Survivin modulates genes with divergent molecular functions and regulates proliferation of hematopoietic stem cells through Evi-1.

The inhibitor of apoptosis protein Survivin regulates hematopoiesis, although its mechanisms of regulation of hematopoietic stem cells (HSCs) remain largely unknown. While investigating conditional Survivin deletion in mice, we found that Survivin was highly expressed in phenotypically defined HSCs, and Survivin deletion in mice resulted in significantly reduced total marrow HSCs and hematopoietic progenitor cells. Transcriptional analysis of Survivin(-/-) HSCs revealed altered expression of multiple genes not previously linked to Survivin activity. In particular, Survivin deletion significantly reduced expression of the Evi-1 transcription factor indispensable for HSC function, and the downstream Evi-1 target genes Gata2, Pbx1 and Sall2. The loss of HSCs following Survivin deletion and impaired long-term HSC repopulating function could be partially rescued by ectopic Evi-1 expression in Survivin -/- HSCs. These data demonstrate that Survivin partially regulates HSC function by modulating the Evi-1 transcription factor and its downstream targets and identify new genetic pathways in HSCs regulated by Survivin.

Plaque type and composition as evaluated non-invasively by MSCT angiography and invasively by VH IVUS in relation to the degree of stenosis.

BACKGROUND: Imaging of coronary plaques has traditionally focused on evaluating degree of stenosis, as the risk for adverse cardiac events increases with stenosis severity. However, the relation between plaque composition and severity of stenosis remains largely unknown. OBJECTIVE: To assess plaque composition (non-invasively by multislice computed tomography (MSCT) angiography and invasively by virtual histology intravascular ultrasound (VH IVUS)) in relation to degree of stenosis. METHODS: 78 patients underwent MSCT (identifying three plaque types; non-calcified, calcified, mixed) followed by invasive coronary angiography and VH IVUS. VH IVUS evaluated plaque burden, minimal lumen area and plaque composition (fibrotic, fibro-fatty, necrotic core, dense calcium) and plaques were classified as fibrocalcific, fibroatheroma, thin-capped fibroatheroma (TCFA), pathological intimal thickening. For each plaque, percentage stenosis was evaluated by quantitative coronary angiography. Significant stenosis was defined >50% stenosis. RESULTS: Overall, 43 plaques (19%) corresponded to significant stenosis. Of the 227 plaques analysed, 70 were non-calcified plaques (31%), 96 mixed (42%) and 61 calcified (27%) on MSCT. Plaque types on MSCT were equally distributed among significant and non-significant stenoses. VH IVUS identified that plaques with significant stenosis had higher plaque burden (67% (11%) vs 53% (12%), p<0.05) and smaller minimal lumen area (4.6 (3.8-6.8) mm(2) vs 7.3 (5.4-10.5) mm(2), p<0.05). Interestingly, no differences were observed in percentage fibrotic, fibro-fatty, necrotic core and dense calcium. Non-significant stenoses were more frequently classified as pathological intimal thickening (46 (25%) vs 3 (7%), p<0.05), although TCFA (more vulnerable plaque) was distributed equally (p = 0.18). CONCLUSION: No evident association exists between the degree of stenosis and plaque composition or vulnerability, as evaluated non-invasively by MSCT and invasively by VH IVUS.

Differential expression of TCL1 during pre-B-cell acute lymphoblastic leukemia progression.

Nonrandom, recurring chromosomal translocations are critical events in the pathogenesis of leukemia. The recently identified TEL/AML1 (CBFA2/EVT6) fusion gene occurs as a result of the t(12;21)(p13;q22) in approximately 25% of children with diagnosed pre-B-cell acute lymphoblastic leukemia (PBC-ALL). To identify changes in gene expression patterns that occur during PBC-ALL disease progression, we used cDNA microarrays to compare expressed sequences from the AT-1 and AT-2 cell lines. These cell lines, from the same patient, were established from two distinct stages of PBC-ALL disease progression, namely, first and second relapse. Analysis of both cell lines with spectral karyotying (SKY) revealed an insertion of chromosome 8 into chromosome 5 and a previously undetected translocation in AT-2 involving chromosomes 1 and 17. Hybridization of cDNA microarrays identified the TCL1 transcript as being overexpressed in the AT-2 cell line relative to AT-1. Northern blot analysis showed an eightfold increase of the TCL1 transcript in AT-2 over AT-1 cells. Western blot analysis showed that the TCL1 protein was expressed more than 50-fold higher in AT-2 than AT-1 cells. TCL1 expression was correlated with TEL expression by reintroducing TEL into AT-2 cells and demonstrating that those cells expressing TEL at high levels showed a decreased expression of endogenous TCL1.

AML1 gene over-expression in childhood acute lymphoblastic leukemia.

The present study was conducted on a series of 41 Egyptian children with newly diagnosed acute lymphoblastic leukemia (ALL) to investigate TEL and AML1 abnormalities. The TEL-AML1 fusion was observed in six patients both by RT-PCR and FISH analyses, with a frequency of 22.2% among the B-lineage group, whereas TEL deletion was seen by FISH analysis in seven patients (17.1%). By FISH analysis, nine patients (22%) showed evidence of extra AML1 copies. In five of these patients the extra copies were due to non-constitutional trisomy 21, whereas in the remaining four cases they were due to tandem AML1 copies on der(21), as evidenced by metaphase FISH. Unexpectedly however, enhanced AML1 expression levels were seen by real-time quantitative RT-PCR in 18 out of the 41 ALL patients (43.9%). This high level of AML1 expression could be an important factor contributing to the pathogenesis and progression of childhood ALL. One key mechanism for over-expression seems to be the extra copies of AML1, but other mechanisms may involve an alteration of the activity of the AML1 promoter. Here, we also report two novel findings. The first is an intragenic deletion of TEL exon 7 in a case of T cell ALL. This deletion creates a frame-shift and results in a truncated protein lacking the C-terminus that includes the ETS domain. This shorter TEL is presumably unable to bind DNA. The second finding is a rearrangement of AML1 in a case of T cell ALL due to t(4;21)(q31;q22). This is the first reported chromosomal translocation where AML1is rearranged in childhood T cell ALL.

Interaction of EVI1 with cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF) results in reversible acetylation of EVI1 and in co-localization in nuclear speckles.

EVI1 is a very complex protein with two domains of zinc fingers and is inappropriately expressed in many types of human myeloid leukemias. Using reporter gene assays, several investigators showed that EVI1 is a transcription repressor, and recently it was shown that EVI1 interacts with the co-repressor carboxyl-terminal binding protein 1 (CtBP1). Earlier, we showed that the inappropriate expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and to increased proliferation. Using biochemical assays, we have identified two groups of transcription co-regulators that associate with EVI1 presumably to regulate gene expression. One group of co-regulators includes the CtBP1 and histone deacetylase. The second group includes the two co-activators cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF), both of which have histone acetyltransferase (HAT) activity. All of these proteins require separate regions of EVI1 for efficient interaction, and they divergently affect the ability of EVI1 to regulate gene transcription in reporter gene assays. Confocal microscopy analysis shows that in the majority of the cells, EVI1 is nuclear and diffused, whereas in about 10% of the cells EVI1 localizes in nuclear speckles. However, in the presence of the added exogenous co-repressors histone deacetylase or CtBP1, all of the nuclei have a diffuse EVI1 staining, and the proteins do not appear to reside together in obvious nuclear structures. In contrast, when CBP or P/CAF are added, defined speckled bodies appear in the nucleus. Analysis of the staining pattern indicates that EVI1 and CBP or EVI1 and P/CAF are contained within these structures. These nuclear structures are not observed when CBP is substituted with a point mutant HAT-inactive CBP with which EVI1 also physically interacts. Finally, we show that the interaction of EVI1 with either CBP or P/CAF leads to acetylation of EVI1. These results suggest that the assembly of EVI1 in nuclear speckles requires the intact HAT activity of the co-activators.

Genetic lesions and perturbation of chromatin architecture: a road to cell transformation.

Differential gene expression is a rigorously precise procedure that defines the developmental program of cells, tissues, organs, and of the entire organism. The correct execution of this program requires the participation of multiple and complex groups of regulators. In addition to transcription factors, which are key tools in ontogenesis by providing sequential switch of different genes, the structure of the chromatin is a dominant determinant leading to gene expression. Through the novel and insightful work of several investigators, it appears that the architecture of the chromatin spanning the genes can and does influence the efficiency of RNA transcription, and therefore of gene expression. Several new enzymatic complexes have been identified that reversibly modify the chromatin architecture by methylation, phosphorylation, and acetylation of the nucleosomal core proteins. These enzymes are crucial for the proper balance and maintenance of gene expression, and are often the target of mutations and alterations in human cancer. Here, we review briefly the current models proposing how some of these enzymes normally modify the chromatin structure and how their functional disruption leads to inappropriate gene expression and cell transformation.

Posttranslational modification of TEL and TEL/AML1 by SUMO-1 and cell-cycle-dependent assembly into nuclear bodies.

The E-26 transforming specific (ETS)-related gene, TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. TEL is a nuclear phosphoprotein that is widely expressed in all normal tissues. TEL contains a DNA-binding domain at the C terminus and a helix-loop-helix domain (also called a pointed domain) at the N terminus. The pointed domain is necessary for homotypic dimerization and for interaction with the ubiquitin-conjugating enzyme UBC9. Here we show that the interaction with UBC9 leads to modification of TEL by conjugating it to SUMO-1. The SUMO-1-modified TEL localizes to cell-cycle-specific nuclear speckles that we named TEL bodies. We also show that the leukemia-associated fusion protein TEL/AML1 is modified by SUMO-1 and found in the TEL bodies, in a pattern quite different from what we observe and report for AML1. Therefore, SUMO-1 modification of TEL could be a critical signal necessary for normal functioning of the protein. In addition, the modification by SUMO-1 of TEL/AML1 could lead to abnormal localization of the fusion protein, which could have consequences that include contribution to neoplastic transformation.

Human AML1/MDS1/EVI1 fusion protein induces an acute myelogenous leukemia (AML) in mice: a model for human AML.

The human t(3;21)(q26;q22) translocation is found as a secondary mutation in some cases of chronic myelogenous leukemia during the blast phase and in therapy-related myelodysplasia and acute myelogenous leukemia. One result of this translocation is a fusion between the AML1, MDS1, and EVI1 genes, which encodes a transcription factor of approximately 200 kDa. The role of the AML1/MDS1/EVI1 (AME) fusion gene in leukemogenesis is largely unknown. In this study, we analyzed the effect of the AME fusion gene in vivo by expressing it in mouse bone marrow cells via retroviral transduction. We found that mice transplanted with AME-transduced bone marrow cells suffered from an acute myelogenous leukemia (AML) 5-13 mo after transplantation. The disease could be readily transferred into secondary recipients with a much shorter latency. Morphological analysis of peripheral blood and bone marrow smears demonstrated the presence of myeloid blast cells and differentiated but immature cells of both myelocytic and monocytic lineages. Cytochemical and flow cytometric analysis confirmed that these mice had a disease similar to the human acute myelomonocytic leukemia. This murine model for AME-induced AML will help dissect the molecular mechanism of AML and the molecular biology of the AML1, MDS1, and EVI1 genes.

AML1 haploinsufficiency, gene dosage, and the predisposition to acute leukemia.

Hematopoiesis is the complex developmental process through which undifferentiated, pluripotent, hematopoietic stem cells come to generate mature, functional blood cells. This process is regulated in large part by specific transcription factors that control expression of genes necessary for the developmental sequence. Leukemias represent one form of disruption of this normal developmental process, and studies over the past few years have shown that many of the genes that underlay leukemogenesis are also essential for normal hematopoiesis. In an interesting recent example, Song et al.((1)) demonstrate that haploinsufficiency of the AML1 gene is the genetic basis of a form of familial thrombocytopenia which predisposes the affected individuals to the development of acute myeloid leukemia. Here we summarize Song's paper and current information describing the interesting dosage effects of this gene and other members of its gene family.

Lineage specificity of CBFA2 fusion transcripts.

The CBFA2 gene on chromosome band 21q22 is one of the most commonly translocated genes in leukemia. As with other translocations, those involving CBFA2 are associated with specific disease phenotypes. Only one of the different translocations involving CBFA2, the t(12;21), has been associated with a non-myeloid lineage. Several different CBFA2 fusion transcripts were expressed in the myeloid 32Dcl3 cell line, and show that unlike the myeloid specific fusion transcripts, the lymphoid specific ETV6/CBFA2 transcript is not compatible with myeloid cell differentiation. It is shown that myeloid cells expressing the ETV6/CBFA2 transcript undergo apoptosis in response to a G-CSF differentiation signal. The molecular differences in the cells we studied are characterized using Western blot analysis to show that t(12;21) expressing cells fail to express the G-CSF receptor.

Forced expression of the leukemia-associated gene EVI1 in ES cells: a model for myeloid leukemia with 3q26 rearrangements.

Chromosome band 3q26 is the locus of two genes, MDS1/EVI1 and EVI1. The proteins encoded by these genes are nuclear factors each containing two separate DNA-binding zinc finger domains. The proteins are identical, aside from the N-terminal extension of MDS1/EVI1, which is missing in EVI1. However, they have opposite functions as transcription factors. In contrast to MDS1/EVI1, EVI1 is often activated inappropriately by chromosomal rearrangements at 3q26 leading to inappropriate expression of the protein in hematopoietic cells and to myeloid leukemias, which are often characterized by abnormal megakaryopoiesis. We previously showed that the two proteins affect replication and differentiation of progenitor hematopoietic cell lines in opposite ways: whereas EVI1 inhibits the response of 32Dc13 cells to G-CSF and TGFbeta1, MDS1/EVI1 has no effect on the G-CSF-induced differentiation of the 32Dc13 cells, and it enhances the growth-inhibitory effect of TGFbeta1. In the present study, we analyzed the endogenous expression of the two genes during in vitro hematopoietic differentiation of murine embryonic stem (ES) cells and evaluated the effects of their forced expression on the ability of ES cells to produce differentiated hematopoietic colonies. We found that the expression of the two genes is independently and tightly controlled during differentiation. In addition, the forced expression of EVI1 led to a much higher rate of cell growth before and during differentiation, whereas the expression of MDS1/EVI1 repressed cell growth and strongly reduced the number of differentiated hematopoietic colonies. Finally, our study also found that the forced expression of EVI1 resulted in the differentiation of abnormally high numbers of megakaryocytic colonies, thus providing one of the first experimental models showing a clear correlation between inappropriate expression of EVI1 and abnormalities in megakaryopoiesis.

The leukemia-associated gene TEL encodes a transcription repressor which associates with SMRT and mSin3A.

The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The TEL protein contains two functional domains that have been partially characterized: a helix-loop-helix (HLH) domain (also known as a pointed domain) at the N-terminus, which physically interacts with itself, with the SUMO-conjugating enzyme UBC9, and with FLI1; and, at the C-terminus, an ETS domain with DNA-binding properties. Little is known about the function of the central region of TEL. The HLH domain and the central region of TEL are consistently maintained in the t(12;21), which is the most frequent chromosomal translocation involving TEL. In this study, we found that the HLH domain and the central region of TEL mediate transcription repression by two distinct mechanisms. The central region involves the recruitment of a repression complex, including SMRT and mSin3A. The HLH domain represses gene transcription through a mechanism that is independent of known corepressors. Thus, TEL belongs to a growing number of transcription factors rearranged by chromosomal translocations that are associated with the corepressor complexes.

Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9.

The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix-loop-helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein-protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an in vivo interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL in vitro and in vivo. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosome-mediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL.

MDS1/EVI1 enhances TGF-beta1 signaling and strengthens its growth-inhibitory effect but the leukemia-associated fusion protein AML1/MDS1/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-beta1.

MDS1/EVI1, located on chromosome 3 band q26, encodes a zinc-finger DNA-binding transcription activator not detected in normal hematopoietic cells but expressed in several normal tissues. MDS1/EVI1 is inappropriately activated in myeloid leukemias following chromosomal rearrangements involving band 3q26. The rearrangements lead either to gene truncation, and to expression of the transcription repressor EVI1, as seen in the t(3;3)(q21;q26) and inv(3)(q21q26), or to gene fusion, as seen in the t(3;21)(q26;q22) which results in the fusion protein AML1/MDS1/EVI1. This fusion protein contains the DNA-binding domain of the transcription factor AML1 fused in-frame to the entire MDS1/EVI1 with the exclusion of its first 12 amino acids. In this report, we have analyzed the response of the hematopoietic precursor cell line 32Dcl3, expressing either the normal protein MDS1/EVI1 or the fusion protein AML1/MDS1/EVI1, to factors that control cell differentiation or cell replication. The 32Dcl3 cells are IL-3-dependent for growth and they differentiate into granulocytes when exposed to G-CSF. They are growth-inhibited by TGF-beta1. We show that whereas the expression of MDS1/EVI1 has no effect on granulocytic differentiation induced by G-CSF, expression of AML1/MDS1/EVI1 blocks differentiation resulting in cell death. This effect is similar to that previously described by others for 32Dcl3 cells that express transgenic Evil. Furthermore, we show that whereas the expression of the fusion protein AML1/MDS1/EVI1 completely abrogates the growth-inhibitory effect of TGF-beta1 and allows 32Dcl3 cells to proliferate, expression of the normal protein MDS1/EVI1 has the opposite effect, and it strengthens the response of cells to the growth-inhibitory effect of TGF-beta1. By using the yeast two-hybrid system, we also show that EVI1 (contained in its entirety in MDS1/EVI1 and AML1/MDS1/EVI1) physically interacts with SMAD3, which is an intracellular mediator of TGF-beta1 signaling. Finally, we have correlated the response of the cells to G-CSF or TGF-beta1 with the ability of the normal and fusion proteins to activate or repress promoters which they can directly regulate by binding to the promoter site. We propose that mutations of MDS1/EVI1 either by gene truncation resulting in the transcription repressor EVI1 or by gene fusion to AML1 lead to an altered cellular response to growth and differentiation factors that could result in leukemic transformation. The different response of myeloid cells ectopically expressing the normal or the fusion protein to G-CSF and TGF-beta1 could depend on the different transactivation properties of these proteins resulting in divergent expression of downstream genes regulated by the two proteins.

CBFA2(AML1) translocations with novel partner chromosomes in myeloid leukemias: association with prior therapy.

CBFA2(AML1) has emerged as a gene critical in hematopoiesis; its protein product forms the DNA-binding subunit of the heterodimeric core-binding factor (CBF) that binds to the transcriptional regulatory regions of genes, some of which are active specifically in hematopoiesis. CBFA2 forms a fusion gene with ETO and MDS1/EVI1 in translocations in myeloid leukemia and with ETV6(TEL) in the t(12;21) common in childhood pre-B acute lymphoblastic leukemia. We have analyzed samples from 30 leukemia patients who had chromosome rearrangements involving 21q22 by using fluorescence in situ hybridization (FISH). Our analysis showed that 7 of them involved CBFA2 and new translocation partners. Two patients had a t(17;21)(q11.2;q22), whereas the other 5 had translocations involving 1p36, 5q13, 12q24, 14q22, or 15q22. Five of these novel breakpoints in CBFA2 occurred in intron 6; this same intron is involved in the t(3;21). One breakpoint mapped to the t(8;21) breakpoint region in intron 5, and 1 mapped 5' to that region. All 7 CBFA2 rearrangements resulted from balanced translocations. All 7 patients had myeloid disorders (acute myeloid leukemia or myelodysplastic syndrome); 2 were de novo and 5 had treatment histories that included topoisomerase II targeting agents. The association of therapy-related disorders with translocations involving CBFA2 was significant by Fisher's exact test (P < .003). These results provide further evidence that this region of CBFA2 is susceptible to breakage in cells exposed to topoisomerase II inhibitors.

BCL6 can repress transcription from the human immunodeficiency virus type I promoter/enhancer region.

The gene BCL6 encodes a zinc finger protein with similarities to transcription factors. We previously reported that a number of viral genomes, including human immunodeficiency virus type I (HIV-1), contain sequences which are similar to the BCL6 DNA-binding consensus in their promoter regions. Electrophoretic mobility shift assays showed that the full-length BCL6 protein extracted from transfected COS cells and a bacterially expressed truncated protein containing the BCL6 zinc fingers can bind specifically to DNA from the U3 promoter/enhancer region of HIV-1. Transient transfections were performed to analyze the effects of the BCL6 protein on luciferase expression driven by the HIV-1 long terminal repeat (LTR) sequences. Full-length BCL6 significantly repressed luciferase activity compared with multiple controls. We conclude that the BCL6 protein can bind to the HIV-1 promoter-enhancer region and contains a domain upstream of its zinc fingers that can repress transcription from the HIV-1 LTR.

Rearrangements of the AML1/CBFA2 gene in myeloid leukemia with the 3;21 translocation: in vitro and in vivo studies.

AML1 is involved at the breakpoint of chromosome 21 band q22 in several recurring chromosomal translocations associated with myeloid and lymphoid leukemias. AML1 corresponds to CBFA2, and encodes one of the DNA-binding subunits of the enhancer core binding factor CBF. Other members of this family of DNA-binding proteins are CBFA1 and CBFA3, also known as AML3 and AML2. The three proteins are characterized by a highly conserved domain (runt domain, > 90% homology) at the amino end that is necessary for DNA-binding and protein dimerization, and by a unique domain at the carboxyl end that is necessary for transactivation. Two recurring chromosomal translocations involving AML1 associated with myeloid leukemias are the t(8;21)(q22;q22), seen in 20% of patients with acute myeloid leukemia (AML) M2, and the t(3;21)(q26;q22), that occurs in myeloid leukemias primarily following treatment with topoisomerase II inhibitors. In five patients with a t(3;21) whom we studied, AML1 is interrupted by the translocation breakpoint between the runt domain and the transactivation domain, and is fused to two genes on chromosome band 3q26: EAP, which encodes the ribosomal protein L22, and MDS1, which encodes a small polypeptide of unknown function. In one of the five patients we studied, a fusion with a third gene EVI1 also occurs. The fusion of EAP to AML1 is not in frame, and leads to a protein that is terminated shortly after the fusion junction by introduction of a stop codon. The fusion of AML1 to MDS1 is in frame, and adds 127 codons to the interrupted AML1. Thus, in the five cases that we studied, the 3;21 translocation results in expression of two coexisting chimeric mRNAs which contain the identical runt domain at the 5' region, but differ in the 3' region. In addition, the chimeric junction AML1/MDS1/EVII has been detected in cells from one of our patients with the 3;21 translocation. Several genes necessary for myeloid lineage differentiation contain the target sequence for AML1 in their regulatory regions. We have compared the normal AML1 to AML1/MDS1 and AML1/EAP as transcriptional regulators of the CSF1R promoter which contains the CBF target sequence. Our results indicate that whereas the normal AML1 can activate the promoter, the chimeric proteins compete with the normal AML1 and repress expression from the CSF1R promoter. To determine the role of the chimeric proteins in cell growth, we expressed their cDNA in rat fibroblasts. When either fusion gene is expressed, the cells lose contact inhibition and form foci over the monolayer. However, only cells expressing AML1/MDS1 grow as large tumors in nude mice. Thus, although both chimeric genes have similar effects in transactivation of the CSF1R promoter, they affect cell growth as tumor promoters differently in vivo.

Functional characterization of ETV6 and ETV6/CBFA2 in the regulation of the MCSFR proximal promoter.

The ETV6/CBFA2 (TEL/AML1) fusion gene occurs as a result of the chromosome translocation t(12;21)(p13;q22) in up to 30% of children diagnosed with B cell precursor (cd10+, cd19+) acute lymphoblastic leukemia. Leukemic cells that have acquired the t(12;21) usually demonstrate loss of the remaining normal ETV6 (TEL) allele. Using reporter gene assays we have functionally characterized both the normal ETV6 and ETV6/CBFA2 fusion proteins in the regulation of the MCSFR proximal promoter. Neither ETV6 or ETV6/CBFA2 has any significant, detectable effect on the promoter by itself. However, both ETV6 and ETV6/CBFA2 inhibit the activation of the promoter by CBFA2B(AML1B) and C/EBPa. We have shown that a 29-bp region of the MCSFR promoter containing the binding sites for CBFA2B and C/EBPa is sufficient for the inhibition by ETV6 and ETV6/CBFA2. Mutational analysis of the MCSFR promoter revealed that binding of both CBFA2B and C/EBPa to their respective sites is necessary for the inhibition by ETV6 and ETV6/CBFA2. Deletion of the helix-loop-helix (HLH) region from the cDNAs of ETV6 and ETV6/CBFA2 decreased but did not completely abrogate the ability of either construct to inhibit promoter activation. We also found that the ETS DNA binding region of ETV6 is necessary for inhibition of the promoter. Addition of ETS1 and FLI1, two ETS family members that have homology in the 5' HLH region, but not Spi1, an ETS family member without the 5' HLH region, also inhibited reporter gene expression. Our data show that the inhibition mediated by ETV6 and ETV6/CBFA2, in the context of the MCSFR promoter, depend on interactions with other proteins, not just CBFA2B. Our results also indicate that the transactivation characteristics of ETV6/CBFA2 are a combination of positive and negative regulatory properties.