RESUMO
HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples, as well as in wild-type versus Hoxa9-deficient marrow, using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors, being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells, indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1, an important regulator of myelopoiesis, was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein, HOXA9 did not appear to modulate Pu.1 expression in murine BM cells.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Proteínas de Homeodomínio/genética , Camundongos , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Regulação para CimaRESUMO
While investigating the mechanism of action of the HOXA9 protein, we serendipitously identified Meis1 as a HOXA9 regulatory target. Since HOXA9 and MEIS1 play key developmental roles, are cooperating DNA binding proteins and leukemic oncoproteins, and are important for normal hematopoiesis, the regulation of Meis1 by its partner protein is of interest. Loss of Hoxa9 caused downregulation of the Meis1 mRNA and protein, while forced HOXA9 expression upregulated Meis1. Hoxa9 and Meis1 expression was correlated in hematopoietic progenitors and acute leukemias. Meis1(+/-) Hoxa9(-/-) deficient mice, generated to test HOXA9 regulation of endogenous Meis1, were small and had reduced bone marrow Meis1 mRNA and significant defects in fluorescence-activated cell sorting-enumerated monocytes, mature and pre/pro-B cells, and functional B-cell progenitors. These data indicate that HOXA9 modulates Meis1 during normal murine hematopoiesis. Chromatin immunoprecipitation analysis did not reveal direct binding of HOXA9 to Meis1 promoter/enhancer regions. However, Creb1 and Pknox1, whose protein products have previously been reported to induce Meis1, were shown to be direct targets of HOXA9. Loss of Hoxa9 resulted in a decrease in Creb1 and Pknox1 mRNA, and forced expression of CREB1 in Hoxa9(-/-) bone marrow cells increased Meis1 mRNA almost as well as HOXA9, suggesting that CREB1 may mediate HOXA9 modulation of Meis1 expression.
Assuntos
Genes Neoplásicos , Hematopoese , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Peso Corporal , Cruzamentos Genéticos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Implantação do Embrião , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Heterozigoto , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide/genética , Masculino , Camundongos , Proteína Meis1 , Proteínas de Neoplasias/genética , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.
