RESUMO
Transcription factor EVI1 is essential for normal hematopoiesis in embryos but is aberrantly elevated in bone marrow cells of myelodysplastic syndrome (MDS) patients. EVI1 and its downstream GATA-2 appear to be a possible therapeutic target of MDS. Here we found that treatment of EVI1-expressing K562 cells with arsenite (As(III)) reduced the mRNA and protein levels of EVI1 and GATA-2. A gel shift assay using the nuclear extract of K562 cells showed that As(III) suppressed the DNA-binding activity of EVI1. The DNA-binding activity of the recombinant EVI1 protein was also suppressed by As(III) but was recovered by excess amounts of dithiothreitol, suggesting the involvement of cysteine residues of EVI1. Since the 7th Zn finger domain of EVI1, having a motif of CCHC, is known to be involved in DNA-binding, the synthetic peptide of 7th Zn finger domain was reacted with As(III) and subjected to MALDI-TOF-MS analysis. The results showed that As(III) binds to this peptide via three cysteine residues. As(III)-induced reduction of the DNA-binding activity of the recombinant EVI1 was abolished by the mutations of each of three cysteine residues to alanine in the 7th Zn finger domain. These results demonstrate that As(III) causes the down-regulation of EVI1 and GATA-2 by inhibiting the transcriptional activity of EVI1 through the binding to the cysteine residues of CCHC-type Zn finger domain.
Assuntos
Arsenitos/farmacologia , Cisteína/metabolismo , Fator de Transcrição GATA2/genética , Proteína do Locus do Complexo MDS1 e EVI1/genética , Compostos de Sódio/farmacologia , Dedos de Zinco/genética , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Núcleo Celular/química , Núcleo Celular/metabolismo , Misturas Complexas/química , Misturas Complexas/metabolismo , Cisteína/genética , Ditiotreitol/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Transcrição GATA2/antagonistas & inibidores , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Humanos , Células K562 , Proteína do Locus do Complexo MDS1 e EVI1/antagonistas & inibidores , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The gene ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI encode zinc-finger proteins that have been recognized as important oncogenes in various types of cancer. In contrast to the established role of EVI and MDS/EVI in cancer development, their potential function during vertebrate postembryonic development, especially in organ-specific adult stem cells, is unclear. Amphibian metamorphosis is strikingly similar to postembryonic development around birth in mammals, with both processes taking place when plasma thyroid hormone (T3) levels are high. Using the T3-dependent metamorphosis in Xenopus tropicalis as a model, we show here that high levels of EVI and MDS/EVI are expressed in the intestine at the climax of metamorphosis and are induced by T3. By using the transcription activator-like effector nuclease gene editing technology, we have knocked out both EVI and MDS/EVI and have shown that EVI and MDS/EVI are not essential for embryogenesis and premetamorphosis in X. tropicalis On the other hand, knocking out EVI and MDS/EVI causes severe retardation in the growth and development of the tadpoles during metamorphosis and leads to tadpole lethality at the climax of metamorphosis. Furthermore, the homozygous-knockout animals have reduced adult intestinal epithelial stem cell proliferation at the end of metamorphosis (for the few that survive through metamorphosis) or during T3-induced metamorphosis. These findings reveal a novel role of EVI and/or MDS/EVI in regulating the formation and/or proliferation of adult intestinal adult stem cells during postembryonic development in vertebrates.-Okada, M., Shi, Y.-B. EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development.
