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• W1986533634 abstract "Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor α (CBFα). In mice, CBFα regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFα regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/β-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFα binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFα co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFα. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFα activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFα to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms. Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor α (CBFα). In mice, CBFα regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFα regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/β-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFα binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFα co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFα. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFα activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFα to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms. Two hereditary human bone marrow failure syndromes leading to leukemia are severe congenital neutropenia (SCN) 1The abbreviations used are: SCN, severe congenital neutropenia; AML, acute myelogenous leukemia; FPD, familial platelet disorder; NE, neutrophil elastase; CBFα, core-binding factor α; LEF-1, lymphoid enhancer factor-1; C/EBP, CCAAT/enhancer-binding protein; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; ChIP, chromatin immunoprecipitation; HMG, high mobility group; PST, proline-, serine-, and threonine-rich; CREB, cAMP-response element-binding protein. and familial platelet disorder with acute myelogenous leukemia (FPD/AML). SCN consists of low numbers of neutrophils, the primary phagocytic white blood cell (1.Zeidler C. Welte K. Semin. Hematol. 2002; 39: 82-88Crossref PubMed Scopus (85) Google Scholar, 2.Horwitz M. Li F.-Q. Albani D. Duan Z. Person R. Meade-White K. Benson K.F. Cancer Invest. 2003; 21: 577-585Crossref Scopus (18) Google Scholar). Examination of the bone marrow reveals that maturation of the myeloid lineage fails at the promyelocytic stage, before the formation of terminally differentiated neutrophils (3.Lightsey A.L. Parmley R.T. Marsh Jr., W.L. Garg A.K. Thomas W.J. Wolach B. Boxer L.A. Am. J. Hematol. 1985; 18: 59-71Crossref PubMed Scopus (16) Google Scholar). Many patients (4.Freedman M.H. Alter B.P. Semin. Hematol. 2002; 39: 128-133Crossref PubMed Scopus (75) Google Scholar, 5.Banerjee A. Shannon K.M. J. Pediatr. Hematol. Oncol. 2001; 23: 487-495Crossref PubMed Scopus (9) Google Scholar, 6.Bernard T. Gale R.E. Evans J.P. Linch D.C. Br. J. Haematol. 1998; 101: 141-149Crossref PubMed Scopus (45) Google Scholar) with SCN develop myelodysplasia or AML. The most common genetic cause of SCN is heterozygous mutation of ELA2 (7.Dale D.C. Person R.E. Bolyard A.A. Aprikyan A.G. Bos C. Bonilla M.A. Boxer L.A. Kannourakis G. Zeidler C. Welte K. Benson K.F. Horwitz M. Blood. 2000; 96: 2317-2322Crossref PubMed Google Scholar), encoding neutrophil elastase (NE). NE (8.Bieth J.G. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego1998: 54-60Google Scholar) is a serine protease, mainly found in the granules of neutrophils, and ELA2 mutations also cause a related disorder, cyclic neutropenia (9.Horwitz M. Benson K.F. Person R.E. Aprikyan A.G. Dale D.C. Nat. Genet. 1999; 23: 433-436Crossref PubMed Scopus (371) Google Scholar). In contrast, FPD/AML is an autosomal dominant bleeding disorder composed of low numbers of functionally defective platelets that also frequently evolves to myelodysplasia or AML (10.Dowton S.B. Beardsley D. Jamison D. Blattner S. Li F.P. Blood. 1985; 65: 557-563Crossref PubMed Google Scholar). FPD/AML results from mutations in AML1 (11.Song W.J. Sullivan M.G. Legare R.D. Hutchings S. Tan X.L. Kufrin D. Ratajczak J. Resende I.C. Haworth C. Hock R. Loh M. Felix C. Roy D.C. Busque L. Kurnit D. Willman C. Gewirtz A.M. Speck N.A. Bushweller J.H. Li F.P. Gardiner K. Poncz M. Maris J.M. Gilliland D.G. Nat. Genet. 1999; 23: 166-175Crossref PubMed Scopus (912) Google Scholar), on chromosome 21, encoding the transcriptional regulator core-binding factor α (CBFα). Somatic mutations of CBFα (12.Osato M. Asou N. Abdalla E. Hoshino K. Yamasaki H. Okubo T. Suzushima H. Takatsuki K. Kanno T. Shigesada K. Ito Y. Blood. 1999; 93: 1817-1824Crossref PubMed Google Scholar, 13.Preudhomme C. Warot-Loze D. Roumier C. Grardel-Duflos N. Garand R. Lai J.L. Dastugue N. Macintyre E. Denis C. Bauters F. Kerckaert J.P. Cosson A. Fenaux P. Blood. 2000; 96: 2862-2869Crossref PubMed Google Scholar) and the t(8;21) translocation, producing the AML1/ETO fusion gene, are among the most common abnormalities of AML (14.Kelly L.M. Gilliland D.G. Annu. Rev. Genomics Hum. Genet. 2002; 3: 179-198Crossref PubMed Scopus (428) Google Scholar, 15.Downing J.R. Higuchi M. Lenny N. Yeoh A.E. Semin. Cell Dev. Biol. 2000; 11: 347-360Crossref PubMed Scopus (52) Google Scholar). The translocation t(12;21), generating the TEL/AML1 fusion gene, and AML1 gene amplification (16.Penther D. Preudhomme C. Talmant P. Roumier C. Godon A. Mechinaud F. Milpied N. Bataille R. Avet-Loiseau H. Leukemia. 2002; 16: 1131-1134Crossref PubMed Scopus (39) Google Scholar) appear often (17.Rubnitz J.E. Pui C.H. Downing J.R. Leukemia. 1999; 13: 6-13Crossref PubMed Scopus (107) Google Scholar, 18.Speck N.A. Stacy T. Wang Q. North T. Gu T.L. Miller J. Binder M. Marin-Padilla M. Cancer Res. 1999; 59: 1789S-1793SPubMed Google Scholar) in acute lymphoblastic leukemia. Somatic AML1 mutations are also frequent in myelodysplasia (19.Imai O. Kurokawa M. Izutsu K. Hangaishi A. Maki K. Ogawa S. Chiba S. Mitani K. Hirai H. Leuk. Lymphoma. 2002; 43: 617-621Crossref PubMed Scopus (7) Google Scholar). CBFα contains a domain homologous to the Drosophila pair rule gene runt (20.Lund A.H. van Lohuizen M. Cancer Cell. 2002; 1: 213-215Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), directing promoter recognition and heterodimerization with the β subunit of core-binding factor, CBFβ, thus stabilizing DNA binding. ELA2 appears to be a downstream target of CBFα, suggesting a possible clue for why leukemia is a feature common to both syndromes. In mice, CBFα regulates (21.Nuchprayoon I. Meyers S. Scott L.M. Suzow J. Hiebert S. Friedman A.D. Mol. Cell. Biol. 1994; 14: 5558-5568Crossref PubMed Google Scholar) expression of the murine neutrophil elastase promoter (mELA2) through a binding site adjacent to recognition sites for the factors PU.1, c-Myb, and C/EBP. The latter three cooperatively regulate the mELA2 promoter in vitro (22.Oelgeschlager M. Nuchprayoon I. Luscher B. Friedman A.D. Mol. Cell. Biol. 1996; 16: 4717-4725Crossref PubMed Scopus (206) Google Scholar, 23.Nuchprayoon I. Simkevich C.P. Luo M. Friedman A.D. Rosmarin A.G. Blood. 1997; 89: 4546-4554Crossref PubMed Google Scholar). Attempts to model these two leukemia syndromes in mice have failed, however. Knock-in mice targeted with an SCN-causing human ELA2 mutation demonstrate normal hematopoiesis (24.Grenda D.S. Johnson S.E. Mayer J.R. McLemore M.L. Benson K.F. Horwitz M. Link D.C. Blood. 2002; 100: 3221-3228Crossref PubMed Scopus (57) Google Scholar). Knock-out mice targeted for heterozygous disruption of AML1 also lack thrombocytopenia, platelet abnormalities, and leukemia (25.Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1604) Google Scholar, 26.Wang Q. Stacy T. Binder M. Marin-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1029) Google Scholar, 27.Sasaki K. Yagi H. Bronson R.T. Tominaga K. Matsunashi T. Deguchi K. Tani Y. Kishimoto T. Komori T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12359-12363Crossref PubMed Scopus (327) Google Scholar). A possible explanation for the shortcomings of the mouse models is that there may be differences in transcriptional control of hematopoiesis. In particular, a CBFα binding site does not appear conserved at the corresponding position in the human neutrophil elastase (hELA2) promoter, and there is evidence for the involvement only of PU.1 (28.Srikanth S. Rado T.A. Biochim. Biophys. Acta. 1998; 1398: 215-223Crossref PubMed Scopus (15) Google Scholar) and C/EBP (29.Verbeek W. Gombart A.F. Chumakov A.M. Muller C. Friedman A.D. Koeffler H.P. Blood. 1999; 93: 3327-3337Crossref PubMed Google Scholar) but not c-Myb (30.Friedman A.D. Int. J. Hematol. 2002; 75: 466-472Crossref PubMed Scopus (31) Google Scholar). To define the regulation of the hELA2 promoter in vivo toward the goal of investigating a potential transcriptional linkage between these two leukemic syndromes, we here utilize bone marrow from patients with these two diseases. One sample comes from an SCN patient demonstrating a novel ELA2 promoter sequence substitution, whereas the other derives from a FPD/AML patient with an AML1 activation domain mutation. We find that CBFα directly interacts with lymphoid enhancer factor-1 (LEF-1). LEF-1 is an effector of Wnt signaling (31.Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (886) Google Scholar), a pathway entrenched in cancer mechanisms. The cooperative activation of ELA2 by LEF-1 and CBFα suggests that there may be a common link between both syndromes that may, in turn, connect them to more general cancer mechanisms. Patients—Bone marrow aspirates from patients and controls, subjected to Institutional Review Board-approved protocols, represent discarded portions of samples obtained for routine care. The -199 C to A ELA2 substitution appears in two SCN cases. One is an 11-year-old girl with SCN presenting at age 4 years following recurrent gingivitis. She died after bone marrow transplant at age 12 years. A 2-year-old brother died of SCN-related sepsis. Her mother lacked this ELA2 variant. Samples from the father and brother were not available. The other is a 46-year-old man, diagnosed at age 40 following years of recurrent aphthous stomatitis; other family members were unavailable. Mutations in other known and candidate SCN genes, including Gfi1, were not detected in either SCN patient. IV:2/pedigree 3 of Michaud et al. (47.Michaud J. Wu F. Osato M. Cotties G.M. Yanagida M. Asou N. Shigesada K. Ito Y. Benson K.F. Raskind W.H. Rossier C. Antonarakis S.E. Israels S. McNicol A. Weiss H. Horwitz M. Scott H.S. Blood. 2002; 99: 1364-1372Crossref PubMed Scopus (319) Google Scholar) is a 19-year-old man with the Y260X AML1 mutation developing M5 AML. Bone marrow was aspirated during first complete remission, when blood cell counts were normal. Vectors and Site-directed Mutagenesis—Human and mouse ELA2 promoters were PCR-amplified from peripheral blood mononuclear cell DNA and subcloned into the pGL3 firefly luciferase reporter (Promega). Human LEF-1HA, dnLEF-1HA, and pt-β-catenin expression constructs have been described (32.Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2594) Google Scholar, 33.Takemaru K.I. Moon R.T. J. Cell Biol. 2000; 149: 249-254Crossref PubMed Scopus (405) Google Scholar). Human CBFα and CBFβ pcDNA3 (Invitrogen) expression vectors were gifts of Dr. H. Scott (Walter and Eliza Hall Institute). cDNA encoding human PU.1, C/EBPϵ, and c-Myb were reverse transcription (RT)-PCR-amplified from HL-60 cells and subcloned into PCS2+ expression vectors (34.Li F.Q. Horwitz M. J. Biol. Chem. 2001; 276: 14230-14241Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). LEF-1 cDNA was subcloned into pCS2+ for in vitro transcription/translation. Mutagenesis with oligonucleotide cassettes was used to construct ELA2 promoter and LEF-1 and CBFα mutations. LEF-1 and CBFα mutants were subcloned into PCDNA3.1 expression vector. pET-23b expression vector (Novagen) was used to generate His-tagged LEF-1 and CBFα. Cell Lines and Cultivation—HEK293T, HeLa, NIH-3T3, U-937, KG-1, and HL-60 cells were purchased from ATCC and grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (first three) or RPMI plus 12.5% fetal bovine serum (latter three). RT-PCR and Real Time PCR Analysis—RNA was isolated from bone marrow with QIAamp and from cell culture with RNeasy (Qiagen). RT-PCR was performed with TaqMan Gold (ABI) using random priming (or oligo(dT), as indicated) for 25 or 40 cycles, as noted. Real time PCR was performed using the ABI 7900HT system in a 50-μl volume for 40 cycles: 50 °C for 2 min; 95 °C for 10 min; 95 °C for 0.15 min; 60 °C for 1 min with 5′-FAM/3′-BHQ-1 ELA2 probe and VIC glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control probe. ELA2 transcript was normalized to GAPDH using the standard curve method (manufacturer's protocol). Expression analysis of LEF/TCF factors used specific human primers with oligo(dT)-primed cDNA for 30 PCR cycles to produce products of the indicated sizes: LEF-1, 251 bp; TCF-1, 247 bp; TCF-3, 250 bp; TCF-4, 264 bp. Transient Expression of Recombinant Proteins and Luciferase Assay—For luciferase assays, 2 × 105 or 4 × 105 cells were seeded per well into 12- or 6-well tissue culture dishes, respectively, cultured overnight, and then transiently transfected using LipofectAMINE Plus (Invitrogen) with 1 or 2 μg of DNA (unless stated otherwise), respectively, according to the manufacturer's instructions. Renilla luciferase (pRL-TK; Promega) was co-transfected as an internal control, and the firefly activities were normalized to Renilla activity. In 12-well dish transfections, a total of 1 μg of the tested activators, 50 ng of reporter and 5 ng of pRL-TK were used (unless stated otherwise), and quantities per well were doubled for 6-well plates. Luminometry (Berthold Technologies) scored luciferase activity 24 h after transfection using the Dual-Luciferase assay (Promega). Transfections were performed in triplicate and repeated three times, and S.E. was reported. Empty vector was added to normalize the total amount of DNA in every transfection, and each transfection was performed in triplicate. 0.1 μg of the 260-bp ELA2 promoter were used as luciferase reporter in all of the assays (unless stated otherwise). Recombinant Protein Expression—His6-tagged LEF-1 and CBFα were produced in Escherichia coli. cDNAs were subcloned into pET-23b (Novagen) and expressed in the pLysS strain with nickel affinity chromatography purification. In vitro transcription/translation was performed using TNT coupled reticulocyte lysate (Promega) with or without [35S]methionine (Amersham Biosciences). Co-immunoprecipitation, Immunoblotting, and in Vitro Association Assay—Transiently co-transfected cells were lysed in 400 μl of iced lysis buffer (20 mm Tris-HCl, pH 8.0, 135 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 1% Triton X-100, and 10% glycerol), followed by 3-s sonication. Lysates were cleared by centrifugation at 15,000 × g for 30 min at 4 °C, adjusted for protein concentration, and incubated with 2 μg of goat polyclonal anti-CBFα antiserum (C-19; Santa Cruz Biotechnology) or rat monoclonal anti-hemagglutin (anti-HA) antiserum (Roche Applied Science) at 4 °C overnight with rotation, followed by the addition of 10 μl of protein G-Sepharose (Sigma) with 1 h of further incubation. Immunoprecipitates were centrifuged and washed three times with 1 ml of ice-cold lysis buffer. 20 μl of loading buffer were added to the drained beads and then resolved by SDS-PAGE with immunoblot detection using ECL (Amersham Biosciences). The concentrations of antibodies for immunoblot were as follows: 3 μg/ml mouse monoclonal antibody to human LEF-1 (REMB1, Exalpha Biologicals); 1:300 C-19; 1:5,000 mouse monoclonal antibody to GAPDH (6C5; Biodesign International); 1:1,000 anti-HA; 1:2,000 mouse monoclonal anti-FLAG antiserum (Sigma). Secondary detection employed 1:10,000 peroxidase-conjugated antibody (Jackson ImmunoResearch) reactive to the primary antibody. In vitro association assays used 25 μl of vector-programmed TNT extract. 35S-Labeled CBFα- or LEF-1-HA-containing extracts were mixed with unlabeled LEF-1 or CBFα extracts with or without CBFβ. Antibodies against HA or CBFα were added with rotation overnight at 4 °C. 20 μl of protein G beads (Sigma) were added and rotated for 1 h, and immunoprecipitates were subjected to SDS-PAGE with autoradiography. Electrophoretic Mobility Shift Assay (EMSA)—1 μg (unless stated otherwise) of nickel affinity-purified, bacterially expressed His-tagged LEF-1 or CBFα was mixed with 1 ng of 32P-5′-radiolabeled annealed double-stranded oligonucleotide probe in an equal volume of 2× buffer (40 mm Hepes, pH 7.6, 100 mm KCl, 2 mm dithiothreitol, 2 mm EDTA, 10% glycerol, and 1 mg/ml poly(dI-dC)), incubated for 30 min on ice, and then electrophoresed on a 4% polyacrylamide gel. Competitive oligonucleotides, at indicated excess, were added 5 min before probe, and antibody was added 30 min before probe. For titration experiments, shifted bands were excised from dried gels and subject to scintillation counting. Antisense Morpholino Oligonucleotide Knockdown—LEF-1, AML1, or scrambled sequence control antisense morpholino oligonucleotides (GeneTools) were transfected to HL-60 or U937 cells according to the manufacturer's protocol. 17 h later, cells were extracted, and NE activity was assayed (34.Li F.Q. Horwitz M. J. Biol. Chem. 2001; 276: 14230-14241Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed as described (35.Duan Z. Horwitz M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5932-5937Crossref PubMed Scopus (89) Google Scholar) using U937 cells. 1 μg of mouse monoclonal antibody to human LEF-1 (REMB1) or goat polyclonal antibody to CBFα (N-20; Santa Cruz Biotechnology) were used to precipitate protein-DNA complexes. About 5 ng of immunoprecipitated DNA served as a template in each reaction in a total volume of 50 μl and corresponded to about 1:10,000 to 1:20,000 of the genomic DNA isolated from 1.5 ml of soluble chromatin. ELA2 Promoter Substitutions in SCN—ELA2 mutations affecting the protein sequence cause the majority of SCN cases (7.Dale D.C. Person R.E. Bolyard A.A. Aprikyan A.G. Bos C. Bonilla M.A. Boxer L.A. Kannourakis G. Zeidler C. Welte K. Benson K.F. Horwitz M. Blood. 2000; 96: 2317-2322Crossref PubMed Google Scholar); promoter mutations have not previously been sought. In 4 of 41 SCN patients lacking ELA2 coding abnormalities, we identified two different single nucleotide substitutions in the promoter after sequencing ∼300 upstream base pairs. Two patients demonstrate a heterozygous C to A transversion 15 bases upstream of the transcription start site in a potential GC-box for the general transcription factor Sp1 (Fig. 1A). Two other SCN patients possess a heterozygous C to A transversion at position -199, within a potential LEF-1/TCF motif that differs from the consensus only by a G to T transition (Fig. 1A). Neither variant appears in any of 47 SCN patients with ELA2 coding mutations. Among 260 controls, the -15 C to A substitution does not occur. The -199 C to A substitution is present in just one of these controls. SCN is usually a monogenic disorder; however, there is a suggestion that in some circumstances multiple inherited factors might be contributory (2.Horwitz M. Li F.-Q. Albani D. Duan Z. Person R. Meade-White K. Benson K.F. Cancer Invest. 2003; 21: 577-585Crossref Scopus (18) Google Scholar). Thus, the greatly elevated frequency of promoter variants among individuals with SCN suggests that these variants may contribute to disease risk. Regardless of their potential contributions to disease, these variants afford an opportunity to investigate ELA2 transcriptional regulation in vivo. Elevated ELA2 Expression in an SCN Patient with the Potential LEF/TCF Binding Site Substitution—To determine whether the -199 C to A substitution affects ELA2 expression, we performed RT-PCR (Fig. 1B) and real time RT-PCR (Fig. 1C) on bone marrow obtained from one of these SCN patients. The ELA2 transcript shows an over 4-fold increase in the SCN patient compared with two normal controls. These results suggest that the -199 C to A substitution found in the LEF-1/TCF binding site of the ELA2 promoter up-regulates expression. LEF-1 Expression in Myeloid Lineages—LEF-1, a LEF/TCF transcription factor family member, is a terminal component of the canonical Wnt pathway (31.Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (886) Google Scholar). The Wnt family of secreted proteins regulates intercellular interactions by binding a cell surface receptor, a Frizzled family member, which, in the canonical pathway, relays the signal through the cytoplasm to stabilize β-catenin protein levels. β-Catenin translocates to the nucleus and forms a complex with LEF/TCF, thereby transcriptionally activating or repressing target genes. The name of LEF-1 derives from its isolation from lymphocytes (36.van de Wetering M. de Lau W. Clevers H. Cell. 2002; 109: 13-19Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), but expression of LEF/TCF family members (37.Thiele A. Wasner M. Muller C. Engeland K. Hauschildt S. J. Immunol. 2001; 167: 6786-6793Crossref PubMed Scopus (33) Google Scholar) and β-catenin (38.Chung E.J. Hwang S.G. Nguyen P. Lee S. Kim J.S. Kim J.W. Henkart P.A. Bottaro D.P. Soon L. Bonvini P. Lee S.J. Karp J.E. Oh H.J. Rubin J.S. Trepel J.B. Blood. 2002; 100: 982-990Crossref PubMed Scopus (126) Google Scholar) also occurs (at least) in monocytes. Given elevated ELA2 expression in the SCN patient with an altered potential LEF/TCF binding site, we tested for the expression of these factors in myelocytic cells. RT-PCR confirms expression of transcripts encoding LEF-1 and TCF-3, but neither TCF-1 nor TCF-4, in human bone marrow (Fig. 1D) and LEF-1 expression (Fig. 1E) in human HL-60 promyelocytes (39.Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2458-2462Crossref PubMed Scopus (1426) Google Scholar) and U-937 promonocytes (40.Nilsson K. Sundstrom C. Int. J. Cancer. 1974; 13: 808-823Crossref PubMed Scopus (122) Google Scholar), both of which (8.Bieth J.G. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego1998: 54-60Google Scholar) express ELA2. (Immunoblot analysis also confirms expression of LEF-1 in HL-60 cells, shown below in Fig. 3A.) We therefore have focused on LEF-1 as probably contributing to the transcriptional effects of the -199 C to A ELA2 promoter substitution. β-Catenin-independent Enhancer Function of the Potential LEF-1 Element—We addressed whether the potential LEF-1 binding site contributes to in vitro ELA2 promoter activity by constructing derivative ELA2 promoters, containing either 560 or 260 bp of the upstream regulatory region, driving a luciferase reporter gene (Fig. 2A). We assayed activity in transiently transfected HEK293T kidney cells, commonly used to investigate Wnt/β-catenin signaling, with or without co-transfection of a LEF-1 expression vector. As shown in Fig. 2B, LEF-1 stimulates the wild type ELA2 promoter (NEP260WT). In contrast, reporters containing a deletion (NEP260ΔLEF-1) or substitutions (NEP260LEF-1BM) within the LEF-1 binding site only weakly react to LEF-1, confirming that this sequence functions as a LEF-1-responsive element in vitro. (Assays in HeLa and NIH-3T3 cells produced similar results (not shown).) TCF-3 also activated the expression of ELA2 (data not shown). Wnt signaling can proceed through noncanonical β-catenin-independent pathways (41.Kuhl M. Sheldahl L.C. Park M. Miller J.R. Moon R.T. Trends Genet. 2000; 16: 279-283Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar), and LEF-1 can activate transcription independently of β-catenin (42.Hsu S.C. Galceran J. Grosschedl R. Mol. Cell. Biol. 1998; 18: 4807-4818Crossref PubMed Scopus (338) Google Scholar, 43.Eastman Q. Grosschedl R. Curr. Opin. Cell Biol. 1999; 11: 233-240Crossref PubMed Scopus (474) Google Scholar). To determine whether β-catenin influences ELA2 transactivation, we tested the effects of pt-β-catenin, a stabilized form in which four aminoterminal serine and threonine residues are substituted with alanines (44.Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1018) Google Scholar). Co-expression of pt-β-catenin and LEF-1 did not significantly increase reporter activity (Fig. 2B). A dominant negative form of LEF-1 (dnLEF-1) lacking the β-catenin binding domain stimulated the ELA2 promoter as efficiency as the full-length pt-β-catenin (data not shown), indicating that LEF-1 activates the ELA2 promoter largely independently of β-catenin. Elevated ELA2 Expression with the -199 C to A Substitution in HEK293T Cells—To determine whether the effect of the -199 C to A ELA2 substitution can be reproduced in vitro, we prepared reporters containing this substitution fused with either 560 bp (NEP560C>A) or 260 bp (NEP260C>A) of the upstream regulatory region (Fig. 2A). This substitution confers increased transcription upon both lengths of sequence in HEK293T cells, whereas there is no significant difference in the basal level (Fig. 2C). The effect is not as strong as what is apparent in the bone marrow of the SCN patient with the corresponding sequence change. There are at least two reasons for this difference. First, the reporter may not include all upstream elements required for co-activation of the more proximal promoter region present in the reporters. Second, HEK293T cells, derived from kidney, are unlikely to express an appropriate repertoire of co-activating transcription factors. Nevertheless, we can conclude that the elevated ELA2 transcription in bone marrow from an individual with this substitution probably results from this single base substitution, as opposed to the possibility of an undetected, additional DNA sequence change outside of the boundaries of the region that we examined. LEF-1 Binding in Electrophoretic Mobility Shift Assays—To corroborate the function of the LEF-1 binding site in the ELA2 promoter, we carried out EMSAs employing recombinant LEF-1 (Fig. 2D). The probes consisted of 32P-5′-labeled doublestranded oligonucleotides spanning the region from -210 to -176 of the ELA2 promoter, corresponding to either the wild type sequence or the -199 C to A substitution. Fig. 2D shows that LEF-1 forms a retarded complex with the wild type sequence (compare lane 1 with lane 4). Anti-LEF-1 antibody (lane 3), but not control IgG (lane 2), supershifts the retarded complex. LEF-1 therefore binds specifically to the sequence found in the ELA2 promoter. LEF-1 also binds specifically to a probe containing the -199 C to A substitution (lane 10). When the cold -199 C to A substituted probe" @default.
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• W1986533634 date "2004-01-01" @default.
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• W1986533634 title "Lymphoid Enhancer Factor-1 Links Two Hereditary Leukemia Syndromes through Core-binding Factor α Regulation of ELA2" @default.
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