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The AML1-CBFbeta transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The promyelocytic leukemia (PML) gene is also frequently involved in AML-associated translocation. Here we report that a specific isoform PML I forms a complex with AML1. PML I was able to recruit AML1 and coactivator p300 in PML nuclear bodies and enhance the AML1-mediated transcription in the presence of p300. A specific C-terminal region of PML I and a C-terminal region of AML1 were found to be required for both their association and colocalization in the nuclear bodies. Overexpression of PML I stimulates myeloid cells to differentiate. These results suggest that PML I could act as a mediator for AML1 and its coactivator p300/CBP to assemble into functional complexes and, consequently, activate AML1-dependent transcription and myeloid cell differentiation.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to the SSX gene on chromosome X. Although the SYT-SSX fusion proteins may trigger synovial sarcoma development, the biological functions of SYT, SSX and SYT-SSX genes are unclear. Transfections of Gal4 DNA binding domain fusion protein constructs demonstrate that SYT protein acts as a transcriptional co-activator at the C-terminal domain and that the activity is repressed through the N-terminus. The N-terminal 70 amino acids of SYT bind not only to BRM, but also to Brg1, both of which are subunits of SWI/SNF chromatin remodelling complexes. Here, we have investigated the functions of BRM and Brg1 on the repression of SYT activity. The negative regulation of SYT transcriptional co-activator activity is dependent on the ATP-hydrolysis of BRM and Brg1 in the protein complexes. This indicates that the SWI/SNF protein complexes regulate SYT activity using the chromatin remodelling activity.

1. Fyn, a member of Src-family tyrosine kinases, is implicated in both brain development and adult brain function. Recent studies have identified some Fyn substrates, however, little is known about the transcriptional targets for Fyn mediated signaling pathways. In the present study, we sought to identify targets downstream of Fyn in vivo. 2. We compared genes expressed in adult hippocampi of wild-type and fyn-deficient mice using gene chips containing more than 12,000 genes. 3. The results showed that 559 transcripts were expressed differentially between these mice. Expression of 20 genes including a substantial number of myelin-associated genes was strongly repressed in fyn-deficient mice. 4. Reduced expression of these myelin-associated genes, such as MBP and MOG, in fyn-deficient mice was also confirmed by real-time PCR and northern blotting, arguing that Fyn is important for function and development of oligodendrocytes. 5. Further analysis of the genes that are differently expressed in fyn-deficient mice may shed light on the molecular mechanism by which Fyn regulates adult neural function.

The AML1-CBF beta transcription factor complex is the most frequent target of specific chromosome translocations in human leukemia. The MOZ gene, which encodes a histone acetyltransferase (HAT), is also involved in some leukemia-associated translocations. We report here that MOZ is part of the AML1 complex and strongly stimulates AML1-mediated transcription. The stimulation of AML1-mediated transcription is independent of the inherent HAT activity of MOZ. Rather, a potent transactivation domain within MOZ appears to be essential for stimulation of AML1-mediated transcription. MOZ, as well as CBP and MOZ-CBP, can acetylate AML1 in vitro. The amount of AML1-MOZ complex increases during the differentiation of M1 myeloid cells into monocytes/macrophages, suggesting that the AML1-MOZ complex might play a role in cell differentiation. On the other hand, the MOZ-CBP fusion protein, which is created by the t(8;16) translocation associated with acute monocytic leukemia, inhibits AML1-mediated transcription and differentiation of M1 cells. These results suggest that MOZ-CBP might induce leukemia by antagonizing the function of the AML1 complex.

AML1-MTG8 fusion protein, which is produced from the rearranged gene formed between AML1 and MTG8 in myeloid leukemia with t(8;21) chromosomal translocation, plays an important role in the pathogenesis of leukemia. We previously showed that ectopically expressed AML1-MTG8 fusion protein is associated with an MTG8-like protein in the mouse myeloid precursor cell line L-G, and this association seemed to be required for AML1-MTG8 to stimulate proliferation. As a candidate cDNA for this MTG8-like protein, a 6.4 kb MTGR1 cDNA encoding human MTGR1b protein of 604 amino acids was isolated. Since this cDNA was shorter than the main mRNA (about 7.5 kb), the 5'-end of the MTGR1 cDNA was extended using Marathon Ready cDNA. When the newly obtained 5'-sequence was combined with the previous cDNA, the resultant MTGR1 cDNA (6995 bp), including exon 3 that the previous cDNA lacked, could encode MTGR1a protein of 575 amino acids. Transcripts of the MTGR1 gene were expressed ubiquitously in the human tissues and cell lines examined. PCR analyses of the cDNAs from human tissues showed the presence of various splicing variants with regard to the 5'-region including exons 1, 2 and 3. The MTGR1 gene consists of 14 exons and spans about 68 kb. The genomic structure of MTGR1 is highly similar to those of other MTG 8-family genes, MTG8 and MTG16. MTG16 was recently cloned from the translocation breakpoint of myeloid malignancies with t(16;21) chromosomal translocation.

To detect a translocation (8;21)(q22;q22) in interphase cells by fluorescence in situ hybridization (FISH), we investigated three probe combinations: single-color hybridization with two cosmid probes (cY8 and cY3), single-color hybridization with four cosmid probes (cY8, cY3, cY107, and cYR4), and dual-color hybridization with two cosmid probes (cY107 and cYR4) from the AML1 gene flanking or overlapping the breakpoint region. Over 95% of nuclei gave sufficient signals in all three probe combinations, and the detection rates were not statistically different among them. Among 18 patients examined at diagnosis, 12 with t(8;21) were also monitored for the number of residual leukemic cells after chemotherapy or bone marrow transplantation (BMT). There were some discrepancies between morphology and genetic (especially FISH) results at partial, or even complete remission. As leukemic cells with t(8;21) can maturate, morphological assessment alone is insufficient to evaluate the residual leukemic cells. Interphase FISH provided more precise information about the clinical status of patients with an 8;21 translocation after treatment.

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2alphaB) gene becomes fused to the MTG8 (ETO) gene. We previously found that the terminal differentiation step leading to mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF) was inhibited by the ectopic expression of the AML1-MTG8 fusion protein in L-G murine myeloid progenitor cells. We show here that overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB-binding protein (CBP) interact with AML1. The C-terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1-dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1-dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFbeta, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells.

We have identified a family with an autosomal dominant platelet disorder with a predisposition for developing myeloid malignancies and have previously demonstrated linkage of this trait to chromosome 21q22.1-22.2. The nearest flanking markers, D21S1265 and D21S167, define the familial platelet disorder (FPD) critical region at a genetic distance of approximately 15.2 centimorgans and physical distance of approximately 6 megabases. This locus is of particular interest as it has previously been implicated in the pathogenesis of acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) through the (8;21), (3;21) and (12;21) chromosomal translocations. In each of these cases, the CBFA2 gene is rearranged. As well, there is a potential association of this locus with the hematologic abnormalities seen in Down syndrome (trisomy 21). To identify the mutant gene in this pedigree, a positional cloning strategy has been undertaken. Several candidate genes map to this locus including: CBFA2, IFNAR1, IFNAR2, CRFB4, GART, SON, KCNE1, SCL5A3 and ATP50. CBFA2, as well as IFNAR1 and CRFB4, were the focus of initial mutational analysis efforts. In this report, we exclude CBFA2 as a candidate by Northern and Southern blotting, RNase protection, single-strand conformational polymorphism (SSCP), direct sequencing and gel-shift analysis. Exons of the IFNAR1 and CRFB4 genes were also analyzed by SSCP and demonstrated no evidence of mutation. SSCP analysis identified a new polymorphism in the second exon of the CRFB4 gene and confirmed a previously described polymorphism in the fourth exon of IFNAR1. Efforts are currently underway to delimit further the FPD critical region and to analyze the other known candidate genes, as well as novel candidate genes, which map to this locus.

The translocation t(8;21)(q22;q22) is found in 40% of cases of acute myeloid leukemia (AML) designated as the subtype M2 in the French-American-British (FAB) classification. The 8;21 translocation is clinically of interest because patients with this subtype have a good prognosis. We describe a masked type of the translocation, t(8;12;21)(q22.1;q24.1;q22.1). The translocation was first interpreted as t(8;12)(q22;q24) based on cytogenetics, but was reevaluated as a result of Southern blot and fluorescence in situ hybridization (FISH) analyses.

In a study of 23 patients with t(8;21)-associated acute myeloid leukaemia the AML1-MTG8 fusion transcript was present in the majority of serial samples obtained from 17 patients followed for up to 34 months after diagnosis, but was absent in samples from all six patients who had been in continuous complete remission for 61 months after allogeneic bone marrow transplantation (BMT), or for 52, 53, 123, 182 and 198 months, respectively, after courses of intensive chemotherapy. Previous studies showed that the AML1-MTG8 fusion transcript was present in most patients with this type of translocation in long-term remission. Our results indicate that blood cells of patients with t(8;21) in remission of over 10 years may not show the AML1-MTG8 fusion transcript, and that those of patients who have undergone allogeneic BMT or intensive chemotherapy may become fusion transcript-negative much earlier. Our study suggests that leukaemic cells with the AML1-MTG8 fusion transcript may survive for some time after courses of chemotherapy or BMT, but that they may eventually be eradicated by immunologic and other antileukaemic mechanisms.

The t(11;22)(q24;q12) and t(21;22)(q22;q12) are specific chromosomal translocations found in the Ewing family of tumors including ES, PNET and Askin tumors. In these translocations, the amino-terminal portion of the EWS gene located in 22q12 fuses to the carboxyl-terminal portion of the FLI-1 gene located in 11q24 or the ERG gene located in 21q22, which belong to the ets oncogene superfamily of transcription activators. We investigated the chimeric mRNAs of 15 ESs (7 cell lines and 8 tumor samples) and 7 PNETs (3 cell lines and 4 tumor samples) using the RT-PCR method and sequencing. We detected 2 types of EWS-ERG chimeric mRNA in 2 ES cell lines and 1 PNET tumor sample in addition to 4 types of EWS-FLI-1 chimeric mRNA in 11 ESs (4 cell lines and 7 tumor samples) and 4 PNETs (2 cell lines and 2 tumor samples). There seemed to be no association between the type of chimeric mRNA and clinical features such as sex, age, primary site and histopathology of the patients. All of the chimeric mRNAs are generated from in-frame junctions and are thought to encode fusion proteins that may be the molecular mechanism involved in the Ewing family of tumors.

The human CBFA2T1 (also known as MTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of the CBFA2T1 gene, Cbfa2t1h. Nucleotide sequence analysis of Cbfa2t1h cDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5'- and 3'-untranslated regions (87.0, 92.0, and 93.7% identities for 5'-untranslated, coding, 3'-untranslated regions, respectively). The 3'-untranslated region of Cbfa2t1h contains a (CA)n dinucleotide repeat, and the polymerase chain reaction amplification of the (CA)n repeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mapped Cbfa2t1h to mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 x Mae) x Mae cross. The chromosomal location was also confirmed by fluorescence in situ hybridization.

We previously isolated the AML1 gene, which is rearranged by the t(8;21) translocation in acute myeloid leukemia. The AML1 gene is highly homologous to the Drosophila segmentation gene runt and the mouse transcription factor PEBP2 alpha subunit gene. This region of homology, called the Runt domain, is responsible for DNA-binding and protein--protein interaction. In this study, we isolated and characterized various forms of AML1 cDNAs which reflect a complex pattern of mRNA species. Analysis of these cDNAs has led to the identification of two distinct AML1 proteins, designated AML1b (453 amino acids) and AML1c (480 amino acids), which differ markedly from the previously reported AML1a (250 amino acids) with regard to their C-terminal regions, although all three contain the Runt domain. The large C-terminal region common to AML1b and AML1c is suggested to be a transcriptional activation domain. AML1c differs from AML1b by only 32 amino acids in the N-terminal. Characterization of the genomic structure revealed that the AML1 gene consists of nine exons and spans > 150 kb of genomic DNA. Northern blot analysis demonstrated the presence of six major transcripts, encoding AML1b or AML1c, which can all be explained by the existence of two promoters, alternative splicing and differential usage of three polyadenylation sites. A minor transcript encoding AML1a which results from alternative splicing of a separate exon can be detected only by reverse transcription-polymerase chain reaction amplification. The distinct proteins encoded by the AML1 gene may have different functions, which could contribute to regulating cell growth and/or differentiation through transcriptional regulation of a specific subset of target genes.

The t(3;21)(q26;q22) translocation, which is one of the consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia (CML), is thought to play an important role in the leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t(8;21)(q22;q22) translocation found in acute myelocytic leukemia, was also rearranged by the t(3;21)(q26;q22) translocation. Screening of a cDNA library of the t(3;21)-carrying leukemic cell line cells (SKH1) resulted in the isolation of two potentially complete AML1-EVI-1 chimeric cDNAs of 6 kb. Two species of AML1-EVI-1 fusion transcripts of 8.2 and 7.0 kb were detected in SKH1 cells. These cells expressed the 180 kDa AML1-EVI-1 fusion protein containing an N-terminal half of AML1 including a runt homology domain which is fused to the entire zinc finger EVI-1 protein. The AML1-EVI-1 fusion transcript was consistent in all three cases of the t(3;21)-carrying leukemia examined by RNA-based PCR. These findings strongly suggest that the t(3;21) translocation results in the formation of a new class of chimeric transcription factor which could contribute to the leukemic progression of CML through interference with cell growth and differentiation.

The t(8;21) translocation is one of the most frequent chromosomal abnormalities in acute myeloid leukaemia and results in gene fusion between AML1 on chromosome 21 and MTG8 (= ETO or CDR) on chromosome 8. AML1 contains a region of sequence homology to the Drosophila runt gene and the mouse polyomavirus enhancer binding protein PEBP2 alpha gene. The rearrangement occurs within a specific intron of the AML1 gene and results in the formation of a chimaeric protein with the consistent feature that the region of sequence homology of AML1 is fused with almost the entire MTG8 protein. MTG8 (ETO, CDR) is predicted to be a transcription activation factor from its sequence with zinc-finger motifs and proline-rich domains. Thus the rearrangement is a fusion between two probable transcription activation factors.

The t(16;21)(p11;q22) translocation is a nonrandom chromosomal abnormality found in several types of myeloid leukemia, which show variable cytomorphological features. We constructed rodent-human somatic cell hybrids containing the der(16) chromosome from leukemic cells of a patient with t(16;21). Using these hybrids, we mapped the translocation breakpoint on the Not I restriction map of chromosome 21 which we had previously constructed. The result showed the proximity of the breakpoint to the ERG gene, a member of the ets oncogene superfamily. Polymerase chain reaction and Southern blot analyses of genomic DNA from the hybrids and from peripheral blood cells and bone marrow cells of patients with t(16;21) showed that the breakpoints were clustered within a single intron in the coding region of the ERG gene. This finding and the results obtained by Northern blot analysis suggested the formation of a chimeric product(s) by fusion of the ERG gene and an unknown counterpart gene on chromosome 16.

The chromosomal translocation, t(8;21), is found frequently in acute myeloid leukemia (AML) with maturation (FAB-M2). We have previously mapped the translocation breakpoints of t(8;21) in a specific intron of the AML1 gene on chromosome 21. In this study, we cloned cDNAs synthesized from a cell line carrying t(8;21) by reverse transcription polymerase chain reaction (RT-PCR) using an AML1-specific primer. The analysis of the cDNAs structure has led to the identification of the fusion of AML1 with a gene named MTG8 on chromosome 8, which seems to be identical to ETO. Northern analysis using MTG8 (ETO) probes detected 7.8-kb and 6.2-kb RNAs and several minor RNAs in the cell line with t(8;21), but failed to detect any transcripts in a cell line without t(8;21). A set of primers were designed to detect the AML1/MTG8(ETO) fusion by PCR. The PCR amplified identical products in all 6 patients and one cell line with t(8;21), suggesting that the AML1/MTG8(ETO) fusion is a constant feature associated with t(8;21) and the junctions of the AML1/MTG8(ETO) fusion are restricted in a unique site. Because the PCR detection of the AML1/MTG8(ETO) fusion at the RNA level is highly sensitive, it can be used as a sensitive method for diagnosis and detection of minimal residual disease in t(8;21) leukemia.

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. It has been shown that the t(8;21) breakpoints on chromosome 21 cluster within a single specific intron of the AML1 gene, which is highly homologous to the Drosophila segmentation gene runt. Here we report that this translocation juxtaposes the AML1 gene with a novel gene, named MTG8, on chromosome 8, resulting in the synthesis of an AML1-MTG8 fusion transcript. The fusion protein predicted by the AML1-MTG8 transcript consists of the runt homology region of AML1 and the most part of MTG8, which contains putative zinc finger DNA binding motifs and proline-rich regions constituting a characteristic feature of transcription factors. The MTG8 gene is not expressed in normal hematopoietic cells, whereas AML1 is expressed at high levels. Our results indicate that the production of chimeric AML1-MTG8 protein, probably a chimeric transcription factor, may contribute to myeloid leukemogenesis.