RESUMO
Heat shock transcription factors (HSFs) are characterized by their ability, upon activation, to bind to heat shock response elements (HSE) present in the promoter of their target genes. HSE are composed of inverted repeats of the pentamer nGAAm. In this study, we compare the embryonic HSF2 protein, purified from F9 embryonal carcinoma cells tumor, and the in vitro synthesized HSF2. We show that the context of HSF2 synthesis influences its thermosensitivity and DNA-binding properties. Therefore, we determined the consensus binding sequence for the purified embryonic HSF2 by the technique of systematic evolution of ligands by exponential enrichment (SELEX). We show that embryonic HSF2 prefers sites containing three or four nGAAm inverted pentamers and that its optimal binding sequence contains the 8-mer palindromic core 5'-TTCTAGAA-3'. The consensus binding sequence for the embryonic HSF2 will be very helpful to identify new targets for this factor, during developmental and differentiation processes.
Assuntos
Proteínas de Choque Térmico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Sequência Consenso , DNA/metabolismo , Biblioteca Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Temperatura Alta , Camundongos , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Células Tumorais CultivadasRESUMO
Heat shock factor 2, one of the four vertebrate HSFs, transcriptional regulators of heat shock gene expression, is active during embryogenesis and spermatogenesis, with unknown functions and targets. By disrupting the Hsf2 gene, we show that, although the lack of HSF2 is not embryonic lethal, Hsf2(-/-) mice suffer from brain abnormalities, and meiotic and gameto genesis defects in both genders. The disturbances in brain are characterized by the enlargement of lateral and third ventricles and the reduction of hippocampus and striatum, in correlation with HSF2 expression in proliferative cells of the neuroepithelium and in some ependymal cells in adults. Many developing spermatocytes are eliminated via apoptosis in a stage-specific manner in Hsf2(-/-) males, and pachytene spermatocytes also display structural defects in the synaptonemal complexes between homologous chromosomes. Hsf2(-/-) females suffer from multiple fertility defects: the production of abnormal eggs, the reduction in ovarian follicle number and the presence of hemorrhagic cystic follicles are consistent with meiotic defects. Hsf2(-/-) females also display hormone response defects, that can be rescued by superovulation treatment, and exhibit abnormal rates of luteinizing hormone receptor mRNAs.
Assuntos
Encéfalo/anormalidades , Encéfalo/metabolismo , Cromossomos/ultraestrutura , Proteínas de Choque Térmico/genética , Infertilidade Feminina/genética , Meiose , Fatores de Transcrição/genética , Alelos , Animais , Apoptose , Western Blotting , Embrião de Mamíferos/metabolismo , Feminino , Fertilidade/genética , Vetores Genéticos , Genótipo , Heterozigoto , Imuno-Histoquímica , Óperon Lac , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Genéticos , Ovário/metabolismo , Regiões Promotoras Genéticas , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Testículo/metabolismo , Fatores de Tempo , beta-Galactosidase/metabolismoAssuntos
Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Actinas/metabolismo , Animais , Apoptose , Cristalinas/química , Cristalinas/genética , Cristalinas/metabolismo , Ectoderma/metabolismo , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/química , Humanos , Filamentos Intermediários/metabolismo , Mesoderma/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Crista Neural/metabolismo , Fosforilação , Pele/metabolismoRESUMO
The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.
