The AML1/ETO fusion protein activates transcription of BCL-2 - PubMed (original) (raw)

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

L Klampfer et al. Proc Natl Acad Sci U S A. 1996.

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 AML1/ETO fusion protein. Both AML1/ETO and the AML1 proteins recognize the same consensus DNA-binding motif (TGT/CGGT), 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, AML1/ETO has been shown to dominantly repress its target genes; however, we found that AML1/ETO 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 AML1/ETO. We demonstrated sequence specific binding of both AML1A and AML1/ETO to the TGTGGT sequence in the BCL-2 promoter and showed that the AML1 binding site is required for responsiveness to AML1/ETO. Interestingly, AML1A and AML1B do not modulate the activity of the BCL-2 promoter. The elevated levels of BCL-2 in cells that express AML1/ETO may prolong their life span and contribute to the development of t(8;21) leukemia.

PubMed Disclaimer

Figures

Figure 2

Figure 2

Schematic representation of the BCL-2 5′untranslated region. The location of the P1 and P2 promoters,_Sac_I fragment, and the potential AML1 binding site (shown in bold and underlined) are indicated. The sequence shown is contained in the wild-type oligonucleotide used for the EMSA.

Figure 1

Figure 1

Expression of BCL-2, MCL-1 and BCL-x proteins in myeloid leukemia cell lines. (A) Whole cell lysates from Kasumi, U937, KG-1, K562, TF-1 and SKNO-1 cells were separated on a 12% SDS/PAGE. The gel was stained with Coomassie blue after transfer to demonstrate equal loading of proteins (not shown). The levels of BCL-2 were detected by Western blot analysis, using antibodies specific for BCL-2 proteins. The membrane was stripped of bound antibody and reprobed with an antibody specific for MCL-1. (B) The cell lysates used in Fig. 1_A_ were tested for the presence of the BCL-x protein by Western blot analysis, using antibodies that specifically recognize BCL-x protein.

Figure 3

Figure 3

AML1A and AML1/ETO bind to the BCL-2 promoter. (A) A 32P-labeled oligonucleotide containing the AML1 binding site from the BCL-2 promoter was run alone (lane 1), or incubated with 5 μl of bacterially expressed AML1A protein (lane 2). The reaction mixture was preincubated with a 100-fold molar excess of unlabeled wild-type oligonucleotide (lane 3) or an oligonucleotide with a mutated AML1-binding site (lane 4), an oligonucleotide containing the AML1 site from the IL-3 promoter (lane 5) or the IL-3 promoter oligonucleotide with a mutated AML1 site (lane 6). In lane 7, the AML1A protein was preincubated with 2 μl of AML1 antiserum, and in lane 8 with 2 μl of preimmune serum. The binding of AML1A to the BCL-2 promoter was analyzed by EMSA. The position of the free probe is indicated by the arrow. (B) The 32P-labeled 40-mer oligonucleotide (shown in Fig. 2) was incubated with 5 μl of bacterially expressed AML1/ETO alone (lane 2), or with a 100-fold molar excess of unlabeled BCL-2 oligonucleotide (lane 3), a 100-fold excess of unlabeled oligonucleotide containing the AML1 binding site from the IL-3 promoter (lane 4) or a 100-fold excess of unlabeled oligonucleotide containing a consensus AP-1 site (lane 5). No protein was added in lane 1. The position of the free probe is indicated by the arrow.

Figure 4

Figure 4

AML1/ETO transactivates the BCL-2 promoter. U937 cells were cotransfected with pGL2-basic or pGL2-BCL-2 reporter gene constructs and with pCMV-AML1A, pCMV-AML1B or pCMV-AML1/ETO expression vectors (together with pGHX5 to normalize transfection efficiency). Promoter activity was measured as the ratio of the luciferase activity and the concentration of growth hormone in the conditioned media. The promoter activity in the presence of the empty CMV5 plasmid was defined as 1 and the promoter activity in the presence of expression vectors for AML1A, AML1B, and AML1/ETO was defined relative to that value. The results shown are the mean of four different experiments (SEs are shown when measurable).

Figure 5

Figure 5

The AML1 binding site in the _Sac_I fragment confers AML1/ETO responsiveness to a heterologous promoter. U937 cells were cotransfected with the pTE2, pTE2/_Sac_I or pTE2/_Sac_Im plasmids and with AML1/ETO expression vector (AML1/ETO sense), with its antisense derivative (AML1/ETO antisense) or with an empty pCIneo vector (0). The activity of the pTE2 plasmid in the presence of an empty vector was assigned a value of one. Results from one of three independent experiments are shown.

Figure 6

Figure 6

(A) Schematic representation and transcriptional activity of the various AML1/ETO deletion mutants. RHD, rhd; TAF, TATA-binding-protein-associated factor-110 homology domain; PST, proline-serine-threonine-rich region; ZF, zinc finger domain. (B) Transcriptional activity of the wild-type AML1/ETO and mutant AML/ETO proteins. U937 cells were cotransfected with the pGL2-BCL2 plasmid and the wild-type CMV5-AML1/ETO expression plasmid or with its deletion mutants as shown in Fig. 6_A_. Luciferase activity in the presence of the CMV5 plasmid was assigned a value of 1. Results represent the mean of four independent experiments.

Similar articles

Cited by

References

    1. Nichols J, Nimer S D. Blood. 1992;80:2953–2963. - PubMed
    1. Erickson P, Gao K S, Chang T, Look T, Whisenant E, Raimondi S, Lasher J, Trujillo J, Rowley J, Drabkin H. Blood. 1992;80:1825–1831. - PubMed
    1. Meyers S, Downing J R, Hiebert S W. Mol Cell Biol. 1993;23:6336–6345. - PMC - PubMed
    1. Bae S C, Ogawa E, Maruyama M, Oka H, Satake M, Shigaseda K, Jenkins N A, Gilbert D J, Copela N G, Ito Y. Mol Cell Biol. 1994;14:3242–3252. - PMC - PubMed
    1. Frank R, Zhang J, Hiebert S W, Meyers S, Nimer S D. Oncogene. 1995;11:2667–2674. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources