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The AMLI gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. MLxing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins.The (8;21)(q22;q22) translocation is one of the most commonly observed chromosomal abnormalities in patients with acute myelogenous leukemia (AML) and involves the AMLI gene on chromosome 21 and the ETO (eight-twenty-one) gene on chromosome 8 (9,12,28,29,36). The fusion of these genes on the der(8) chromosome results in the production of a novel chimeric gene, AMLJ/ETO (9,29,40). A reciprocal translocation product from the der(21) chromosome has yet to be identified, and analysis of complex translocations suggests that the critical genetic rearrangement for cellular transformation is the der(8) (29, 37). A second, more rare, chromosomal abnormality occurring in patients with AML, t(3;21), also involves the AMLI locus (31,38). Thus, cumulative evidence suggests that the AMLI gene is a frequent target for chromosomal aberrations leading to leukemia.Northern (RNA) blot analysis demonstrates four distinct AML1 mRNAs, in a number of cell types, which appear to result from alternative splicing (9,28,29). An AMLl cDNA cloned by Miyoshi and coworkers (28) encodes a protein of 250 amino acids with a predicted molecular mass of 33 kDa which contains a consensus ATP binding sequence but no known DNA-binding motifs (9,28,29). However, AML1 does contain a region of 118 amino acids with 69% homology to the Drosophila pair-rule gene, runt (5, 13). Genetic evidence suggests that runt is an early-acting segmentation protein that regulates the expression of other segmentation genes such as hairy, even-skipped, and fushi tarazu (8,11,13,21,23,26). Additionally, runt may act as a positionspecific numerator element required for expression of the sex-determining gene, sex-lethal (7, 44). Unlike most segmentation genes, runt lacks an identifiable DNA-binding motif and has not been shown to bind DNA (13). Thus, its role in the regulation of gene expression remains unclear.DNA sequence analysis of AMLI/ETO chimeric cDNAs * Corresponding author.indicated t...

The AML-1/CBF␤ transcription factor complex is targeted by both the t(8;21) and the inv(16) chromosomal alterations, which are frequently observed in acute myelogenous leukemia. AML-1 is a site-specific DNAbinding protein that recognizes the enhancer core motif TGTGGT. The t(8;21) translocation fuses the first 177 amino acids of AML-1 to MTG8 (also known as ETO), generating a chimeric protein that retains the DNA-binding domain of AML-1. Analysis of endogenous AML-1 DNA-binding complexes suggested the presence of at least two AML-1 isoforms. Accordingly, we screened a human B-cell cDNA library and isolated a larger, potentially alternatively spliced, form of AML1, termed AML1B. AML-1B is a protein of 53 kDa that binds to a consensus AML-1-binding site and complexes with CBF␤. Subcellular fractionation experiments demonstrated that both AML-1 and AML-1/ETO are efficiently extracted from the nucleus under ionic conditions but that AML-1B is localized to a salt-resistant nuclear compartment. Analysis of the transcriptional activities of AML-1, AML-1B, and AML-1/ETO demonstrated that only AML-1B activates transcription from the T-cell receptor ␤ enhancer. Mixing experiments indicated that AML-1/ETO can efficiently block AML-1B-dependent transcriptional activation, suggesting that the t(8;21) translocation creates a dominant interfering protein.The second-most-frequently observed genetic alteration associated with acute myelogenous leukemia is the t(8;21) translocation, in which the AML1 gene on chromosome 21 is juxtaposed with the MTG8 (ETO) gene on chromosome 8 (9, 28-30). The t(8;21) breakpoint occurs within the same intron of AML1 in every case studied to date (8,32), and the resultant fusion transcript encodes a chimeric protein of 83 kDa containing the first 177 amino acids (aa) of the 250-aa AML-1 protein and 575 aa of the 604-aa ETO protein (28). AML-1 contains a 117-aa region with a high level of homology to the Drosophila segmentation gene runt (the runt homology domain [rhd]) (28,29). This rhd mediates both the DNA-binding and protein-protein interactions of AML-1 (27) and is retained in the t(8;21) translocation protein, suggesting that the fusion protein could function through enhancers or promoters containing the AML-1-binding site.AML-1 is expressed in hematopoietic tissues and recognizes the DNA sequence TGT/cGGT (27). This sequence is the enhancer core motif, originally described for the simian virus 40 enhancer, which is required for tissue-specific expression of a number of viral enhancers (5, 19, 24-26, 34, 36, 41, 44). The identification of the AML-1 DNA-binding site suggested that AML-1 was related to the murine enhancer core-binding factor (CBF) and to the polyomavirus enhancer-binding protein (PEBP2), which recognize the consensus site TGPGGTA/P (P, pyrimidine). Purification and cloning of CBF and PEBP2 revealed a DNA-binding component (termed ␣) and a non-DNA-binding component (termed ␤) (33, 43). The ␣ clones encoded rhd-containing proteins and are highly related to AML-1 (3, 34). Two types ...

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t(8;21) and t(16;21) create two fusion proteins, AML-1-ETO and AML-1-MTG16, respectively, which fuse the AML-1 DNA binding domain to putative transcriptional corepressors, ETO and MTG16. Here, we show that distinct domains of ETO contact the mSin3A and N-CoR corepressors and define two binding sites within ETO for each of these corepressors. In addition, of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. We also show that the murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. In addition, we show that expression of AML-1-ETO causes disruption of the cell cycle in the G 1 phase. Disruption of the cell cycle required the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, ⌬469, which removes the majority of the corepressor binding sites, had no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restored cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein.The acute myeloid leukemia 1 (AML-1) gene is one of the most frequently mutated genes in human leukemia and is disrupted by multiple chromosomal translocations in AML, including t(8;21) and t(16;21) (9, 35, 38). t(8;21) is the most frequent of these translocations, and it contains the AML-1 DNA binding domain fused to a transcriptional corepressor, ETO (also known as MTG8) (4, 5, 34). t(16;21), although rarer, fuses the AML-1 DNA binding domain to an ETOrelated protein, MTG16 (9). AML-1 is also indirectly affected by inv(16), which fuses CBF␤, an allosteric regulator of AML-1, to a smooth muscle myosin heavy chain (25).ETO is highly related to MTG16 and a third family member, MTGR1, in mammalian cells and Nervy in Drosophila (6). The mammalian family members are highly conserved throughout the proteins, with four domains conserved in Nervy. These regions are an N-terminal domain that is also homologous to the transcriptional coactivator TAF110 (17), a hydrophobic heptad repeat (HHR) that mediates dimerization (3, 21), a domain of unknown function termed the Nervy domain, and a domain containing two zinc finger motifs that are required for contacting the central domain of N-CoR (29). The murine homologue of MTG16 was identified by low-stringency screening of a cDNA library by using an ETO cDNA as a probe (3). It shares 77% overall identity with human ETO, but within three of four conserved domains, these proteins are 92 to 96% identical, implying that they function similarly. The Nervy domain is the least conserved domain among family members and is 86% identical between these two proteins.ETO is a component of a high-molecular-weight complex containing ...