AML1 Site Research Articles

Research resource: Aorta- and liver-specific ERα-binding patterns and gene regulation by estrogen.

Estrogen has vascular protective effects in premenopausal women and in women younger than 60 years who are receiving hormone replacement therapy. However, estrogen also increases the risks of breast and uterine cancers and of venous thromboses linked to up-regulation of coagulation factors in the liver. In mouse models, the vasculoprotective effects of estrogen are mediated by the estrogen receptor α (ERα) transcription factor. Here, through next-generation sequencing approaches, we show that almost all of the genes regulated by 17β-estradiol (E2) differ between mouse aorta and mouse liver, ex vivo, and that this difference is associated with a distinct genomewide distribution of ERα on chromatin. Bioinformatic analysis of E2-regulated promoters and ERα binding site sequences identify several transcription factors that may determine the tissue specificity of ERα binding and E2-regulated genes, including the enrichment of NF-κB, AML1, and AP1 sites in the promoters of E2 down-regulated inflammatory genes in aorta but not liver. The possible vascular-specific functions of these factors suggest ways in which the protective effects of estrogen could be promoted in the vasculature without incurring negative effects in other tissues.

Epigenetic Down-Regulation Of AML2 Expression By EZH2 Contributes To NF-E2 Overexpression In Myeloproliferative Neoplasms

The transcription factor NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPN). In Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) NF-E2 levels are elevated independent of the presence or absence of the JAK2V617Fmutation. We have recently shown that NF-E2 overexpression in a murine model leads to an MPN phenotype followed by spontaneous transformation to acute leukemia in a subset of mice.We have demonstrated that increased NF-E2 transcription is mediated by the transcription factor and proto-oncogene AML-1, which is overexpressed in MPN patients, again irrespective of the JAK2V617Fstatus. AML-1 binds to a conserved enhancer region located 3500 bp upstream of the NF-E2 transcriptional start site. Since AML-1 responsive genes are often also inversely regulated by the tumor suppressor AML-2 (RUNX3), we investigated whether NF-E2 expression is affected by AML-2 as well.Using chomatin immunoprecipitation assays (ChIP) we show that AML-2 binds the NF-E2 enhancer in vivo at a site distinct from but in proximity to the three AML-1 sites in the -3500bp region. AML-2 binding strongly represses transcription off the NF-E2 enhancer in reporter gene assays. This repression is abrogated by site directed mutagenesis of the AML-2 recognition sequence. Likewise, lentivirally induced AML-2 expression drastically reduces the amount of NF-E2 protein in erythroid and myeloid cells. These data clearly demonstrate that NF-E2 expression is directly regulated by AML-2. AML-2 thus serves as a repressor on the hematopoietic NF-E2 gene, a function previously noted mainly in solid tumors.In primary cells from patients with polycythemia vera (PV) AML-2 mRNA expression is significantly reduced. Moreover, ChIP assays demonstrate that in primary PV cells, significantly less AML-2 is bound to the NF-E2 enhancer than in healthy controls. Decreased repression by AML-2 thus cooperates with increased AML-1 induced transcription to elevate NF-E2 levels in PV patients.It has been demonstrated that AML-2 expression can be regulated by two distinct epigenetic mechanisms. For one, DNA methylation of the AML-2 promoter has been reported to silence AML-2 expression in gastric, colorectal and bladder cancers. On the other hand, aberrant histone methylation in the promoter region can also silence AML-2 expression. Here we show that DNA methylation of the AML-2 promoter is unaltered in PV patients. Rather, PV patients display aberrant histone methylation in the AML-2 promoter. Compared to healthy controls, H3K27me3 is significantly increased and H3K4me3 is significantly decreased in primary PV cells. This results in an inactive chromatin conformation on the AML-2 promoter in PV patients. Moreover, we show here that the histone-lysine-methyl-transferase “enhancer of zeste homologue 2” (EZH2), which confers the K3K27me3 mark, binds to the AML-2 promoter and decreases AML-2 expression. PV patients demonstrate significantly increased levels of EZH2 binding to the AML-2 promoterTreatment of primary PV cells and MPN cell lines with 2'-deoxy-5-azacytidine (DAC, Decitabine) ex vivo reverses the altered histone methylation, restoring the pattern found in healthy controls and decreases EZH2 binding to the AML-2 promoter. At the same time, Decitabine treatment induces AML-2 protein expression, decreases EZH2 expression and reduces the elevated NF-E2 levels. Moreover, a post-PV AML patient receiving Decitabine, displayed normalization of AML-2 promoter histone methylation and EZH2 binding on day 8 and day 15 of treatment.Taken together our data demonstrate that epigenetic silencing of AML-2 contributes to the elevated NF-E2 expression observed in MPN patients. Treatment with Decitabine restores physiological histone modifications to the AML-2 locus and decreases NF-E2 levels by reactivating AML-2 expression. These data provide a molecular rationale for extending the clinical investigation of epigenetic modifiers such as Decitabine or Azacitidine, currently used in MDS and AML, to patients with MPNs. A phase I study using Azacitidine in high risk PMF patients is currently being planned by the MPD-RC. Disclosures:No relevant conflicts of interest to declare.

Autoregulation of the Transcription Factor PU.1 Via Its Evolutionarily Conserved Upstream Regulatory Element Is Critical in Adult Mouse Hematopoiesis.

Abstract 1468Poster Board I-491 Background:Levels of the Ets transcription factor PU.1 control normal hematopoietic differentiation and even modest alterations can lead to leukemia and lymphoma. Regulation of PU.1 levels at different stages of hematopoiesis requires multiple interactions between several regulatory elements and transcription factors. Our previous studies identified a potential autoregulatory mechanism of the PU.1 gene through the combined activity of the proximal promoter and an evolutionarily conserved upstream regulatory element (URE), located at –14 kb relative to the transcription start site in mice. PU.1 binds to a conserved PU.1 site in the PU.1 URE both in vitro and in vivo. Approach:To ask at which stages of hematopoietic differentiation autoregulation of PU.1 via binding to its URE might play a role, we developed a mouse model with targeted disruption of the PU.1 binding site in the PU.1 URE. Results:Targeted mutation of the PU.1 autoregulatory site in PU.1 URE abolished PU.1 binding as verified by Chromatin Immuno-precipitation (ChIP). PU.1 URE activity was manifestly reduced resulting in a variety of lineage-specific abnormalities. As shown here in adult mice, the absence of the autoregulatory PU.1 site affected PU.1 expression in a lineage dependent manner. PU.1 expression was markedly decreased in phenotypic long term hematopoietic stem cells (LT-HSC: CD150+/CD48−/ c-kit+/sca-1+/lin−) and short term HSCs (ST-HSCs: CD150−/CD48+/ c-kit+/sca-1+/lin−) and, to a lesser extent, in Common Myeloid Progenitors (CMPs: lin−/c-kit+/Sca-1−/CD34+/FcrRlow), and Megakaryocyte/Erythrocyte Progenitors (MEPs: lin−/c-kit+/Sca-1−/CD34−/FcrRhigh). Within the lymphoid linage, PU.1 levels were unchanged in Common Lymphoid Progenitors (CLPs: lin−/c-kitlow/Sca-1low /IL-7Ra+/Thy1.1−) and pre-B-cells (B220+/ CD43−), up in pro-B-cells (B220+/CD43+), and down in mature B cells. Myeloid cells appeared to be unaffected. Interestingly, while PU.1 levels were decreased in LT- and ST-HSC populations, only phenotypic LT-HSCs were reduced in number. To further analyze HSC function of PU.1 site mutated mice we performed limiting dilution transplantation assays and measured the frequency of competitive repopulation units (CRU) using the congenic Ly5.1/Ly5.2 system. Our preliminary data indicated a decrease of LT-HSC function in PU.1 site mutated mice, although their homing and engraftment functions were not affected. This was also observed in mice with targeted disruption of all three AML-1 sites that are in close proximity of the PU.1 site at the PU.1 URE. While AML-1 itself appeared not to influence LT-HSC function (M. Ichikawa, T. Asai et al. Nature Medicine, 2004), we found that the conformational changes of the URE present in mice with disrupted AML-1 binding sites, as measured by Quantitative Chromosome Conformation Capture, impede PU.1 binding to its autoregulatory site. Conclusion:PU.1 indeed autoregulates its expression via binding to the -14kb URE in a lineage specific manner in vivo. Our data point to a critical role of PU.1 autoregulation especially for long-term hematopoietic stem cell function. Disclosures:No relevant conflicts of interest to declare.

Functional characterization of the murine Tnk1 promoter

Tnk1/Kos1 is a non-receptor protein tyrosine kinase found to be a tumor suppressor. It negatively regulates cell growth by indirectly suppressing Ras activity. We identified and characterized the critical cis-elements required for Tnk1/Kos1's promoter activity. Results indicate that the murine Tnk1 promoter lacks a conventional TATA, CAAT or initiator element (Inr) but contains multiple transcription start sites. Transcription is initiated by a TATA-like element composed of an AT rich sequence at − 30 (30 bp upstream) from the major transcription start site and an Inr-like element that overlaps the multiple start sites. Deletion analysis of the m-Tnk1 promoter reveals the presence of both positive (− 25 to − 151) and negative (− 151 to − 1201) regulatory regions. The three GC boxes which bind Sp1 and Sp3 with high affinity, an AP2 site (that overlaps with an AML1 site) and a MED1 site comprise the necessary cis-elements of the proximal promoter required for both constitutive and inducible Tnk1/Kos1 expression. Importantly, results reveal that cellular stress reverses the repression of Tnk1/Kos1 and induces its expression through increased high affinity interactions between nuclear proteins Sp1, Sp3, AP2 and MED1 for the m-Tnk1 promoter. These findings provide a mechanism by which the m-Tnk1 promoter can be dynamically regulated during normal growth.

Genetics and Epigenetics of the PU.1 Upstream Regulatory Element: AML1 Binding Sites Are Critical for Leuekmia Induced by the AML/ETO9a Fusion Oncogene

The level of expression of the transcription factor PU.1 is a critical determinant of lineage commitment in normal hematopoiesis, and dysregulation of PU.1 leads to development of leukemia. In mice with targeted disruption of the PU.1 upstream regulatory element (URE), expression of PU.1 is decreased to 20% of wild type levels and results in development of acute myeloid leukemia (AML). These data suggests that tightly regulated PU.1 expression is important to maintain normal hematopoiesis and prevent leukemogenesis. Previously, we reported that AML1 (RUNX1) regulated PU.1 expression. Here we demonstrate that AMLl regulates PU.1 through 3 AML1 binding sites in the URE. Mice with targeted mutations in the 3 AML1 binding sites have decreased PU.1 expression in multiple hematopoietic lineages at multiple different developmental stages. Conditional targeting of AML1 in transgenic mice in which the URE homology region 2 (H2, containing all 3 AML1 binding sites) is used to drive expression of a reporter decreased reporter gene expression, suggesting that AML1 regulates PU.1 through these 3 sites in URE homology region 2. Using a second mouse model with a targeted mutation in the PU.1 binding site in the PU.1 URE (which is flanked by the 3 AML1 sites), we demonstrated that PU.1 indeed autoregulates itself through the URE. These results demonstrated that AML1 regulates PU.1 through the 3 AML1 sites in the URE. However, while low levels of PU.1 lead to leukemia, we have not observed frank leukemia development in AML1 conditional knockouts or in mice with targeted disruption of the 3 AML1 sites in the PU.1 URE. We hypothesized that this might be the case because disruption of AML1 or the AML1 sites reduces PU.1 levels to about 40% of wild type, but not as great as that found in PU.1 URE knockouts, which do progress to AML (20% of wild type). We hypothesized that downregulation of PU.1 as a result of binding of AML1/ETO fusion proteins to the URE might result in further reductions of PU.1 expression, and contribute to leukemogenesis. Therefore, we predicted that development of leukemia might be delayed in mice with mutations in the PU.1 URE AML1 DNA binding sites, and this was indeed the case in a modle using a retrovirus expressing the AML1/ETO9a form. We further explored the effect of AML1 and PU.1 binding on chromatin strucutre using chromatin immunoprecipitation (Chip) in the AML1 and PU.1 site URE knockin models, and found that AML1 is involved in H3K4me3 and H3/H4 acetylation of histone tails in the PU.1 URE, while PU.1 is involved in H3/H4 acetylation but not H3K4me3; H3K4 methylation and H3 acetylation decreased in AML1 sites mutant knockin mice and H3 acetylation decreased in PU.1 site mutant knockin mice. Mutation of the AML1 site in mice not only altered the chromatin structure of the URE region, but also interefered with the physical interaction between the URE and PU.1 promoter, as assessed by chromosome capture configuration (3C) assays. Interestingly, the AML1/ETO9a fusion oncogene has a unique role on the epigenetic status of the PU.1 URE in addition to its dominant effect on the 3 AML1 sites. AML1-ETO9 blocks the autoregulation of PU.1 through the PU.1 site in the URE. In summary, our data suggests that AML1 regulates PU.1 expression through 3 AML1 binding sites in the PU.1 URE by modifying chromatin structure in the URE region. In addition, PU.1 can autoregulate itself by facilitating similar epigenetic changes. Dysregulation of the epigenetic status by chromosome translocation products such as AML1-ETO might play an important role in leukemogenesis.First two authors contribute equally to this work.

Identification of Gfi1 as a Direct Target of the Leukemogenic Fusion Protein AML1-ETO9a

AML1-ETO9a (AE9a), a C-terminal splice variant of t(8;21), is highly leukemogenic in mice. Using combined gene expression and promoter occupancy (ChIP-on-chip) profiling of an AE9a induced murine AML model, we identified Gfi1, a transcriptional repressor important for neutrophil differentiation, as a direct target of AE9a. The level of Gfi1 mRNA was down-regulated following leukemia development. We further show that t(8;21) AML-M2 patient samples have lower Gfi1 mRNA levels compared to non-t(8;21) AML-M2 samples. In addition, Gfi1 protein was down-regulated in the human hematopoietic cell line U937 upon AE or AE9a expression. Two consensus AML1 sites (TGTGGT) located at positions −1750 and +405 from the Gfi1 transcriptional initiation site, respectively, are found in the proximity of the peaks of ChIP-on-chip profiling. We confirmed direct DNA binding of AE9a to these regions by ChIP assays. Furthermore, we showed that the activity of the Gfi1 promoter region −1880 to +481 is repressed by AE and AE9a in luciferase reporter assays. To address the effects of down-regulation of Gfi1 on proliferation, we overexpressed it in a primary AML1-ETO C-terminal mutant (AML1-ETOtr) leukemia cell line. Its overexpression slowed down growth and promoted apoptosis and differentiation of this cell line. In this report, we provide evidence that AE9a directly down-regulates Gfi1, thereby inhibiting hematopoietic differentiation, which may contribute to leukemia development.

C/EBP and C-Myb sites are important for the functional activity of the human myeloperoxidase upstream enhancer

Myeloperoxidase (MPO), an enzyme active against bacterial and fungal infections, is expressed specifically in myeloblasts and promyelocytes and minimal in other cell types. We recently identified and partially characterized an upstream enhancer located between −4100 and −3844 bp of the MPO gene. We showed that an AML1 site contributes to enhancer activity and specificity. We now demonstrate three additional footprints within the MPO enhancer and provide evidence that C/EBP and c-Myb sites contribute to its functional, tissue-specific activity. This distal enhancer appears to play an important role in the control of MPO transcription during differentiation of myeloid cells.

PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis

Both PU.1 (also called SFPI1), an Ets-family transcription factor, and AML1 (also called RUNX1), a DNA-binding subunit of the CBF transcription factor family, are crucial for the generation of all hematopoietic lineages, and both act as tumor suppressors in leukemia. An upstream regulatory element (URE) of PU.1 has both enhancer and repressor activity and tightly regulates PU.1 expression. Here we show that AML1 binds to functionally important sites within the PU.1 upstream regulatory element and regulates PU.1 expression at both embryonic and adult stages of development. Analysis of mice carrying conditional AML1 knockout alleles and knock-in mice carrying mutations in all three AML1 sites of the URE proximal region demonstrated that AML1 regulates PU.1 both positively and negatively in a lineage dependent manner. Dysregulation of PU.1 expression contributed to each of the phenotypes observed in these mice, and restoration of proper PU.1 expression rescued or partially rescued each phenotype. Thus, our data demonstrate that PU.1 is a major downstream target gene of AML1.

Targeting fusion protein/corepressor contact restores differentiation response in leukemia cells

The AML1/ETO and PML/RARalpha leukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARalpha and AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARalpha-N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis.

PU.1 Is a Critical Downstream Target of AML1.

PU.1 is expressed in hematopoietic stem cells (HSC), progenitors and differentiating blood cells except terminally differentiated T cells, erythrocytes and megakaryocytes. PU.1 is required for commitment of HSC to multiple lineages. PU.1 −/− embryos die perinatally and fail to generate myeloid and B cells. We previously reported that a DNase I hypersensitive site located 14 kb upstream of the PU.1 transcription start site (−14 DHS) confers myelomonocytic specific gene expression. Targeted deletion this DHS fragment in mice results in a decrease in PU.1 expression in bone marrow to 20% of wild type levels, subsequently leading to a profound decrease in macrophages and B cells. Within the DHS fragment is a “core” consisting of a distal (296bp) and a proximal (253bp) region, which are highly conserved among different species. The PU.1 promoter by itself cannot direct gene expression in vivo. However, −14 DHS confers to the promoter the ability to direct expression of a reporter gene in granulocytes, monocytes, and B-cells of transgenic mice. The proximal region can itself direct high-level gene expression. The proximal region contains 3 AML1 sites. These results, along with data indicating that PU.1 expression is selectively absent from Aml1 −/− embryos (Okada, et al, Oncogene. 1998), suggested that AML1 is likely to be upstream of PU.1. Electro-mobility gel shift assays and chromatin immunoprecipitation assays confirmed that AML1 binds to all 3 AML1 sites both in vitro and in vivo. Mutation of the 3 AML1 sites dramatically reduced the DHS activity of conferring gene expression. We used real time PCR to quantitatively measure PU.1 expression in both embryonic and adult hematopoiesis. We found that PU.1 expression was completely lost in the 9.5 dpc yolk sac, 10.5 dpc AGM and fetal liver of Aml1−/− embryos, suggesting that AML1 is required for PU.1 expression during embryonic hematopoiesis. To evaluate the effects of AML1 loss in the adult hematopoiesis, we employed a conditional Aml1 knockout allele in which LoxP flanked Aml1 (Aml1F/F) was excised by Mx1 promoter driven Cre expression following injection of pIpC. These mice show that Aml1 is not required for maturation of myeloid lineages in adult mice. However, these mice develop a mild myeloproliferative phenotype characterized by increasing in bone marrow and peripheral blood (PB) neutrophils, a 5 fold increasing in HSC, and 2–3 fold increasing myeloid progenitors. Spleen and liver contain infiltration by myeloid cells. These mice also display a dramatic decrease (~80%) in PB platelets and bone marrow megakaryocytes. Furthermore, there are significant blocks in lymphoid development, including reduced numbers of pre-B, pro-B and mature B cells, as well a block in T cell maturation at the DN2 (CD4−;CD8−;CD44+;CD25+) stage. We observed a 70% reduction of PU.1 expression in sorted HSC, progenitors, Gr1+/Mac1+ and B-cells from these mice relative to control mice. In contrast, upregulation of 3–5 fold expression in Ter119+, CD41+, and T cells in these mice compared to controls. Our data shows that PU.1 is a critical target gene of AML1, and AML1 regulates PU.1 in both positive and negative way. We are currently testing the ability of restoration of PU.1 expression to rescue specific defects in Aml1F/F; Tg (Mx1-cre) mice, as well as investigating the role of decreased PU.1 expression in human AML in which the function of AML1 is disrupted.

Granulocyte-macrophage colony-stimulating factor enhancer activation requires cooperation between NFAT and AP-1 elements and is associated with extensive nucleosome reorganization.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is activated by an NFAT-dependent enhancer forming an inducible DNase I hypersensitive (DH) site. The enhancer core comprising the DH site contains the GM330 and GM420 elements that bind NFAT and AP-1 cooperatively. Here we demonstrate that both elements are essential for enhancer activity and that Sp1 and AML1 sites in the enhancer become occupied in vivo only after activation. Chromatin structure analysis revealed that the GM-CSF enhancer core elements are divided between two adjacent nucleosomes that become destabilized and highly accessible after activation. Inducible chromatin reorganization was not restricted to the enhancer core but extended across a 3-kb domain of mobilized nucleosomes, within which the nucleosome repeat length was compressed from approximately 185 to 150 bp. The GM420 element is a high-affinity site that binds NFAT independently of AP-1 but depends on the linked AP-1 site for enhancer function. Nevertheless, just the NFAT motif from the GM420 element was sufficient to form a DH site within chromatin even in the absence of the AP-1 site. Hence, NFAT has the potential to cooperate with other transcription factors by promoting chromatin remodelling and increasing accessibility at inducible regulatory elements.

AML1/RUNX1 Increases During G1 to S Cell Cycle Progression Independent of Cytokine-dependent Phosphorylation and Induces Cyclin D3 Gene Expression

AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G(2)/M phase cells compared with G(1) cells. In addition, G(1) arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G(1) phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFbeta-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G(1) to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation.

Shared features of transcription: mutational analysis of the eosinophil/basophil Charcot-Leyden crystal protein gene promoter.

The lineage-specific Charcot-Leyden crystal (CLC) protein is found in human eosinophils and basophils where it comprises 7-10% of the cellular protein content. Previous work from our laboratory has identified the motif GGAGA[A/G] as a powerful enhancer of gene transcription ill two eosinophil ribonuclease genes. To evaluate a potentially larger role for this motif in the transcriptional regulation of eosinophil genes, we have isolated 1504 nucleotides 5' to the transcriptional start site of the gene encoding CLC protein and identified a functionally active promoter that includes three distinct copies of the GGAGAA motif. Destruction of only one of the three motifs by site-directed mutagenesis resulted in loss of promoter activity (73 +/- 6% reduction), suggesting that this core motif is necessary but not sufficient to support enhanced transcriptional activity. Sequence comparisons and site-specific mutagenesis has permitted further delineation of this enhancer element which, as a result of this work, is now defined as GGAGA[A/G]NNNA. Electromobility shift assays demonstrated specific binding of nuclear protein(s) from an eosinophilic clone-15 nuclear extract to this extended motif. Similar analysis of a GATA-1 binding site demonstrated enhancer activity, with mutagenesis resulting in a 94 +/- 1.4% reduction in activity, whereas the AML1 site functioned as a gene silencer.

AML1, the target of chromosomal rearrangements in human leukemia, regulates the expression of human complement receptor type 1 ( CR1) gene

The human CR1 gene is expressed specifically in hematopoietic cells. It is suggested that some cell-type specific factors which involve in gene-specific activation or repression exist in cells according to the result that the gene expression varies differently depend on differentiation stage. Here, we demonstrate that the integrity of a polyomavirus enhancer core sequence, 5′-TGTGGT-3′, is critical to the human CR1 promoter activity. AML1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. We show that the AML1 binds specifically to this site and activates the human CR1 promoter. Furthermore, we demonstrate that the Ets binding site (GGAA) located 2 bp upstream of the AML1 site is also involved in the regulation of the human CR1 promoter activity. Point mutations of either the AML1 or the Ets binding site that abolish the binding of the respective factors result in significant decreases of the human CR1 promoter activity. These results suggest that AML1 and Ets proteins direct the expression of the human CR1 promoter.

Identification of an upstream enhancer containing an AML1 site in the human myeloperoxidase (MPO) gene

Myeloperoxidase (MPO) is an important antibacterial enzyme found only in granulocytes and monocytes. The human MPO gene is transcribed early during myelogenesis but MPO RNA synthesis ceases at the end of the promyelocyte stage of myeloid maturation. We recently identified a basal MPO promoter and several adjacent cis-elements in the proximal 5′-flanking region of this gene. Transfection studies using constructs containing several kb of 5′-flanking MPO DNA revealed the presence of a DNA segment located between bp (base pair) −4200 and bp −3800 with enhancer activity for the endogenous basal MPO promoter. Deletion studies revealed the core enhancer activity to lie between bp −4100 and bp −3844. The percentage enhancement of promoter activity is greater in MPO-expressing myeloid cells than in MPO-non-expressing myeloid cells or non-myeloid cells. Furthermore, the enhancer confers TPA- or DMSO-responsiveness upon either endogenous or exogenous promoters. DNase I footprinting and transfection experiments identified an AML1 site as a functionally important element within the enhancer. Gelshift competition and supershift experiments demonstrated the binding of the alpha subunit of the transcription factor AML1 to this site in HL-60 cells. This distal enhancer appears likely to play an important role in the control of MPO transcription during myeloid differentiation.

Increased lymphomagenicity and restored disease specificity of AML1 site (core) mutant SL3-3 murine leukemia virus by a second-site enhancer variant evolved in vivo.

SL3-3 is a highly T-lymphomagenic murine retrovirus. The major genetic determinant of disease is the transcriptional enhancer, which consists of a repeated region with densely packed binding sites for several transcription factors, including AML1 (also known as core binding factor and polyoma enhancer-binding protein 2) and nuclear factor 1 (NF1). Previously, we examined the enhancer structure of proviruses from murine tumors induced by SL3-3 with mutated AML1 (core) sites and found a few cases of second-site alterations. These consisted of deletions involving the NF1 sites and alterations in overall number of repeat elements, and they conferred increased enhancer strength in transient transcription assays. We have now tested the pathogenicity of a virus harboring one such second-site variant enhancer in inbred NMRI mice. It induced lymphomas with a 100% incidence and a significantly shorter latency than the AML1 mutant it evolved from. The enhancer structure thus represents the selection for a more tumorigenic virus variant during the pathogenic process. Sequencing of provirus from the induced tumors showed the new enhancer variant to be genetically stable. Also, Southern blotting showed that the tumors induced by the variant were T-cell lymphomas, as were the wild-type-induced lymphomas. In contrast, tumors induced by the original core/AML1 site I-II mutant appeared to be of non-T-cell origin and several proviral genomes with altered enhancer regions could be found in the tumors. Moreover, reporter constructs with the new tumor-derived variant could not be transactivated by AML1 in cotransfection experiments as could the wild type. These results emphasize the importance of both core/AML1 site I and site II for the pathogenic potential of SL3-3 and at the same time show that second-site alterations can form a viral variant with a substantial pathogenic potential although both AML1 sites I and II are nonfunctional.

Stability of AML1 (core) site enhancer mutations in T lymphomas induced by attenuated SL3-3 murine leukemia virus mutants.

Murine retrovirus SL3-3 is highly T lymphomagenic. Its pathogenic properties are determined by the transcriptional enhancer of the U3 repeat region which shows preferential activity in T cells. Within the U3 repeats, the major determinant of T-cell specificity has been mapped to binding sites for the AML1 transcription factor family (also known as the core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF-1]). SL3-3 viruses with AML1 site mutations have lost a major determinant of T-cell-specific enhancer function but have been found to retain a lymphomagenic potential, although disease induction is slower than for the SL3-3 wild type. To compare the specificities and mechanisms of disease induction of wild-type and mutant viruses, we have examined lymphomas induced by mutant viruses harboring transversions of three consecutive base pairs critical to AML1 site function (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström. J. Virol. 65:4177-4181, 1991). Our results show that the mutated AML1 sites are genetically stable during lymphomagenesis and that ecotropic provirus numbers in DNA of tumors induced by wild-type and mutant viruses fall within the same range. Moreover, proviruses were found to be integrated at the c-myc locus in similar proportions of wild-type and mutant SL3-3-induced tumors, and the mutated AML1 sites of proviruses at c-myc are unaltered. In some cases, however, including one c-myc-integrated provirus, a single-base pair change was detected in a second, weaker AML1 binding site. By DNA rearrangement analysis of the T-cell receptor beta-locus, tumors induced by the AML1 site mutants are found to be of the T-cell type. Thus, although the AML1 site mutants have weakened T-cell-specific enhancers they are T-lymphomagenic, and wild-type- and mutant-virus-induced tumor DNAs are similar with respect to the number of overall ecotropic and c-myc-integrated clonal proviruses. The SL3-3 wild-type and AML1 site mutant viruses may therefore induce disease by similar mechanisms.

Second-site proviral enhancer alterations in lymphomas induced by enhancer mutants of SL3-3 murine leukemia virus: negative effect of nuclear factor 1 binding site.

SL3-3 is a highly T-lymphomagenic murine retrovirus. Previously, mutation of binding sites in the U3 repeat region for the AML1 transcription factor family (also known as core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF1]) were found to strongly reduce the pathogenicity of SL3-3 (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström, J. Virol. 65:4177-4181, 1991). We have now examined the few cases in which tumors developed harboring proviruses that besides the AML1 (core) site mutations carried second-site alterations in their U3 repeat structures. In three distinct cases we observed the same type of alteration which involved deletions of regions known to contain binding sites for nuclear factor 1 (NF1) and the addition of extra enhancer repeat elements. In transient-expression experiments in T-lymphoid cells, these new U3 regions acted as stronger enhancers than the U3 regions of the original viruses. This suggests that the altered proviruses represent more-pathogenic variants selected for in the process of tumor formation. To analyze the proviral alterations, we generated a series of different enhancer-promoter reporter constructs. These constructs showed that the additional repeat elements are not critical for enhancer strength, whereas the NF1 sites down-regulate the level of transcription in T-lymphoid cells whether or not the AML1 (core) sites are functional. We therefore also tested SL3-3 viruses with mutated NF1 sites. These viruses have unimpaired pathogenic properties and thereby distinguish SL3-3 from Moloney murine leukemia virus.

The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability.

AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.