RESUMO
Pioneer factors, which can directly bind to nucleosomes, have been considered to change chromatin conformations. However, the binding impact on the nucleosome is little known. Here, we show how the pioneer factor GATA3 binds to nucleosomal DNA and affects the conformation and dynamics of nucleosomes by using a combination of SAXS, molecular modeling, and molecular dynamics simulations. Our structural models, consistent with the SAXS data, indicate that only one of the two DNA binding domains, N- and C-fingers, of GATA3 binds to an end of the DNA in solution. Our MD simulations further showed that the other unbound end of the DNA increases the fluctuation and enhances the DNA dissociation from the histone core when the N-finger binds to a DNA end, a site near the entry or exit of the nucleosome. However, this was not true for the binding of the C-finger that binds to a location about 15 base pairs distant from the DNA end. In this case, DNA dissociation occurred on the bound end. Taken together, we suggest that the N-finger and C-finger bindings of GATA3 commonly enhance DNA dissociation at one of the two DNA ends (the bound end for the C-finger binding and the unbound end for the N-finger binding), leading to triggering a conformational change in the chromatin.
Assuntos
Fator de Transcrição GATA3 , Nucleossomos , Cromatina/química , DNA/química , Simulação de Dinâmica Molecular , Nucleossomos/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Ligação Proteica , Fator de Transcrição GATA3/química , Domínios ProteicosRESUMO
Although the T helper 2 (Th2) subset is a critical player in the humoral immune response to extracellular parasites and suppression of Th1-mediated inflammation, Th2 cells have been implicated in allergic inflammatory diseases such as asthma, allergic rhinitis, and atopic dermatitis. GATA binding protein 3 (GATA3) is a primary transcription factor that mediates Th2 differentiation and secretion of Th2 cytokines, including IL-4, IL-5, and IL-13. Here, a nucleus-deliverable form of GATA3-transcription modulation domain (TMD) (ndG3-TMD) was generated using Hph-1 human protein transduction domain (PTD) to modulate the transcriptional function of endogenous GATA3 without genetic manipulation. ndG3-TMD was shown to be efficiently delivered into the cell nucleus quickly without affecting cell viability or intracellular signaling events for T cell activation. ndG3-TMD exhibited a specific inhibitory function for the endogenous GATA3-mediated transcription, such as Th2 cell differentiation and Th2-type cytokine production. Intranasal administration of ndG3-TMD significantly alleviated airway hyperresponsiveness, infiltration of immune cells, and serum IgE level in an OVA-induced mouse model of asthma. Also, Th2 cytokine secretion by the splenocytes isolated from the ndG3-TMD-treated mice substantially decreased. Our results suggest that ndG3-TMD can be a new therapeutic reagent to suppress Th2-mediated allergic diseases through intranasal delivery.
Assuntos
Asma , Fator de Transcrição GATA3 , Hipersensibilidade Respiratória , Animais , Humanos , Camundongos , Administração Intranasal , Asma/terapia , Núcleo Celular/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Fator de Transcrição GATA3/administração & dosagem , Fator de Transcrição GATA3/química , Camundongos Endogâmicos BALB C , Ovalbumina , Hipersensibilidade Respiratória/terapia , Células Th2RESUMO
PU.1 (encoded by Spi1), an ETS-family transcription factor with many hematopoietic roles, is highly expressed in the earliest intrathymic T cell progenitors but must be down-regulated during T lineage commitment. The transcription factors Runx1 and GATA3 have been implicated in this Spi1 repression, but the basis of the timing was unknown. We show that increasing Runx1 and/or GATA3 down-regulates Spi1 expression in pro-T cells, while deletion of these factors after Spi1 down-regulation reactivates its expression. Leveraging the stage specificities of repression and transcription factor binding revealed an unconventional but functional site in Spi1 intron 2. Acute Cas9-mediated deletion or disruption of the Runx and GATA motifs in this element reactivates silenced Spi1 expression in a pro-T cell line, substantially more than disruption of other candidate elements, and counteracts the repression of Spi1 in primary pro-T cells during commitment. Thus, Runx1 and GATA3 work stage specifically through an intronic silencing element in mouse Spi1 to control strength and maintenance of Spi1 repression during T lineage commitment.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA3/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/metabolismo , Transativadores/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem da Célula , Subunidade alfa 2 de Fator de Ligação ao Core/química , Fator de Transcrição GATA3/química , Deleção de Genes , Perfilação da Expressão Gênica , Inativação Gênica , Loci Gênicos , Íntrons/genética , Camundongos Endogâmicos C57BL , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Fator de Transcrição GATA3/química , Nucleossomos/química , Nucleossomos/genética , Motivos de Aminoácidos/genética , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/ultraestrutura , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Dedos de Zinco/genéticaRESUMO
BACKGROUND: The tissue-specific transcriptional programs during normal development require tight control by distal cis-regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence-specific DNA-binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T-cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown. RESULTS: Here we describe the identification of a novel bipartite Gata3 lens-specific enhancer located â¼18 kb upstream from its transcriptional start site. We also found that a 5-kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP-1, Ets-, and Smad1/5-binding sites as well as binding sites for lens-associated DNA-binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2. CONCLUSIONS: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up-regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186-1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Assuntos
Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Cristalino/embriologia , Animais , Sítios de Ligação , Proteína Morfogenética Óssea 4/fisiologia , Proteínas de Ligação a DNA , Fator 2 de Crescimento de Fibroblastos/fisiologia , Fator de Transcrição GATA3/química , Fator de Transcrição GATA3/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
Sleep apnea syndrome (SAS) is characterized by intermittent hypoxia (IH) during sleep. SAS and obesity are strongly related to each other. Here, we investigated the effect of IH on the expression of major appetite regulatory genes in human neuronal cells. We exposed NB-1, SH-SY5Y, and SK-N-SH human neuronal cells to IH (64 cycles of 5â¯min hypoxia and 10â¯min normoxia), normoxia, or sustained hypoxia for 24â¯h and measured the mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), galanin, galanin-like peptide, ghrelin, pyroglutamylated RFamide peptide, agouti-related peptide, neuropeptide Y, and melanocortin 4 receptor by real-time RT-PCR. IH significantly increased the mRNA levels of POMC and CART in all the neuronal cells. Deletion analysis revealed that the -705 to -686 promoter region of POMC and the -950 to -929 region of CART were essential for the IH-induced promoter activity. As possible GATA factor binding sequences were found in the two regions, we performed real-time RT-PCR to determine which GATA family members were expressed and found that GATA2 and GATA3 mRNAs were predominantly expressed. Therefore, we introduced siRNAs against GATA2 and GATA3 into NB-1 cells and found that GATA2 and GATA3 siRNAs abolished the IH-induced up-regulation of both POMC and CART mRNAs. These results indicate that IH stress up-regulates the mRNA levels of anorexigenic peptides, POMC and CART, in human neuronal cells via GATA2 and GATA3. IH can have an anorexigenic effect on SAS patients through the transcriptional activation of POMC and CART in the central nervous system.
Assuntos
Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/metabolismo , Núcleo Arqueado do Hipotálamo/metabolismo , Núcleo Arqueado do Hipotálamo/patologia , Sítios de Ligação , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Fator de Transcrição GATA2/antagonistas & inibidores , Fator de Transcrição GATA2/química , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA3/antagonistas & inibidores , Fator de Transcrição GATA3/química , Fator de Transcrição GATA3/genética , Deleção de Genes , Genes Reporter , Humanos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Pró-Opiomelanocortina/química , Pró-Opiomelanocortina/genética , Regiões Promotoras Genéticas , Síndromes da Apneia do Sono/metabolismo , Síndromes da Apneia do Sono/patologia , Fatores de Tempo , Regulação para CimaRESUMO
Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.
Assuntos
Fator de Transcrição GATA3/metabolismo , Memória Imunológica , Interferon gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Th2/imunologia , Sequência de Aminoácidos , Animais , Ativação Enzimática , Feminino , Fator de Transcrição GATA3/química , Histona Desacetilase 2/metabolismo , Humanos , Interleucina-4/biossíntese , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas com Domínio T , Dedos de ZincoRESUMO
Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during molluscan evolution and development. However, although the morphological changes during larval shell formation have been well described, the underlying molecular mechanisms remain poorly understood. In this study, we focused on the potential involvement of a GATA gene in shell formation because GATA genes are often downstream genes of BMP (bone morphogenetic protein) signaling pathways, which have been suggested to participate in molluscan shell formation. In the Pacific oyster Crassostrea gigas, we observed that the expression of a GATA2/3 homolog (cgi-gata2/3) was clearly restricted to the edge of the shell field in early larval stages (trochophore and D-veliger). This expression pattern supports the notion that cgi-gata2/3 gene plays conserved roles in bilaterian ectodermal development. It is possible that cgi-gata2/3 is one shell-formation gene under the regulation of BMP signaling pathways. In addition, cgi-gata2/3 was also detected in the ventral side of embryos. The expression of cgi-gata2/3 away from the shell field may be involved in hematopoiesis. Our results provide fundamental support for studies into the molecular mechanisms of larval shell formation and the functions of molluscan GATA genes.
Assuntos
Exoesqueleto/crescimento & desenvolvimento , Crassostrea/genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA3/genética , Sequência de Aminoácidos , Exoesqueleto/metabolismo , Animais , Crassostrea/anatomia & histologia , Crassostrea/crescimento & desenvolvimento , Fator de Transcrição GATA2/química , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA3/química , Fator de Transcrição GATA3/metabolismo , Larva/anatomia & histologia , Larva/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA3/metabolismo , Hematopoese , Fator de Transcrição Ikaros/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Células Cultivadas , Quinase 9 Dependente de Ciclina/química , Fator de Transcrição GATA1/química , Fator de Transcrição GATA2/química , Fator de Transcrição GATA3/química , Fator de Transcrição Ikaros/química , Camundongos , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
GATA-3 is a master transcription factor of the Th2 cells. We have identified GATA-3 cDNA and its splice variant in Atlantic cod. Cod GATA-3 (GmGATA-3) has a 1320 b p open reading frame encoding a polypeptide of 440 amino acids with two zinc finger domains that are well conserved within teleosts and higher vertebrates. The GATA-3 cDNA splice variant without zinc finger domains was shown to contain an 828 b p open reading frame encoding a polypeptide of 276 amino acids. Both GATA-3 proteins fused with RFP-tag were identified in or close to the nuclei 48 h after the plasmids were transfected in CHSE-214 cells. The full length GATA-3 with two zinc finger domains has a transcriptional function confirmed by transfection with GATA-3 reporter vector along with expression constructs of GATA-3 plasmids in CHSE-214 cells, whereas the GATA-3 splice variant without zinc finger domain did not enhance the activity of the GATA-3 reporter vector, and no interference was found between these two GATA-3 variants. RT-PCR analysis revealed that the two Atlantic cod GATA-3 variants were strongly expressed in the gills and infection with live Vibrio anguillarum induced the spleen expression of both GmGATA-3L and GmGATA-3S. Unexpectedly, PMA increased the expression of the GATA-3 splice variant in vivo and especially in vitro, with an increase of more than 100,000-fold in head kidney leukocytes at 24 and 48 h. On the other hand, there were no significant increases at the transcript level of full length GATA-3 between Poly I:C and ß-glucan treatment groups compared to controls.
Assuntos
Proteínas de Peixes/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Gadus morhua/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Proteínas de Peixes/análise , Proteínas de Peixes/química , Proteínas de Peixes/genética , Fator de Transcrição GATA3/análise , Fator de Transcrição GATA3/química , Rim Cefálico/metabolismo , Dados de Sequência Molecular , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Baço/metabolismo , Transcrição GênicaRESUMO
GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage and differentiation of T helper type 2 cells. We have identified and characterized the full-length Atlantic salmon GATA-3 cDNA (3074bp), having two zinc finger domains which are fully conserved within teleosts and higher vertebrates. RT-PCR analysis revealed that the Atlantic salmon GATA-3 (AsGATA-3) is strongly expressed in gills, thymus, and brain. Moreover, the involvement of GATA-3 in Atlantic salmon immune response was demonstrated by investigating the early time dependent expression profile of GATA-3 in spleen and head kidney following intraperitoneal injection of live Aeromonas salmonicida, LPS, and beta-glucan. Furthermore, we have determined 1.9kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites and the AsGATA-3 promoter is a TATA-less promoter. Activities of presumptive regulatory regions of this gene were assessed by transfecting different 5' deletion constructs and the result showed the basal promoter and positive transcriptional regulator activity of AsGATA-3 gene is determined by sequences located between +58 and -199bp upstream of the transcriptional start site (TSS). This study provides further insights into the transcriptional regulation of AsGATA-3.
Assuntos
Aeromonas salmonicida , Fator de Transcrição GATA3/biossíntese , Infecções por Bactérias Gram-Negativas/veterinária , Regiões Promotoras Genéticas/genética , Salmo salar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Fator de Transcrição GATA3/agonistas , Fator de Transcrição GATA3/química , Genes Reporter/genética , Genes Reporter/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Células HeLa , Humanos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , Salmo salar/imunologia , Alinhamento de Sequência , Transfecção , beta-Glucanas/farmacologiaRESUMO
Many advances in our understanding of the molecules that regulate the development, differentiation and function of T cells have been made over the past few years. One important regulator of T-cell differentiation is the transcription factor GATA-binding protein 3 (GATA3). Although the main function of GATA3 is to act as a master transcription factor for the differentiation of T helper 2 (T(H)2) cells, new research has helped to uncover crucial functions of GATA3 in T cells that go beyond T(H)2-cell differentiation and that are important at earlier stages of haematopoietic and lymphoid-cell development. This Review focuses on the functions of GATA3 from early thymocyte development to effector T-cell differentiation. In addition, we discuss the interactions between GATA3 and other transcription factors and signalling pathways, and highlight the functional significance of the GATA3 protein structure.
Assuntos
Linhagem da Célula/imunologia , Fator de Transcrição GATA3/metabolismo , Células Th2/imunologia , Sequência de Aminoácidos , Animais , Fator de Transcrição GATA3/química , Humanos , Linfopoese , Dados de Sequência Molecular , Transdução de Sinais , Timo/imunologiaRESUMO
The GATA family of transcription factors (GATA1-6) binds selected GATA sites in vertebrate genomes to regulate specific gene expression. Although vertebrate GATA factors have two highly conserved zinc finger motifs, how the two fingers act together to recognize functional DNA elements is not well understood. Here we determined the crystal structures of the C-terminal zinc finger of mouse GATA3 bound to DNA containing two variously arranged GATA binding sites. Our structures and accompanying biochemical analyses reveal two distinct modes of DNA binding by GATA to closely arranged sites. One mode involves cooperative binding by two GATA factors that interact with each other through protein-protein interactions. The other involves simultaneous binding of the N-terminal zinc finger (N-finger) and the C-terminal zinc finger of the same GATA factor. Our studies represent the first crystallographic analysis of GATA zinc fingers bound to DNA and provide new insights into the DNA recognition mechanism by the GATA zinc finger. Our crystal structure also reveals a dimerization interface in GATA that has previously been shown to be important for GATA self-association. These findings significantly advance our understanding of the structure and function of GATA and provide an important framework for further investigating the in vivo mechanisms of GATA-dependent gene regulation.
Assuntos
DNA/metabolismo , Fator de Transcrição GATA3/química , Fator de Transcrição GATA3/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
GATA-3, the only T cell-specific member of the GATA family of transcription factors, is essential for the intrathymic development of CD4+ T cells and for the differentiation of Th2 cells. However, whether distinct biochemical features, unique to GATA-3 compared with other GATA family members, are required to drive T cell transcriptional programs or whether the T cell-specific functions of GATA-3 can simply be ascribed to its expression pattern is unclear. Nor do we understand the protein structural requirements for each individual function of GATA-3. In this study, we report that a heterologous GATA factor, GATA-4, was competent in supporting the development of CD4+ T cells but could not fully compensate for GATA-3 in regulating the expression of Th cytokines. Specifically, GATA-3 was more potent than GATA-4 in driving the production of IL-13 due to a mechanism independent of DNA binding or chromatin remodeling of the IL-13 locus. The difference was mapped to a partially conserved region C-terminal to the second zinc finger. Converting a single proline residue located in this region of GATA-4 to its counterpart, a methionine of GATA-3, was sufficient to enhance the IL-13-promoting function of GATA-4 but had no effect on other cytokines. Taken together, our data demonstrate that the unique function of GATA-3 is conferred by both its cell type-specific expression and distinct protein structure.
Assuntos
Fator de Transcrição GATA3/química , Fator de Transcrição GATA3/metabolismo , Ativação Linfocitária/genética , Células Th2/imunologia , Timo/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Antígenos CD4/análise , Diferenciação Celular/genética , Sequência Conservada , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA4/química , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-3/genética , Interleucina-3/metabolismo , Metionina/química , Metionina/genética , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Prolina/química , Prolina/genética , Regiões Promotoras Genéticas , Células Th2/citologiaRESUMO
Pig GATA-3 cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR), using in silico cloning strategy based on pig dbEST. The length of pig GATA-3 cDNA is 1,760 bp containing a 1,335 bp open reading frame (ORF), which encodes a 444 amino acid protein. Semi-quantitative RT-PCR analysis of GATA-3 mRNA expression was done using the total RNAs from different normal tissues of a large white pig. The GATA-binding family of transcription factors comprised of a subgroup of DNA-binding proteins that both bound the consensus GATA motif and contained the class IV zinc finger motif. The molecular evolution tree was constructed based on the GATA-3 amino acid sequence and class IV zinc finger motif using mega 3.1. Phylogeny analysis of GATA factors isolated from vertebrates suggested that the six distinct vertebrate GATAs had descended from a common ancestral sequence, and the topology also suggested multiple modes of evolution including gene duplication and class IV zinc finger motif recombination. These data helped the authors in illuminating the pathways of divergent and convergent evolution of the GATA-binding family of transcription factors.