The AML1/ETO fusion protein blocks transactivation of the GM-CSF promoter by AML1B.
The t(8;21) translocation, commonly found in acute myelogenous leukemia (AML), generates a fusion protein containing N-terminal AML1 and C-terminal ETO amino acids. The human AML1 gene encodes several related proteins that specifically bind to the sequence TGT/cGGT, located in the promoter regions of a variety of hematopoietic growth factor genes. To examine the abilities of the AML1B protein (which contains 479 amino acids), a shorter AML1A isoform (which contains amino acids 1-250), and the AML1/ETO fusion protein (which contains AML1A amino acids 1-177) to stimulate transcription from the GM-CSF promoter, we performed co-transfection experiments in T cells using a human GM-CSF promoter-CAT reporter gene plasmid and expression vectors that contain the cDNAs for one of the above proteins. Our data demonstrate that AML1B, but not AML1A or AML1/ETO transactivates the GM-CSF promoter, requiring the TGTGGT sequence contained between base pairs -68 and -53. Furthermore, we show that AML1/ETO, but not AML1A, inhibits the ability of AML1B to stimulate CAT expression. Electrophoretic mobility shift assays demonstrated the specific binding of AML1 proteins to the GM-CSF promoter TGTGGT sequence, which does not require GM-CSF sequences immediately upstream of this binding site. Our data support a role for AML1B as a transcriptional activator and establish that the AML1/ETO fusion protein can act as a dominant negative protein on the human GM-CSF promoter. Although AML1/ETO does not stimulate the transcription of GM-CSF, it may function by inhibiting the normal activity of AML1B in AML cells with the t(8;21) translocation.
Laboratory of Molecular Aspects of Hematopoiesis, Sloan-Kettering Institute, New York, NY 10021, USA.
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Frank
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Zhang
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Uchida
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Meyers
S W
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Hiebert
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Nimer
D001483:Base Sequence; D002460:Cell Line; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D000075142:RUNX1 Translocation Partner 1 Protein; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D000075142:RUNX1 Translocation Partner 1 Protein; C493728:RUNX1 protein, human; C082222:RUNX1T1 protein, human; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor