Shari Meyers

Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

The AML1 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. Mixing 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.

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter.

Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation.

The AML-1/CBF beta 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 DNA-binding 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 beta. 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 beta 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.

ETO, a Target of t(8;21) in Acute Leukemia, Makes Distinct Contacts with Multiple Histone Deacetylases and Binds mSin3A through Its Oligomerization Domain

ABSTRACT 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 G1 phase. Disruption of the cell cycle required the ability of AML-1–ETO to repress transcription because a mutant of AML-1–ETO, Δ469, 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 t (12;21) translocation converts AML-1B from an activator to a repressor of transcription

The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt- and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal domain of AML-1B that is required for transactivation of the T-cell receptor beta enhancer, it fails to activate transcription but rather inhibits the basal activity of this enhancer. TEL/AML-1B efficiently interferes with AML-1B dependent transactivation of the T-cell receptor beta enhancer, and coexpression of wild-type TEL does not reverse this inhibition. The N-terminal TEL helix-loop-helix domain is essential for TEL/AML-1B-mediated repression. Thus, the t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML-1 genes is critical for B-cell leukemogenesis.

Gfi‐1 attaches to the nuclear matrix, associates with ETO (MTG8) and histone deacetylase proteins, and represses transcription using a TSA‐sensitive mechanism

Gfi‐1 and Gfi‐1B can repress transcription and play important roles in hematopoietic cell survival and differentiation. Although these proteins are known to bind DNA through a C‐terminal zinc‐finger domain and may require an N‐terminal SNAG domain (SNAIL/Gfi‐1) to repress transcription, the mechanism by which Gfi‐1 and Gfi‐1B act is unknown. A first step towards understanding the mechanism by which these proteins repress transcription is to identify interacting proteins that could contribute to transcriptional repression. ETO (also termed MTG8), was first identified through its involvement in the (8;21) translocation associated with acute myelogenous leukemia. It attaches to the nuclear matrix and associates with histone deacetylases and the co‐repressors N‐CoR, SMRT, and mSin3A, and may act as a co‐repressor for site‐specific transcriptions factors. In this report we demonstrate that Gfi‐1 interacts with ETO and related proteins both in vitro and in vivo and with histone deacetylase proteins in vivo. We observed that a portion of Gfi‐1 and Gfi‐1B associated with the nuclear matrix, as is the case with ETO. Moreover, Gfi‐1 and ETO co‐localize to punctate subnuclear structures. When co‐expressed in mammalian cells, Gfi‐1 associates with histone deacetylse‐1 (HDAC‐1), HDAC‐2, and HDAC‐3. These data identify ETO as a partner for Gfi‐1 and Gfi‐1B, and suggest that Gfi‐1 proteins repress transcription through recruitment of histone deacetylase‐containing complexes. J. Cell. Biochem. 89: 1005–1018, 2003.

Mtgr1 Is a Transcriptional Corepressor That Is Required for Maintenance of the Secretory Cell Lineage in the Small Intestine

ABSTRACT Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.

ETO-2, a new member of the ETO-family of nuclear proteins

The t(8;21) is associated with 12 – 15% of acute myelogenous leukemias of the M2 subtype. The translocation results in the fusion of two genes, AML1 (CBFA2) on chromosome 21 and ETO (MTG8) on chromosome 8. AML1 encodes a DNA binding factor; the ETO protein product is less well characterized, but is thought to be a transcription factor. Here we describe the isolation and characterization of ETO-2, a murine cDNA that encodes a new member of the ETO family of proteins. ETO-2 is 75% identical to murine ETO and shares very high sequence identities over four regions of the protein with ETO (domain I – III and zinc-finger). Northern analysis identifies ETO-2 transcripts in many of the murine tissues analysed and in the developing mouse embryo. ETO-2 is also expressed in myeloid and erythroid cell lines. We confirmed the nuclear localization of ETO-2 and demonstrated that domain III and the zinc-finger region are not required for nuclear localization. We further showed that a region within ETO, containing domain II, mediates dimerization among family members. This region is conserved in the oncoprotein AML-1/ETO. The recent identification of another ETO-like protein, myeloid translocation gene-related protein 1, together with the data presented here, demonstrates that at least three ETO proteins exist with the potential to form dimers in the cell nucleus.

Nuclear import and subnuclear localization of the proto-oncoprotein ETO (MTG8)

ETO (MTG8) was first described due to its involvement in the (8;21) translocation frequently observed in acute myeloid leukemias. In the t(8;21) the AML1 gene on chromosome 21 is fused to ETO on chromosome 8. The resultant hybrid protein is comprised of the DNA binding domain of AML-1 and the majority of ETO. This study examines the subnuclear distributions of ETO, AML-1B and AML-1/ETO proteins fused to green fluorescence protein in living cells using fluorescence microscopy. Further, we identified a 40 amino acid portion of ETO (amino acids 241–280) that was sufficient to cause nuclear import of green fluorescent protein. Mutational analysis demonstrated that lysine 265 and/or arginine 266 were required for nuclear import of ETO, but that the surrounding basic residues were not critical. ETO interacted with the nuclear import proteins importin-α and β in vitro, and mutations in ETO that abolish nuclear localization also abolished the in vitro interaction with importin-α and β. These data suggest that ETO enters the nucleus via an importin-mediated pathway. Additionally, ETO and AML-1/ETO co-localized to punctate nuclear bodies distinct from those containing promyelocytic leukemia protein. Nuclear body formation was dependent upon a region of ETO N-terminal to the nuclear localization signal. Thus, ETO and AML-1/ETO reside in potentially novel subnuclear compartments.

Transcriptional Regulation by the t(8;21 )Fusion Protein, AML-1/ETO

Certain chromosomal translocations are characteristically associated with acute myeloid leukemia (AML), suggesting that alteration of specific genes at the translocation breakpoints contribute to the genesis or maintenance of leukemia. The t(8;21) is the second most frequent chromosomal abnormality associated with AML, occurring in 12–15% of cases (Downing et al. 1993). The t(8;21) breakpoint was cloned and the disrupted gene on chromosome 21 was termed AML1 (Miyoshi et al. 1991). Cytogenetic evidence from t(8;21)-containing leukemia cells with complex translocations indicates that the der(8) chromosome is conserved in each case; tightly linking this chromosomal aberration to the pathogenesis of AML (Rowley 1982). In addition, AML1 is interrupted in the less commonly observed (3;21) and (12;21) translocations found in chronic myeloid leukemia and acute lymphocytic leukemia, respectively (Nucifora et al. 1993; Golub et al. 1995), suggesting that AML-l may be a principal regulatory protein.