RESUMO
The parasitic nematode Ascaris suum successfully adapts to a significant decrease in oxygen availability during its life cycle by altering its metabolic system dramatically. However, little is known about the regulatory mechanisms of adaptation to hypoxic environments in A. suum. In multicellular organisms, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1ß subunits, is a master regulator of genes involved in adaptation to hypoxia. In the present study, cDNAs encoding HIF-1α and HIF-1ß were cloned from A. suum and characterized. The full-length A. suum hif-1α and hif-1ß cDNAs contain open reading frames encoding proteins with 832 and 436 amino acids, respectively. In the deduced amino acid sequences of A. suum HIF-1α and HIF-1ß, functional domains essential for DNA-binding, dimerization, and oxygen-dependent prolyl hydroxylation were conserved. The interaction between A. suum HIF-1α and HIF-1ß was confirmed by the yeast two-hybrid assay. Both A. suum hif-1α and hif-1ß mRNAs were expressed at all stages examined (fertilized eggs, third-stage larvae, lung-stage larvae, young adult worms, and adult muscle tissue), and most abundantly in the aerobic free-living third-stage larvae, followed by a gradual decrease after infection of the host. hif-1 mRNA transcription was not sensitive to the oxygen environment in either third-stage larvae or adult worms (muscle tissue), and was regulated in a stage-specific manner. High expression of hif-1 mRNAs in third-stage larvae suggests its contribution to pre-adaptation to a hypoxic environment after infection of their host. Sequence analysis of 5'-upstream regions of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) genes, which show stage-specific expression and play an important role in oxygen adaptation during the life cycle, revealed that all subunits except for the adult-type flavoprotein subunit (Fp) possess putative hypoxia-responsive elements (HREs), suggesting that they are hif-1 target genes.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Ascaris suum/crescimento & desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Estágios do Ciclo de Vida , Oxigênio/metabolismo , Sequência de Aminoácidos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Ascaris suum/genética , Clonagem Molecular , DNA Complementar , DNA de Helmintos/genética , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Feminino , Flavoproteínas/genética , Flavoproteínas/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
DNA supercoiling factor (SCF) is a protein capable of generating negative supercoils in DNA in conjunction with topoisomerase II. To clarify the biological functions of SCF, we introduced a heritable SCF RNAi into Drosophila. Upon knockdown of SCF, we observed male lethality and male-specific reduction in the expression levels of X-linked genes. SCF functionally interacts with components of the MSL complex, which are required for dosage compensation via hypertranscription of the male X chromosome. Moreover, SCF colocalizes with the MSL complex along the male X chromosome. Upon overexpression of SCF, the male X chromosome had a bloated appearance. This phenotype was dependent on the histone acetyltransferase MOF and was suppressed by simultaneous overexpression of ISWI. These findings demonstrate that SCF plays a role in transcriptional activation via alteration of chromatin structure and provide evidence that SCF contributes to dosage compensation.
