RESUMO
The RUNX3 gene belongs to the runt domain family of transcription factors that act as master regulators of gene expression in major developmental pathways. In mammals the family includes three genes, RUNX1, RUNX2 and RUNX3. Here, we describe a comparative analysis of the human chromosome 1p36.1 encoded RUNX3 and mouse chromosome 4 encoded Runx3 genomic regions. The analysis revealed high similarities between the two genes in the overall size and organization and showed that RUNX3/Runx3 is the smallest in the family, but nevertheless exhibits all the structural elements characterizing the RUNX family. It also revealed that RUNX3/Runx3 bears a high content of the ancient mammalian repeat MIR. Together, these data delineate RUNX3/Runx3 as the evolutionary founder of the mammalian RUNX family. Detailed sequence analysis placed the two genes at a GC-rich H3 isochore with a sharp transition of GC content between the gene sequence and the downstream intergenic region. Two large conserved CpG islands were found within both genes, one around exon 2 and the other at the beginning of exon 6. RUNX1, RUNX2 and RUNX3 gene products bind to the same DNA motif, hence their temporal and spatial expression during development should be tightly regulated. Structure/function analysis showed that two promoter regions, designated P1 and P2, regulate RUNX3 expression in a cell type-specific manner. Transfection experiments demonstrated that both promoters were highly active in the GM1500 B-cell line, which endogenously expresses RUNX3, but were inactive in the K562 myeloid cell line, which does not express RUNX3.
Assuntos
Proteínas de Ligação a DNA/genética , Genes/genética , Fatores de Transcrição/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Subunidade alfa 3 de Fator de Ligação ao Core , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Regulação da Expressão Gênica , Humanos , Íntrons , Células K562 , Luciferases/genética , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas , Fatores de Transcrição/biossíntese , Animais , Desenvolvimento Ósseo , Osso e Ossos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core , Sistema Hematopoético/embriologia , Imuno-Histoquímica , Camundongos , Fatores de Tempo , Distribuição TecidualRESUMO
AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.
Assuntos
Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas , Capuzes de RNA/genética , Ribossomos/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Proteínas Virais , Regiões 5' não Traduzidas/genética , Diferenciação Celular , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Regulação da Expressão Gênica , Genes Reporter , Hematopoese/genética , Humanos , Células K562 , Megacariócitos/metabolismo , Proteínas Nucleares , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , TransfecçãoRESUMO
The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.
Assuntos
Proteínas de Ligação a DNA , Hematopoese , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Sequência de Bases , Sítios de Ligação , Subunidade alfa 2 de Fator de Ligação ao Core , Primers do DNA/química , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The human chromosome 21 acute myeloid leukemia gene AML1 is frequently rearranged in the leukemia-associated translocations t(8;21) and t(3;21), generating fused proteins containing the amino-terminal part of AML1. In normal blood cells, five size classes (2-8 kb) of AML1 mRNAs have been previously observed. We isolated seven cDNAs corresponding to various AML1 mRNAs. Sequencing revealed that their size differences were mainly due to alternatively spliced 5' and 3' untranslated regions, some of which were vast, exceeding 1.5 kb (5') and 4.3 kb (3'). These untranslated regions contain sequences known to control mRNA translation and stability and seem to modulate AML1 mRNA stability. Further heterogeneity was found in the coding region due to the presence of alternatively spliced stop codon-containing exons. The latter led to production of polypeptides that were smaller than the full-length AML1 protein; they lacked the trans-activation domains but maintained DNA binding and heterodimerization ability. The size of these truncated products was similar to the AML1 segment in the fused t(8;21) and t(3;21) proteins. In thymus, only one mRNA species of 6 kb was detected. Using in situ hybridization, we showed that its expression was confined to the cortical region of the organ. The 6-kb mRNA was also prominent in cultured peripheral blood T cells, and its expression was markedly reduced upon mitogenic activation by phorbol myristate acetate (TPA) plus concanavalin A (ConA). These results and the presence of multiple coding regions flanked by long complex untranslated regions, suggest that AML1 expression is regulated at different levels by several control mechanisms generating the large variety of mRNAs and protein products.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Processamento Alternativo , Animais , Sequência de Bases , Subunidade alfa 2 de Fator de Ligação ao Core , Primers do DNA/química , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Genes , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Neoplásico/genética , TimoRESUMO
cDNAs corresponding to three human runt domain containing genes, AML1, AML2, and AML3, were isolated and characterized. In addition to homology in the highly conserved runt domain, extensive sequence similarities were also observed in other parts of the proteins. All three carried an identical, putative ATP binding site -GRSGRGKS-, and their C-terminal halves were particularly rich in proline and serine residues. While AML1 cDNAs were cloned by others, AML2 represents a new member, not previously described, of the runt domain gene family, and AML3 was identified as the human homologue of mouse PEB-P2 alpha A. The chromosomal location of AML1 to chromosome 21q22 was confirmed, while AML2 and AML3 were mapped to chromosome regions 1p36 and 6p21, respectively. Analysis of AML1 and AML2 expression in hematopoietic cell lines revealed a distinct pattern of expression.
Assuntos
DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Família Multigênica , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 6 , Clonagem Molecular , Síndrome de Down/genética , Proteínas de Drosophila , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares , Homologia de Sequência de AminoácidosRESUMO
The homeobox gene Hox-2.4 is transcriptionally activated in cells of the mouse myeloid leukemia WEHI-3B. The constitutive Hox-2.4 expression in WEHI-3B cells is due to insertion of a transposable element belonging to the family of intracisternal A particles. In this study, we demonstrated the oncogenic potential of this activated homeobox gene. NIH 3T3 fibroblast clones bearing the activated Hox-2.4 gene produced fibrosarcomas in nude mice.
