RESUMO
MEIS1 is a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1 is rate-limiting in MLL-associated leukemias and potently interacts with Hox and NUP98-HOX genes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by Meis1, we assessed the function of a novel engineered fusion form of Meis1, M33-MEIS1, designed to confer transcriptional repression to Meis1 target genes that are otherwise up-regulated in normal and malignant hematopoiesis. Retroviral overexpression of M33-Meis1 resulted in the rapid and complete eradication of M33-Meis1-transduced normal and leukemic cells in vivo. Cell-cycle analysis showed that M33-Meis1 impeded the progression of cells from G(1)-to-S phase, which correlated with significant reduction of cyclin D3 levels and the inhibition of retinoblastoma (pRb) hyperphosphorylation. We identified cyclin D3 as a direct downstream target of MEIS1 and M33-MEIS1 and showed that the G(1)-phase accumulation and growth suppression induced by M33-Meis1 was partially relieved by overexpression of cyclin D3. This study provides strong evidence linking the growth-promoting activities of Meis1 to the cyclin D-pRb cell-cycle control pathway.
Assuntos
Ciclo Celular , Ciclina D3/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Transplante de Medula Óssea , Transformação Celular Neoplásica , Imunoprecipitação da Cromatina , Ciclina D3/metabolismo , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Perfilação da Expressão Gênica , Hematopoese , Proteínas de Homeodomínio/genética , Imunoprecipitação , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , TransfecçãoRESUMO
The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Modelos Biológicos , Proteína Meis1 , Fator de Transcrição 1 de Leucemia de Células Pré-B , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The monocytic leukemia zinc finger protein MOZ and the related factor MORF form tetrameric complexes with ING5 (inhibitor of growth 5), EAF6 (Esa1-associated factor 6 ortholog), and the bromodomain-PHD finger protein BRPF1, -2, or -3. To gain new insights into the structure, function, and regulation of these complexes, we reconstituted them and performed various molecular analyses. We found that BRPF proteins bridge the association of MOZ and MORF with ING5 and EAF6. An N-terminal region of BRPF1 interacts with the acetyltransferases; the enhancer of polycomb (EPc) homology domain in the middle part binds to ING5 and EAF6. The association of BRPF1 with EAF6 is weak, but ING5 increases the affinity. These three proteins form a trimeric core that is conserved from Drosophila melanogaster to humans, although authentic orthologs of MOZ and MORF are absent in invertebrates. Deletion mapping studies revealed that the acetyltransferase domain of MOZ/MORF is sufficient for BRPF1 interaction. At the functional level, complex formation with BRPF1 and ING5 drastically stimulates the activity of the acetyltransferase domain in acetylation of nucleosomal histone H3 and free histones H3 and H4. An unstructured 18-residue region at the C-terminal end of the catalytic domain is required for BRPF1 interaction and may function as an "activation lid." Furthermore, BRPF1 enhances the transcriptional potential of MOZ and a leukemic MOZ-TIF2 fusion protein. These findings thus indicate that BRPF proteins play a key role in assembling and activating MOZ/MORF acetyltransferase complexes.
Assuntos
Histona Acetiltransferases/metabolismo , Complexos Multiproteicos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA , Histona Acetiltransferases/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
MEIS proteins form heteromeric DNA-binding complexes with PBX monomers and PBX.HOX heterodimers. We have shown previously that transcriptional activation by PBX.HOX is augmented by either protein kinase A (PKA) or the histone deacetylase inhibitor trichostatin A (TSA). To examine the contribution of MEIS proteins to this response, we used the chromatin immunoprecipitation assay to show that MEIS1 in addition to PBX1, HOXA1, and HOXB1 was recruited to a known PBX.HOX target, the Hoxb1 autoregulatory element following Hoxb1 transcriptional activation in P19 cells. Subsequent to TSA treatment, MEIS1 recruitment lagged behind that of HOX and PBX partners. MEIS1A also enhanced the transcriptional activation of a reporter construct bearing the Hoxb1 autoregulatory element after treatment with TSA. The MEIS1 homeodomain and protein-protein interaction with PBX contributed to this activity. We further mapped TSA-responsive and CREB-binding protein-dependent PKA-responsive transactivation domains to the MEIS1A and MEIS1B C termini. Fine mutation of the 56-residue MEIS1A C terminus revealed four discrete regions required for transcriptional activation function. All of the mutations impairing the response to TSA likewise reduced activation by PKA, implying a common mechanistic basis. C-terminal deletion of MEIS1 impaired transactivation without disrupting DNA binding or complex formation with HOX and PBX. Despite sequence similarity to MEIS and a shared ability to form heteromeric complexes with PBX and HOX partners, the PREP1 C terminus does not respond to TSA or PKA. Thus, MEIS C termini possess transcriptional regulatory domains that respond to cell signaling and confer functional differences between MEIS and PREP proteins.