varying degrees and which grew distally. The blastemas of both these RA-treated limb types consisted of a proximal, low-density cell population that formed the girdle of the regenerate and a distal, high-density cell population that formed the free limb. In the free limb portion of the blastema, the density of the mesenchymal cell population was higher than in controls. Blastemas of RA-treated normal and double anterior zeugopodia appeared similar in size and proportions to controls at the medium bud stage, but subsequently took on the characteristics of stylopodial blastemas. These observations suggest that the extra pattern induced by RA in regenerating urodele limbs may be correlated with an increase in the number of defifferentiated cells per unit of blastema volume.
RESUMO
A novel inhibitor of apoptosis protein family member termed SIX was identified in Xenopus containing a single baculoviral IAP repeat (BIR) domain and no COOH-terminal RING finger domain. It exhibited striking amino acid sequence similarity with human survivin, mouse TIAP, and recently found Xenopus survivin, especially a part of BIR domain was highly conserved. Interestingly, SIX interacted with RXRalpha through the AF2 domain in the absence of ligand, which was weakened when the ligand was present. Northern blot analysis demonstrated that SIX mRNA was not detectable in adult with exception of the ovary and testis, and whole-mount in situ hybridization and Northern blot analyses revealed strong and homogeneous expression of SIX in the developing oocytes. In the embryos, the expression of SIX was observed in the animal hemisphere from one-cell to yolk plug stages and high level of expression was detected in the future brain and dorsal region of the neural tube at the neurula stage and early tail-bud stage. These results strongly support the fact that survivin is evolutionarily conserved in structure and SIX is likely to be the Xenopus counterpart of human and mouse survivin.
Assuntos
Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Inibidores de Cisteína Proteinase/genética , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Humanos , Hibridização In Situ , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Proteínas de Neoplasias , Ligação Proteica , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Alinhamento de Sequência , Survivina , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Orphan nuclear receptors belong to the nuclear receptor superfamily of liganded transcription factors, whose ligands either do not exist or remain to be identified. We report here the cloning and characterization of the chicken orphan nuclear receptor, cTR2 (chicken testicular receptor 2). The cTR2 gene encodes a protein of 569 amino acids which shows approximately 72% overall identity with TR2 (NR2C1) and 95% identity in the DNA-binding domain (DBD). The cTR2 gene is expressed in almost all adult tissues and embryonic stages examined unlike its mammalian relative TR2, which is specifically expressed in testis. Electrophoretic mobility shift assays demonstrate that cTR2 binds the canonical direct repeat DNA recognition sequences spaced by one, four, and five nucleotides (DR1, DR4, and DR5), and in consistence with the results with canonical DNA-binding sequences, cTR2 forms specific DNA-protein complex with chicken phenobarbital response elements containing DR4 motifs. Both in vitro and in vivo interaction studies demonstrate that cTR2 forms homodimer. Moreover, transient transfection studies reveal its capability to transactivate canonical DR1, DR4, and DR5 sequences and the constitutive activity of cTR2 is mapped to the N-terminal region of this orphan receptor. Finally, cTR2 represses transactivation of estrogen receptor in a dose-dependent manner.
Assuntos
Galinhas/genética , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Galinhas/metabolismo , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Membro 1 do Grupo C da Subfamília 2 de Receptores Nucleares , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Distribuição Tecidual , Ativação TranscricionalRESUMO
We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR. We also identified a variant of FOR1, called FOR2, which has 15 additional C-terminal amino acids. Both variants include an unusual insertion of 33 amino acids in the helix 7 region of the canonical ligand binding domain sequence, suggesting a unique structure for FOR. Northern blot analysis demonstrates that the FOR gene is highly expressed in adult and tadpole liver, kidney, and tail bud stage of the embryo. Detailed expression analysis using in situ hybridization indicates that FOR expression is first detectable at stage 30/31 in the presumptive liver region lasting until stage 41 with a peak level evident at stage 35/36. FOR forms heterodimeric complexes with retinoid X receptor (RXR) as demonstrated by biochemical and mammalian two-hybrid approaches. Gel mobility shift assays demonstrate that FORs form specific DNA-protein complexes on an FXR binding element consisting of an inverted repeat DNA element with 1 nucleotide spacing (IR1) from the phospholipid transfer protein gene promoter. Finally, although FORs do not exhibit constitutive transcriptional activity, frog gallbladder extract significantly augments the transcriptional activities of FORs.