RESUMO
Genes that control the early stages of adipogenesis remain largely unknown. Here, we show that murine GATA-2 and GATA-3 are specifically expressed in white adipocyte precursors and that their down-regulation sets the stage for terminal differentiation. Constitutive GATA-2 and GATA-3 expression suppressed adipocyte differentiation and trapped cells at the preadipocyte stage. This effect is mediated, at least in part, through the direct suppression of peroxisome proliferator-activated receptor gamma. GATA-3-deficient embryonic stem cells exhibit an enhanced capacity to differentiate into adipocytes, and defective GATA-2 and GATA-3 expression is associated with obesity. Thus, GATA-2 and GATA-3 regulate adipocyte differentiation through molecular control of the preadipocyte-adipocyte transition.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA2 , Fator de Transcrição GATA3 , Expressão Gênica , Camundongos , Mutação , Obesidade/genética , Obesidade/metabolismo , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Células-Tronco/citologia , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , Dedos de ZincoRESUMO
In this report we have investigated the role of the Ets-1 transcription factor in the differentiation of the NK cell lineage in mice. Splenic NK cells express high levels of Ets-1. Ets-1-deficient mice produced by gene targeting developed mature erythrocytes, monocytes, neutrophils, and T and B lymphocytes. However, spleens from the Ets-1-deficient mice contained significantly reduced numbers of natural killer (NK) cells, and splenocytes from these mice lacked detectable cytolytic activity against NK cell targets in vitro. Moreover, unlike wild-type animals, Ets-1-deficient mice developed tumors following subcutaneous injection of NK-susceptible RMA-S cells. These NK cell defects could not be correlated with defects in the expression of IL-12, IL-15, and IL-18 or the IL-2 or IL-15 receptors. Thus, Ets-1 defines a novel transcriptional pathway that is required for the development of the NK cell lineage in mice.
Assuntos
Células Matadoras Naturais/imunologia , Proteínas Proto-Oncogênicas/imunologia , Fatores de Transcrição/imunologia , Animais , Diferenciação Celular , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Hematopoese/genética , Hematopoese/imunologia , Técnicas In Vitro , Células Matadoras Naturais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/imunologia , Fenótipo , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Receptores de Citocinas/imunologia , Baço/citologia , Baço/imunologia , Fatores de Transcrição/genéticaRESUMO
THE zinc-finger transcription factor GATA-3 is expressed in haematopoietic cells and in the developing kidney and nervous system. Within the haematopoietic lineages, expression of GATA-3 is restricted to thymocytes and T cells. Functionally important GATA-3 binding sites have been identified in multiple T-cell-specific genes. Mice containing homozygous null mutations of the GATA-3 gene die on embryonic day 12, precluding a detailed assessment of the role of GATA-3 in haematopoietic development. Here we have used murine embryonic stem (ES) cells containing homozygous mutations in the GATA-3 gene (GATA-3(-/-)) in conjunction with the RAG-2(-/-) (ref. 10) and C57BL/6 complementation systems to study the role of GATA-3 in mammalian haematopoiesis. Our results show that GATA-3(-/-) ES cells can contribute to the development of the mature erythroid, myelomonocytic and B-cell lineages, but fail to give rise to thymocytes or mature peripheral T cells. The differentiation of GATA-3(-/-) T cells is blocked at or before the earliest double-negative (CD4-/CD8-) stage of thymocyte development, such that the GATA-3(-/-) ES cells are unable to contribute measurably to the double-negative thymocyte population. These findings suggest that GATA-3 is an essential and specific regulator of early thymocyte development.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Hematopoese/fisiologia , Linfócitos T/citologia , Transativadores/fisiologia , Dedos de Zinco , Animais , Blastocisto , Células da Medula Óssea , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Quimera , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Fator de Transcrição GATA3 , Hematopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/genética , Deleção de Sequência , Baço/citologia , Timo/citologia , Transativadores/genéticaRESUMO
The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Troponina/genética , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/genética , DNA/metabolismo , Elementos Facilitadores Genéticos , Fator de Transcrição GATA4 , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Ativação Transcricional , Troponina CRESUMO
A dominant mutation was generated in transgenic mice as a consequence of insertional mutation. Heterozygous mice from transgenic line 9257 (Tg9257) are hyperactive with bidirectional circling behavior and have a distinctive facial appearance due to hypoplasia of the nasal bone. Morphological analysis of the inner ear revealed asymmetric abnormalities of the horizontal canal and flattening or invagination of the crista ampullaris, which can account for the circling behavior. The sensory epithelium appeared to be normal. The transgene insertion site was localized by in situ hybridization to the B1 band of mouse chromosome 18. Genetic mapping in an interspecific backcross demonstrated the gene order centromere--Tg9257--8.8 +/- 3.4--Grl-1, Egr-1, Fgf-1, Apc--14.7 +/- 4.3--Pdgfr. The phenotype and the mapping data suggest that the transgene may be inserted at the Twirler locus. Homozygosity for the transgene results in prenatal lethality, but compound heterozygotes carrying the Tw allele and the transgene are viable. The function of the closely linked ataxia locus is not disrupted by the transgene insertion. This insertional mutant will provide molecular access to genes located in the Twirler region of mouse chromosome 18.
Assuntos
Amilases/genética , Mapeamento Cromossômico , Elementos de DNA Transponíveis , Ossos Faciais/anormalidades , Camundongos Transgênicos/genética , Polimorfismo de Fragmento de Restrição , Crânio/anormalidades , Vestíbulo do Labirinto/anormalidades , Amilases/biossíntese , Animais , Cruzamentos Genéticos , Feminino , Genes Dominantes , Genes Recessivos , Marcadores Genéticos , Heterozigoto , Humanos , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Atividade Motora , Muridae , Comportamento EstereotipadoRESUMO
The human PCK1 gene encoding phosphoenolpyruvate carboxykinase (GTP) (PEPCK) was isolated and sequenced. There is 91% amino acid sequence identity (567/622 residues) between the human and the rat proteins, with conservation of intron/exon borders. A polymorphic dinucleotide microsatellite with the structure (CA)16(TA)5(CA) was identified in the 3' untranslated region of the cloned human PCK1 gene. This highly informative genetic marker has an estimated PIC value of 0.79 and heterozygosity of 0.81. Analysis of the RW pedigree demonstrated recombination between PCK1 and the MODY gene on chromosome 20. Multipoint linkage analysis of the reference pedigrees of the Centre d'Etude du Polymorphisme Humain localized PCK1 on the genetic map of chromosome 20 at a position distal to markers that are closely linked to MODY. PCK1 is part of a conserved linkage group on mouse Chromosome 2 with identical gene order but expanded length in the human genome.
Assuntos
Cromossomos Humanos Par 20 , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , DNA , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Polimorfismo Genético , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de AminoácidosRESUMO
Analysis of the structures of the human amylase genes has demonstrated that this multigene family contains at least five tandem gene copies, closely related in sequence but with distinct tissue specific expression. The structures of the genes demonstrate that the human salivary amylase gene was derived from a preexisting pancreatic amylase gene. Insertion of a retrovirus upstream of the amylase gene is responsible for the alteration in tissue specificity. A parotid specific enhancer has been identified within the retrovirus by expression studies in transgenic mice. The independent origin of salivary amylase in rodents and primates suggests that there has been strong evolutionary selection for amylase in saliva. The amylase genes demonstrate a novel mechanism for evolution of new patterns of tissue specific gene expression.
Assuntos
Amilases/genética , Saliva/enzimologia , Proteínas e Peptídeos Salivares/genética , Animais , Evolução Biológica , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Camundongos TransgênicosRESUMO
The human salivary amylase genes are associated with two inserted elements, a gamma-actin-processed pseudogene and an endogenous retroviral-like element. To test the contribution of these inserted elements to tissue specificity, 25 lines of transgenic mice carrying 10 amylase constructs were established. A 1-kb fragment of AMY1C (-1003 to +2) was found to be sufficient for parotid-specific expression of a human growth hormone reporter gene. The 1-kb fragment is entirely derived from inserted sequences. Deletion from -1003 to -826 resulted in reduced levels of transgene expression and loss of tissue specificity. The fragment -1003 to -327 was sufficient to transfer parotid specificity to the thymidine kinase promoter. The data demonstrate that the functional tissue-specific promoter of human AMY1C is derived from inserted sequences and that parotid expression can be conferred by sequences derived solely from the retrovirus. A role for retrotransposition in the evolution of gene regulation is indicated by these and other recent observations.
Assuntos
Amilases/genética , Regulação Viral da Expressão Gênica/genética , Camundongos Transgênicos/genética , Glândula Parótida/enzimologia , Provírus/genética , Actinas/genética , Animais , Sequência de Bases , Elementos de DNA Transponíveis/genética , Hormônio do Crescimento/genética , Humanos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Glândula Parótida/microbiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Radioimunoensaio , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica/genéticaRESUMO
Expression of an amylase/CAT hybrid gene was analyzed in transgenic mice. The amylase promoter was derived from a pancreatic amylase gene whose expression is repressed in diabetic animals. Pancreas-specific expression of the amylase/chloramphenicol acetyl-transferase (CAT) construct was observed in two independent transgenic lines. Correct initiation of transcription was demonstrated by protection of an anti-sense riboprobe. To evaluate the insulin dependence of the hybrid gene, diabetes was induced by treatment with streptozotocin. As a result of this treatment, pancreatic CAT activity was reduced to undetectable levels. Subsequent administration of insulin restored CAT activity to normal levels. The abundance of CAT transcripts was also greatly reduced in diabetic pancreas. These studies localize the determinants of pancreas specificity and insulin dependence to the region between -208 and +19 of the mouse pancreatic Amy-2.2 gene. The results are consistent with an effect of insulin on amylase transcription, rather than post-transcriptional regulation of mRNA processing or stability.