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Title: AML1/ETO Oncoprotein Is Directed to AML1 Binding Regions and Co-Localizes with AML1 and HEB on Its Targets}
Article:
Abstract:
A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.
Introduction:
Chromosomal translocations generating fusion genes are the genetic hallmark of Acute Myeloid Leukemia( AML)[1]. Approximately 10–15% of AML cases carry the t( 8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein. AML1 is a DNA-binding transcription factor required for hematopoiesis[2],[3] while ETO is a co-repressor molecule expressed in a variety of tissues[4]. In hematopoietic cells, the fusion protein determines a stage specific arrest of maturation and increases cell survival, thus predisposing to leukemia[5]. Full length AML1/ETO is not sufficient to induce AML in mice, and requires treatment with mutagens to induce leukemic transformation[6]–[9]. An alternatively spliced isoform, AML1/ETO9a, isolated from AML patients bearing t( 8;21), is instead strongly leukemogenic in mice[10]. AML1/ETO9a includes ETO exon 9a, which leads to a frameshift of the original coding sequence and consequently to a C-terminally truncated protein. Co-expression of AML1/ETO and AML1/ETO9a results in earlier onset of AML and blocks myeloid cell differentiation at a more immature stage, suggesting the two isoforms could cooperate in patients to induce leukemia. AML1 and AML1/ETO were originally characterized as DNA binding proteins that recognize, in vitro, the conserved core sequence TGT/cGGT[11]. A recent study of AML1 and AML1/ETO binding sites using a 25 bp random oligonucleotide library revealed that AML1/ETO preferentially binds to DNA sequences containing multiple AML1 binding sites[12], suggesting the fusion protein may selectively regulate a subset of AML1 target genes. AML1/ETO functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA through its ETO moiety[4] and blocks AML1-dependent transactivation in various promoter reporter assays, suggesting it may function as a dominant negative regulator of wild-type AML1[13]–[15]. However, AML1/ETO can also induce the expression of specific target genes: it was initially reported to transactivate M-CSFR and BCL-2 promoters[16],[17] and subsequently, upregulation of other genes was proposed to be crucial for leukemogenesis[18]–[20]. The mechanism for fusion protein-dependent transcriptional activation is not known. AML1/ETO was recently hypothesized to target DNA through E-box motifs as the result of physical interactions with transcription factors of the E-protein family, in particular HEB/TCF12[21]. E-proteins( E2A, HEB, and E2. 2) are regulators of lymphocytic differentiation, and are involved in acute lymphoblastic leukemias( ALL)[22],[23], while their role in myeloid differentiation and AML has not, to date, been described. The large-scale determinants of DNA binding by AML1/ETO and the correlation to global transcriptional effects remain to be elucidated. In this study, we present a comprehensive analysis of the DNA binding pattern of AML1/ETO, and its correlation to AML1 and HEB binding sites. The fusion protein preferentially binds to regions that are occupied by the wild-type AML1 transcription factor, without necessarily displacing it. The DNA binding pattern of HEB is reorganized following AML1/ETO expression, and the E-protein re-localizes to AML1/ETO binding regions. Our study provides an accurate description of the genomic distribution of AML1/ETO, and identifies AML1 and HEB as crucial elements for its transcriptional regulatory function.
Discussion:
The analysis of transcription factor binding patterns using genome-wide approaches can serve not only to identify direct target genes, but also to discover interactions among transcription factors that could help in defining disease-linked regulatory networks. We investigated the genome-wide binding profile of AML1/ETO at human promoters and in a contiguous genomic region, and its correlation with AML1 and HEB, with the aim of understanding the determinants of its transcriptional regulatory function. In U937 cells, AML1/ETO binds to the promoter regions of 2, 513 non-redundant genes, and causes transcriptional changes in 358 of them. Of these, 70% are repressed, including many genes involved in neutrophilic/myeloid differentiation( i. e. MPO, HCK, FYN, GADD45B, ITGAM, ITGB2). On the other hand, 30% of target genes are induced, including HOX genes( HOXA10, HOXA5, HOXC6) and the cell cycle regulators CDKN1A and CDKN1B. CDKN1A is of particular interest since it is know to regulate maintenance of quiescent hematopoietic stem cells[30] and also modulates leukemic development in mouse models of t( 8;21)([31], PG Pelicci, subitted for publication). The AML1 consensus sequence is only enriched in downregulated genes, whereas upregulated genes show enrichment in binding sites for other transcription factors. Therefore, a proportion of AML1/ETO binding to DNA may be achieved independently from the AML1 motif recognition. AML1/ETO has been hypothesized to function as dominant negative of its wild type component AML1[13]–[15]. Analysis of the DNA binding pattern of AML1 showed that the two transcription factors often bind to the same DNA regions. In many cases AML1/ETO target genes represent native AML1 targets, but the presence of the fusion protein does not displace the wild-type protein. In other cases, the two proteins are present in genomic regions where AML1 is normally not found. Therefore, although the two proteins do not physically associate( data not shown), AML1/ETO expression is capable of driving AML1 to new sites on the genome, perhaps by rendering them accessible. The functional consequences of the co-localization of AML1 and AML1/ETO on transcriptional regulation remain to be elucidated. The E-protein HEB has been described as an interactor of both AML1/ETO and ETO[21]. It was hypothesized that through this interaction, AML1/ETO could be redirected to E-protein target genes, possibly deregulating their expression. We found that the binding pattern of HEB in U937 cells is disrupted upon expression of AML1/ETO, and the fusion protein recruits HEB to its binding regions. Co-occurrence of HEB and AML1/ETO in promoters was also detected in the SKNO-1 cell line, demonstrating that this phenomenon is not peculiar to the cellular system under analysis. The specific repositioning of HEB to AML1/ETO binding sites is also supported by sequence analysis, which identifies the AML1 motif in HEB binding regions only in cells expressing AML1/ETO. More information concerning the complexity of AML1/ETO binding can be obtained by looking simultaneously at the binding patterns of AML1 and HEB in the presence and absence of AML1/ETO. The first observation is that these two transcription factors share a proportion of target genes in wild-type conditions, pointing to common regulatory functions in hematopoiesis. When they bind to common target sites, AML1 and HEB are less prone to be displaced by AML1/ETO expression, and these regions represent preferential binding sites for the fusion protein. Furthermore, regulation of gene expression by AML1/ETO is significantly associated with the co-occurrence of AML1 and HEB, suggesting that common binding regions for the three transcription factors are of relevance to AML1/ETO function. A proportion of genes regulated by AML1/ETO did not display binding of the fusion protein in proximity of the gene. Our results demonstrate that some of these genes contain regions bound by HEB in wild-type conditions, and that this binding was no longer present in cells expressing AML1/ETO. Therefore, indirect transcriptional regulation determined by AML1/ETO may be partially due to displacement of HEB from its native target genes, and interference with HEB-dependent gene expression. In the hematopoietic system, HEB has been studied in lymphoid differentiation: loss of function experiments have shown that HEB is relevant in the early stages of B-cell and T-cell development[32]. We show that HEB is expressed in the Lin− compartment of murine bone marrow, and is absent in more differentiated myeloid cells. HEB expression appears to be necessary for survival of cells expressing the fusion protein( A. Gardini and M. Alcalay, unpublished), suggesting that the translocation may only be viable in cells that express HEB, such as early progenitors or stem cells. Furthermore, HEB protein levels increase in the presence of AML1/ETO, whereas HEB mRNA levels remain unchanged, implying that expression of the fusion protein is associated with stabilization of HEB through unknown post-translational mechanisms. Therefore, it is possible that the HEB-expressing subpopulation of progenitor/stem cells might contain the target cell for AML1/ETO, which would then expand the compartment of HEB-expressing cells and sustain HEB protein levels by modulating post-translational regulatory mechanisms. Our study represents the first comprehensive analysis of AML1/ETO global genomic occupancy and provides a correlation to its effect on gene expression. Our results point to a pivotal role for AML1 and HEB in t( 8;21) myeloid leukemogenesis.
Acute myeloid leukemias ( AML ) are a group of hematologic malignancies initiated by chromosomal abnormalities that often give origin to oncogenic proteins with transcriptional regulatory functions . These aberrant transcription factors bind to specific sequences on DNA and influence the activity of adjacent genes . The result is that leukemic blasts display abnormalities in their gene expression programs , which are ultimately responsible for the malignant phenotype . In this study , genome-wide approaches were exploited not only to identify target genes , but also to discover interactions among different transcription factors , with the aim of defining disease-linked regulatory networks . We performed a detailed analysis of the DNA binding pattern of an oncogenic transcription factor , AML1/ETO , which is responsible for approximately 10–15% of AML . We identified a specific signature , which is characterized by the presence of binding regions for AML1/ETO and for other transcription factors , AML1 and HEB , and found that the DNA binding pattern of AML1 and HEB is significantly affected in cells expressing AML1/ETO . Our results , therefore , describe genes regulated by AML1/ETO and demonstrate that this oncogenic protein can significantly interfere with the function of other transcriptional regulators .