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
HOXA9 expression is a common feature of acute myeloid leukemia, and high-level expression is correlated with poor prognosis. Moreover, HOXA9 overexpression immortalizes murine marrow progenitors that are arrested at a promyelocytic stage of differentiation when cultured and causes leukemia in recipient mice following transplantation of HOXA9 expressing bone marrow. The molecular mechanisms underlying the physiologic functions and transforming properties of HOXA9 are poorly understood. This study demonstrates that HOXA9 is phosphorylated by protein kinase C (PKC) and casein kinase II and that PKC mediates phosphorylation of purified HOXA9 on S204 as well as on T205, within a highly conserved consensus sequence, in the N-terminal region of the homeodomain. S204 in the endogenous HOXA9 protein was phosphorylated in PLB985 myeloid cells, as well as in HOXA9-immortalized murine marrow cells. This phosphorylation was enhanced by phorbol ester, a known inducer of PKC, and was inhibited by a specific PKC inhibitor. PKC-mediated phosphorylation of S204 decreased HOXA9 DNA binding affinity in vitro and the ability of the endogenous HOXA9 to form cooperative DNA binding complexes with PBX. PKC inhibition significantly reduced the phorbol-ester induced differentiation of the PLB985 hematopoietic cell line as well as HOXA9-immortalized murine bone marrow cells. These data suggest that phorbol ester-induced myeloid differentiation is in part due to PKC-mediated phosphorylation of HOXA9, which decreases the DNA binding of the homeoprotein.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Mieloides/fisiologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Caseína Quinase II , Linhagem Celular , Ativação Enzimática , Isoenzimas/metabolismo , Leucemia Mieloide , Camundongos , Dados de Sequência Molecular , Células Mieloides/citologia , Ésteres de Forbol/metabolismo , Fosforilação , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismoRESUMO
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.
Assuntos
Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Regulação para Baixo , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors, and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis, we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner, pre-B-cell leukemia transcription factor-1 (PBX1). Additionally, we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo, HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4, a region frequently deleted in HOXA9-induced AMLs. In vitro, HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1.
Assuntos
Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Transformação Celular Neoplásica/patologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Leucemia Mieloide/sangue , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Doença Aguda , Animais , Diferenciação Celular/genética , Linhagem Celular Transformada , Proliferação de Células , Eritropoese/genética , Feminino , Proteínas de Homeodomínio/fisiologia , Cariotipagem , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Linfopoese/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteínas de Neoplasias/fisiologia , Fenótipo , Fatores de TempoRESUMO
Although HOXB6 and other HOX genes have previously been associated with hematopoiesis and leukemias, the precise mechanism of action of their protein products remains unclear. Here we use a biological model in which HOXB6 represses alpha- and gamma-globin mRNA levels to perform a structure/function analysis for this homeodomain protein. HOXB6 protein represses globin transcript levels in stably transfected K562 cells in a DNA-binding dependent fashion. However, the capacity to form cooperative DNA-binding complexes with the PBX co-factor protein is not required for HOXB6 biological activity. Neither the conserved extreme N-terminal region, a polyglutamic acid region at the protein C terminus, nor the Ser(214) CKII phosphorylation site was required for DNA binding or activity in this model. We have previously reported that HOX proteins can inhibit CREB-binding protein (CBP)-histone acetyltransferase-mediated potentiation of reporter gene transcription. We now show that endogenous CBP is co-precipitated with exogenous HOXB6 from nuclear and cytoplasmic compartments of transfected K562 cells. Furthermore, endogenous CBP co-precipitates with endogenous HOXB6 in day 14.5 murine fetal liver cells during active globin gene expression in this tissue. The CBP interaction motif was localized to the homeodomain but does not require the highly conserved helix 3. Our data suggest that the homeodomain contains most or all of the important structures required for HOXB6 activity in blood cells.