Multiple regions of ETO cooperate in transcriptional repression.
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In acute myeloid leukemias (AMLs) with t(8;21), the transcription factor AML1 is juxtaposed to the zinc finger nuclear protein ETO (Eight-Twenty-One), resulting in transcriptional repression of AML1 target genes. ETO has been shown to interact with corepressors, such as N-CoR and mSin3A to form complexes containing histone deacetylases. To define regions of ETO required for maximal repressor activity, we analyzed amino-terminal deletions in a transcriptional repression assay. We found that ETO mutants lacking the first 236 amino acids were not affected in their repressor activity, whereas a further deletion of 85 amino acids drastically reduced repressor function and high molecular weight complex formation. This latter mutant can still homodimerize and bind to N-CoR but shows only weak binding to mSin3A. Furthermore, we could show that a "core repressor domain" comprising nervy homology region 2 and its amino- and carboxyl-terminal flanking sequences recruits mSin3A and induces transcriptional repression. These results suggest that mSin3A and N-CoR bind to ETO independently and that both binding sites cooperate to maximize ETO-mediated transcriptional repression. Thus, ETO has a modular structure, and the interaction between the individual elements is essential for the formation of a stable repressor complex and efficient transcriptional repression. (+info)
Breakage and fusion of the TEL (ETV6) gene in immature B lymphocytes induced by apoptogenic signals.
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TEL-AML1 fusion resulting from the t(12;21)(p13;q22) is one of the most common genetic abnormalities in childhood acute lymphoblastic leukemia. Recent findings that site-specific cleavage of the MLL gene can be induced by chemotherapeutic agents such as topoisomerase-II inhibitors suggest that apoptogenic agents can cause chromosomal translocations in hematopoietic cells. This study demonstrates a possible relationship between exposure to apoptogenic stimuli, TEL breaks, and the formation of TEL-AML1 fusion in immature B lymphocytes. Short-term culture of immature B cell lines in the presence of apoptogenic stimuli such as serum starvation, etoposide, or salicylic acid induced double-strand breaks (DSBs) in intron 5 of the TEL gene and intron 1 of the AML1 gene. TEL-AML1 fusion transcripts were also identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in cell lines treated by serum starvation or aminophylline. DSBs within the TEL gene were also associated with fusion to other unknown genes, presumably as a result of chromosomal translocation. We also examined 67 cord blood and 147 normal peripheral blood samples for the existence of in-frame TEL-AML1 fusion transcripts. One cord blood sample (1.5%) and 13 normal peripheral blood samples (8.8%) were positive as detected by nested RT-PCR. These data suggest that breakage and fusion of TEL and AML1 may be relatively common events and that sublethal apoptotic signals could play a role in initiating leukemogenesis via the promotion of DNA damage. (+info)
Type B leukemogenic virus has a T-cell-specific enhancer that binds AML-1.
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Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except that TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determine if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experiments showed that upregulation of reporter gene activity by the TBLV triplication was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-cell lines but not in fibroblasts, B cells, or mammary cells, suggesting that enhancer function is cell type dependent. To analyze the transcription factor binding sites that are important for TBLV enhancer function, we prepared substitution mutations in a reconstituted C3H MMTV LTR that recapitulates the deletion observed in the TBLV LTR. Transient transfections showed that a single mutation (556M) decreased TBLV enhancer activity at least 20-fold in two different T-cell lines. This mutation greatly diminished AML-1 (recently renamed RUNX1) binding in gel shift assays with a mutant oligonucleotide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as judged by competition and supershift experiments. The 556 mutation also reduced TBLV enhancer binding of two other protein complexes, called NF-A and NF-B, that did not appear to be related to c-Myb or Ets. AML-1 overexpression in a mammary cell line enhanced expression from the TBLV LTR approximately 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, likely in combination with other factors, is necessary for optimal enhancer function. (+info)
Chloride binding by the AML1/Runx1 transcription factor studied by NMR.
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It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings. (+info)
Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation.
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The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of definitive hematopoiesis and osteogenesis. The RUNX genes encode the alpha subunit of the transcription factor PEBP2/CBF. The beta subunit consists of the non-RUNX protein PEBP2beta. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. When PEBP2beta is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2beta and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2beta(-/-) mouse. CBF(PEBP2)beta- SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more efficiently than PEBP2beta. These results reveal a hitherto unknown and major role of PEBP2beta, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFbeta-SMMHC. (+info)
Histone deacetylase-targeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia.
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Histone deacetylase (HDAC)-dependent transcriptional repression of the retinoic acid (RA)-signaling pathway underlies the differentiation block of acute promyelocytic leukemia. RA treatment relieves transcriptional repression and triggers differentiation of acute promyelocytic leukemia blasts, leading to disease remission. We report that transcriptional repression of RA signaling is a common mechanism in acute myeloid leukemias (AMLs). HDAC inhibitors restored RA-dependent transcriptional activation and triggered terminal differentiation of primary blasts from 23 AML patients. Accordingly, we show that AML1/ETO, the commonest AML-associated fusion protein, is an HDAC-dependent repressor of RA signaling. These findings relate alteration of the RA pathway to myeloid leukemogenesis and underscore the potential of transcriptional/differentiation therapy in AML. (+info)
Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFbeta.
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The core binding factor (CBF) heterodimeric transcription factors comprised of AML/CBFA/PEBP2alpha/Runx and CBFbeta/PEBP2beta subunits are essential for differentiation of hematopoietic and bone cells, and their mutation is intimately related to the development of acute leukemias and cleidocranial dysplasia. Here, we present the crystal structures of the AML1/Runx-1/CBFalpha(Runt domain)-CBFbeta(core domain)-C/EBPbeta(bZip)-DNA, AML1/Runx-1/CBFalpha(Runt domain)-C/EBPbeta(bZip)-DNA, and AML1/Runx-1/CBFalpha(Runt domain)-DNA complexes. The hydrogen bonding network formed among CBFalpha(Runt domain) and CBFbeta, and CBFalpha(Runt domain) and DNA revealed the allosteric regulation mechanism of CBFalpha(Runt domain)-DNA binding by CBFbeta. The point mutations of CBFalpha related to the aforementioned diseases were also mapped and their effect on DNA binding is discussed. (+info)
Fusion AML1 transcript in a radiation-associated leukemia results in a truncated inhibitory AML1 protein.
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AML1 is a transcription factor that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of radiation-associated secondary acute myelogenous leukemia that had AML1 translocations. Using polymerase chain reaction, the AML1 fusion transcript was isolated from the patient who had a t(19;21) radiation-associated leukemia. The AML1 gene is fused out of frame to chromosome 19 sequences, resulting in a truncated AML protein bearing the DNA binding domain but not the transcriptional activation domain. This fusion AML1 protein functions as an inhibitor of the normal AML1 protein. (Blood. 2001;97:2168-2170) (+info)