The AML1/ETO fusion protein activates transcription of BCL-2

Abstract

The AML1 gene, located on chromosome 21, is involved in several distinct chromosomal translocations in human leukemia. In t(8;21) acute myelogenous leukemia (AML), the AML1 gene is juxtaposed to the ETO gene located on chromosome 8, generating an AML1͞ETO fusion protein. Both AML1͞ETO and the AML1 proteins recognize the same consensus DNA-binding motif (TGT͞CGGT), which is found in the promoters of several genes involved in hematopoiesis. We found that two myeloid leukemia cell lines with the t(8;21) translocation, Kasumi and SKNO-1, have elevated levels of BCL-2 protein compared with other myeloid cell lines. In addition, we identified a consensus AML1 binding site in the BCL-2 promoter. Thus far, AML1͞ETO has been shown to dominantly repress its target genes; however, we found that AML1͞ETO activates transcription of the BCL-2 gene in U937 cells. This activation requires the presence of both the runt homology domain (rhd) and the C-terminal portion of AML1͞ ETO. We demonstrated sequence specific binding of both AML1A and AML1͞ETO to the TGTGGT sequence in the BCL-2 promoter and showed that the AML1 binding site is required for responsiveness to AML1͞ETO. Interestingly, AML1A and AML1B do not modulate the activity of the BCL-2 promoter. The elevated levels of BCL-2 in cells that express AML1͞ETO may prolong their life span and contribute to the development of t(8;21) leukemia.Chromosomal translocations found in human leukemia frequently involve genes that code for transcription factors (1). In acute myeloid leukemia (AML) with the t(8;21) chromosomal translocation, which occurs in Ϸ40% cases of AML with the M2 French-American-British subtype, coding sequences of the AML1 gene (on chromosome 21) are juxtaposed to coding sequences of the ETO gene (on chromosome 8) generating an AML1͞ETO fusion protein (2, 3). The AML1 family of transcription factors recognize the binding sequence 5Ј-TGT͞ CGGT-3Ј (2) through an 117-amino acid region that is highly homologous to the Drosophila segmentation gene runt (2, 3), and has been called the runt homology domain (rhd). This domain is necessary for DNA binding, as well as for proteinprotein interactions (3). At least three forms of AML1 protein are produced by alternative splicing (4). The AML1-B isoform (479 amino acids) contains the rhd and a putative C-terminal transcriptional activation domain; the AML1-A isoform (250 amino acids) contains the DNA binding domain, but lacks the potential transcriptional activation domain. AML1-B, but not A ML-1A, can transactivate the human granulocyte͞ macrophage colony-stimulating factor (GM-CSF) promoter (5) and the T cell receptor ␤ enhancer (6), whereas both isoforms can transactivate the human interleukin 3 (IL-3) gene (H.U., S.Z., and S.D.N., unpublished work).The genes encoding AML1 or its dimerization partner CBF␤, have been shown to be involved in several other translocations in human acute leukemia (7). The AML1 gene is fused to the TEL gene in t(12;21) acute lymphoblastic leukemia (8). In the t(3;21) translocation, seen ...

CBFβ-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells

“…Consistent with this model, when Kasumi AML cells, which harbor t(8;21), were treated with an AML1-ETO antisense oligonucleotide they di erentiated and their proliferation rate slowed (Sakakura et al, 1994). The possibility that CBF oncoproteins also contribute to tumor progression or phenotype by activating a subset of gene regulated by CBFs was recently raised by the observation that AML1-ETO can activate the BCL-2 gene promoter (Klampfer et al, 1996). Again, such gene activation might be potentiated by initial genetic hits which enable higher level expression of AML1-ETO and other CBF oncoproteins.…”

“…Blockade of di erentiation is not absolute, as the majority of leukemic blasts harboring these oncoproteins express di erentiation markers such as MPO. The possibility that CBF oncoproteins activate genes leading to tumor progression, such as the BCL-2 gene (Klampfer et al, 1996), or partial di erentiation must also be considered 1993). For oligonucleotide competition, unlabeled competitors were added 5 min prior to the radiolabeled probe.…”

The t(8;21) fusion protein, AML1/ETO, transforms NIH3T3 cells and activates AP-1

“…Consistent with a negative e ect on AML1 function, AML1/ETO has been shown to block both the di erentiation of myeloid progenitor cell lines and the ability of AML1 to activate myeloid speci®c promoters (Kitabayashi et al, 1998;Westendorf et al, 1998). In contrast to its role as a dominant inhibitor, AML1/ETO can activate the BCL-2 promoter in U937 cells, an e ect not seen when AML1 or ETO are overexpressed in these cells (Klampfer et al, 1996). AML1/ETO expression in a knock-in model, and in retrovirally transduced murine bone marrow stem cells, caused increased progenitor cell self-renewal capacity and dysplastic hematopoiesis (Okuda et al, 1998).…”

Three distinct domains in TEL-AML1 are required for transcriptional repression of the IL-3 promoter

“…The arrows in a and b indicate the location of the speci®c PCR products for IL-3, and GAPDH, respectively CSFR promoter) and AML1/EV1-can activate AP-1 activity (Mitani et al, 1994). The transcriptional activities of AML1/ETO require the Rhd of AML1, and varying amino acids contributed by ETO (Klampfer et al, 1996;Lenny et al, 1995;Rhoades et al, 1996) whereas the activation of AP-1 activity by AML1/EVI-1 requires EVI-1 amino acids and not the Rhd (Mitani et al, 1994). We, and others, have now demonstrated that AML1/ETO and TEL-AML1 act as repressors of basal transcription of the IL-3 promoter and TCR-b enhancer in T cell lines (Uchida et al, 1997), or in C33a cervical carcinoma cells Meyers et al, 1995), respectively.…”

“…TCRb, GM-CSF, IL-3) (Frank et al, 1995;Meyers et al, 1995;Uchida et al, 1997) and activate the promoter of other genes (e.g. BCL-2, M-CSFR) (Klampfer et al, 1996;Rhoades et al, 1996). Similarly, the TEL-AML1 fusion protein has been shown to repress T-cell receptor b enhancer activity , but have no e ect by itself on M-CSF receptor promoter activity (Fears et al, 1997).…”