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Originally published In Press as doi:10.1074/jbc.M007262200 on September 26, 2000

J. Biol. Chem., Vol. 275, Issue 51, 40588-40593, December 22, 2000
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Human alpha (1,3)-Fucosyltransferase IV (FUTIV) Gene Expression Is Regulated by Elk-1 in the U937 Cell Line*

Donald A. WithersDagger § and Sen-itiroh HakomoriDagger

From the Dagger  Pacific Northwest Research Institute, Seattle, Washington 98122 and the  Department of Pathobiology, University of Washington, Seattle, Washington 98195

Received for publication, August 10, 2000, and in revised form, September 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The alpha 1,3-fucosyltransferase IV (FucTIV) encoded by its gene (FUTIV) is responsible for synthesis of Lex (Galbeta 4[Fucalpha 3]GlcNAcbeta 3Galbeta 1,R), which causes compaction in the morula stage of the preimplantation mouse embryo, as well as alpha 1,3-fucosylation at multiple internal GlcNAc of unbranched poly-N-acetyllactosamine, termed "myeloglycan," the physiological epitope of E-selectin. Since myeloglycan-type structure is also expressed in various types of human cancer and may mediate E-selectin-dependent metastasis, expression of FUTIV is oncodevelopmentally regulated. The mechanisms controlling FUTIV expression remain to be clarified. In this report, we further characterize FUTIV gene structure and define a non-TATA box-dependent transcriptional start region just upstream from the translational start. FUTIV promoter/reporter fusion constructs defined a "full-length" promoter and highly active fragments in the macrophage-derived U937 and myeloid HL60 cell lines. One highly active fragment contains a consensus binding site for the Ets-1 transcription factor (Withers, D. A., and Hakomori, S. (1997) Glycoconj. J. 14, 764). Gel shift analysis shows specific binding to this site in nuclear extracts from U937 cells. Mutation of the Ets consensus site significantly reduces FUTIV promoter activity in both cell lines. Gel supershift and dominant negative cotransfection experiments identified the Ets family member Elk-1 as one component binding and regulating the FUTIV promoter in U937 cells. The significance of FUTIV regulation by Elk-1 is discussed.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Among nine fucosyltransferases (FucTs)1 expressed in animal cells (FucTI-FucTIX; see "Discussion"), FucTIV is of great biological interest based on results from two areas of study. One area is early embryogenesis, in which expression of Lex epitope becomes maximal at morula-stage preimplantation embryo, and declines quickly thereafter, by conversion to Ley expressed at the surface of blastocyst. Recent studies indicate that Lex expression in embryonal carcinoma F9 cells is due to expression of the FUTIV gene (1). Lex at morula stage is thought to cause compaction, the first cell adhesion event during embryogenesis, in which Lex-Lex interaction plays a major role (2, 3) prior to uvomorulin (cadherin)-dependent adhesion (4). Cell adhesion based on Lex glycolipid was confirmed recently (5). FucTIV is also involved in synthesis of the cell surface epitope, so-called myeloglycan, consisting of unbranched long chain type 2 poly-N-acetyllactosamine having alpha 1right-arrow3 fucosylation at multiple internal G1cNAc with alpha 2right-arrow3 sialylation at terminal Gal (6). Myeloglycan is considered the major physiological E-selectin epitope controlling tethering and rolling of neutrophils on solid phase E-selectin, particularly under dynamic flow conditions (7). Expression of FucTVII is responsible for alpha 1right-arrow3 fucosylation at penultimate G1cNAc, but penultimate fucosylation by FucTVII is inhibited by internal alpha 1right-arrow3 fucosylation by FucTIV. Thus, FucTIV is responsible mainly for synthesis of myeloglycan without fucosylation at penultimate GlcNAc, i.e., without sialyl-Lex (8). Various types of tumor express myeloglycan-type structures (9), which may mediate E-selectin-dependent metastasis (for review, see Ref. 10).

Expression of the alpha 1,3-FUT gene family has been examined for lung (11), colorectal (12, 13), and gastric (14) tumors. In all cases, FUTIV expression is significantly higher in tumors than in adjacent non-tumorous tissue. In lung tumors, FUTIV and FUTVII expression levels were inversely correlated with patient prognosis (11). FUTIV expression has also been studied in purified myeloid lineage cells and cell lines induced to differentiate (15-17). Typically, differentiated cells lose surface Lex expression accompanied by a decrement in FUTIV transcript levels as compared with undifferentiated cells. Conversely, the colon adenocarcinoma cell line HT-29 shows elevated FUTIV mRNA levels when apoptosis is induced (18).

The collective data suggest that Lex, determined by "myeloid type" fucosyltransferase (FucTIV) expression, is an oncofetal marker. Expression is regulated during normal development (16, 19, 20) and in tumors (11-13). We sought to identify the trans-acting factors responsible for up-regulating FUTIV expression in tumor cell lines. Preliminary studies localized upstream genomic DNA fragments with high promoter activity in U937 and HL60 tumor cell lines (21).2 The data presented here identifies the Ets family member Elk-1 as a positive regulatory factor acting on the FUTIV promoter in U937 cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of FUTIV Genomic DNA, Plasmid Construction, and Sequence Analysis-- A FUTIV genomic clone, lambda 20, was isolated from a human placental genomic DNA library (Stratagene) while screening with a FUTIII probe. The EcoRI/NcoI 3.1-kb and BamHI/NcoI 1.65-kb fragments from lambda 20 were cloned into the luciferase reporter vector pGL3basic (Promega) to make pG-3092Eco and pG-1647Bam, respectively. Using unique restriction sites, deletion derivatives of these constructs were made by simple digestion and recircularization.

The consensus Ets-1 site at -350 (from the translational start site) was mutated in promoter fusion constructs using the recombinant PCR technique (22). Primary PCR reactions used the -518 (XhoI/NcoI) construct in the pOGH vector (Nichols Institute, San Juan Capistrano, CA) as template. 3'-Primary PCR used a mutated Elk-1 site primer, 3'ELKmut (-361 to -338; 5'-GGGCGCGGCCCTTCAGCCCTCGGG-3', mutated bases underlined), and a downstream vector primer (5'-TGCAGCTAGGTGAGCTGTC-3'). 5'-Primary PCR used 5'ELKmut primer (complementary to 3'ELKmut) and an upstream vector primer (5'-CCCAGTCACGACGTTGTAAAACG-3'). Secondary PCR used primary PCR fragments as templates and the flanking vector primers. The resulting fragment was digested with BstXI and SfiI and cloned into pG-1647Bam deleted for BstXI/SfiI. Resulting clones were sequenced through the BstXI/SfiI interval. DNA was sequenced either manually using Sequenase version 2.0 (Amersham Pharmacia Biotech) or with an ABI310 automated sequencer. Putative promoter sequences were analyzed for consensus transcription factor binding sites using Transcription Element Search Software (TESS) (23).

RT-PCR was performed using the TitanTM one-tube RT-PCR kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. HL60 RNA samples were treated with RQ1 DNase (RNase-free; Promega Corp.) or RNase (prepared by boiling for 5 min; DNase-free) to confirm absence of DNA-based PCR. PCR used the following primers; 3'-1 (5'-CAAGTACTATCTGTTCCC-3') in combination with either 3'-3 (5'-AAACCTGAACTCTTTCCC-3') or 3'-2 (5'-CTATACCACTGCATTGAC-3') for FUTIV RT-PCR and FT7us (5'-GCACCCCAGCCCACGATCACC-3') and FT7ds (5'-CTCAGGCCTGAAACCAACCC-3') for FUTVII control RT-PCR. Amplification was for 40 cycles of 1 min at 95 °C, 2 min at 54 °C (for FUTIV primer combinations) or 58 °C (for FUTVII), followed by 3 min at 72 °C.

3'-RACE was performed using U937 cDNA essentially as described (24) except that Elongase (Life Technologies, Inc.) was substituted for Taq polymerase. Nested PCR using first primer 3'-4 (5'-GTATTAAAGTGTGGGCAG-3') and then primer 3'-5 (5'-GAGTTCAGGTTTTGAAGG-3') sequentially in combination with the RACE adapter primer, amplified a 2.2-kb fragment. 5'-RACE was modified to include 2% Me2SO and 1 M betaine as described (25). U937 RNA was reverse-transcribed, then amplified using first primer 5'-1 (5'-GCGCCGTACACGTCAAG-3') and then primer 5'-2 (5'-AGCACACAGACGGTCCATGG-3') sequentially in combination with the RACE adapter primer. Race products were cloned into pBluescript (Stratagene) and sequenced.

Cell Culture and Transient Transfections-- U937 and HL60 cell lines were originally purchased from ATCC and maintained in this laboratory. Cells were cultured in RPMI containing 10% heat-inactivated fetal bovine serum at cell density <= 8 × 105 cells/ml. Cultures were split 1 day prior to electroporation and collected at a culture density of ~5 × 105 cells/ml. For U937 electroporation, 5 × 106 cells were resuspended in 250 µl of room temperature RPMI containing 10% fetal bovine serum and 10 µg of plasmid DNA and transferred to a 0.4-cm gap cuvette. HL60 cells (2 × 106) were resuspended in 400 µl of medium containing 20 µg of DNA. Each electroporation included 1 µg of pXGH5 (growth hormone expression vector) to control for transfection efficiency. Electroporation at 242 V and 975 microfarads (U937) or 260 V and 1,050 microfarads (HL60) was done with a Gene Pulser II electroporator (Bio-Rad). After electroporation, cells were cultured in a 5-ml final volume of RPMI with 10% fetal bovine serum for 48 h. For cotransfection experiments, the Elk-1 dominant negative-expressing plasmid in the pCMV5 vector was used. This plasmid encodes the DNA binding, but lacks the transactivation domain of Elk-1. pCMV5 or pGL3 vectors were used as controls. Cotransfection experiments used 8 µg of reporter plasmid, 2 µg of control or experimental plasmid (pGL3basic, pCMV5, or pCMV5-Elk-Dominant negative), and 1 µg of pXGH5.

Luciferase, Growth Hormone, and Protein Quantitation from Transient Transfections-- Electroporated cell extracts were prepared and assayed using the luciferase assay system (Promega) according to the manufacturer's instructions. Firefly luciferase levels were determined on an EG&G Berthold Lumat LB 9507 luminometer with a 100-µl D-luciferin injection. Growth hormone levels were assayed from the culture medium using the HGH-TGES kit purchased from Nichols Institute (San Juan Capistrano, CA). Extract protein levels were determined by the bicinchonic acid method (Pierce). Luciferase activity was normalized by both growth hormone (transfection efficiency control) and protein levels (cell number control).

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared by the method of Dent and Latchman (26). EMSA and "supershift" experiments were performed essentially as described (26), except that Tris-glycine (25 mM Tris, pH 8.5, 190 mM glycine, 1 mM EDTA) was used as gel running buffer. Antibodies for supershift were purchased from Santa Cruz Biotechnology, Inc.

RNA isolation used the Ultraspec (Biotecx Laboratories, Inc.) acid-phenol procedure, and poly(A)+ RNA was selected using the poly(A)tract mRNA isolation system IV kit from Promega Corp. Northern blots were prepared as described (27).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FUTIV Gene Structure-- The FUTIV gene produces three transcripts of 2.3, 3.0, and 6.0 kb as detected on Northern blots (15, 28, 29). The 2.3- and 6.0-kb transcripts are typically detected at much higher abundance than the 3.0-kb species (Fig. 2B). cDNAs corresponding to the 2.3- and 3.0-kb transcripts were described previously (28) (see Fig. 1). The derivation of the 6.0-kb transcript, however, is not known and has been suggested to come from an as yet undescribed FUT gene. This possibility led us to further investigate FUTIV gene structure. DNA sequence of a FUTIV 3'-genomic DNA fragment and the FUTIV 3'-untranslated region (Fig. 1) were used to synthesize downstream and upstream primers, respectively, for RT-PCR amplification of the putative 6.0-kb transcript. This experiment, shown in Fig. 2A, clearly detects the presence of another FUTIV transcript containing sequences common to the 3.0- and 2.3-kb FUTIV transcripts (since the upstream primer falls within the 3'-untranslated region of the 2.3- and 3.0-kb transcripts) as well as additional 3'-untranslated sequences. In addition, this RT-PCR-generated fragment detects only a ~6.0-kb transcript on Northern blots (Fig. 2B).



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  		Fig. 1.   Map of the 11q21 chromosome region encoding FUTIV transcripts. Center line represents genomic DNA containing restriction sites for EcoRI (E), HindIII (H), BamHI (B), NotI (N), and EcoRV (V); large central arrow represents protein coding region. Large arrows above genomic map represent previously described cDNAs (28). The large arrow below the genomic map represents a cDNA described within. Primers used for RT-PCR and RACE experiments are shown as small arrows. Derivation of probes used to detect FUTIV transcripts by Northern analysis (Fig. 2B) is shown at the bottom. Sequences of the 3'-untranslated region of the ~6.0-kb transcript and the promoter region have been deposited with GenBank under accession numbers AF305083 and AF305082, respectively.



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  		Fig. 2.   A) FUTIV 3' RT-PCR. Total RNA from HL60 cells was treated with RQ1 DNase (RNase-free; Promega Corp.), then used in the TitanTM one-tube RT-PCR system (Roche Molecular Biochemicals). Each primer combination included a control amplification after treatment with RNase (DNase-free) to demonstrate the absence of DNA-based PCR. M, DNA size marker; reaction 1, FUTIV primers 3'-1 and 3'-2; reaction 2, FUTIV primers 3'-1 and 3'-3; reaction 3, FUTVII primers. B, Northern blot using 2.0 µg of poly(A)+ RNA/sample hybridized with FUTIV coding region probe (left panel) and 3'-untranslated probe (right panel). Derivation of probes is shown in Fig. 1.

Using 3'-RACE analysis (24), the remainder of this cDNA was cloned. Sequence analysis showed a 3,098-base pair extension from the 3'-most sequences of the 3.0-kb transcript (ELFT-L) to the poly(A) addition site. This cDNA is colinear with genomic DNA (data not shown). The 6.0-kb transcript, which matches previously unassigned clones in the human expressed sequence tag data base, uses an alternate poly(A) addition site from the shorter FUTIV transcripts and appears to differ from them only by the length of 3'-untranslated sequences.

RACE was also used to locate the 5' end of FUTIV transcripts in RNA from U937 cells. Results from this experiment (Fig. 3) show that ~80% of recovered RACE clones extended to a region approximately 85 base pairs upstream from the start of translation. Two further upstream start sites at -129 and -145 were also detected by this method. An additional low frequency FUTIV start site, corresponding to the 3.0-kb transcript, is located approximately 550 base pairs upstream from the start of translation (28). As is typically true for genes with scattered transcriptional start sites, the FUTIV promoter contains no canonical TATA box.



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  		Fig. 3.   Sequence of 3' portion of the FUTIV promoter. Numbering corresponds to distance 5' to the start of translation (underlined). Boldface sequence shows consensus binding sites for known transcription factors (see Ref. 23 for description of transcription factors). Arrows indicate transcriptional start sites mapped by 5'-RACE. Bold arrows show high frequency start sites (9 of 11 independent RACE clones for -86 and -88 sites combined), and thin arrows show low frequency start sites at -129 and -145 (one RACE clone each).

Promoter Analysis-- A genomic clone containing FUTIV was isolated from a placental DNA library (CLONTECH) using FUTIII gene sequences as probe. 3.1 kb extending upstream from the start of translation was subcloned and sequenced (Fig. 3). This fragment matches the restriction map of the FUTIV upstream region (29-31) and contains 5' sequences from the FUTIV 2.3- and 3.0-kb cDNAs (28) (Fig. 1), thus confirming its identity as FUTIV. Comparison of the FUTIV upstream sequence to a transcription factor consensus binding site data base revealed many matches to described factor binding sites (Fig. 3).

The 3.1-kb fragment was cloned into the luciferase reporter vector (pGL3basic) to make "pG-3092Eco." This "full-length" fragment and deleted or mutated derivatives were transfected into FUTIV-expressing cell lines U937 and HL60, and luciferase reporter levels were assayed. The longest constructs (pG-3092Eco, pG-2444Nsi, and pG-2145Pac) have undetectable promoter activity in both cell lines (data not shown). Deletion derivatives, such as pG-1800HindIII (data not shown) and pG-1647Bam have high activity in U937 (Fig. 4) and HL60 (data not shown) cell lines. In addition, the internal deletion removing sequences from -424 to -273 from the full-length promoter has significantly reduced activity in U937 cells. Mutation of a consensus Ets-1 binding site at position -350 reduces promoter activity further (Fig. 4). This region was further studied using EMSA in order to localize potential transcription factor binding sites.



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  		Fig. 4.   Bar graph showing relative activity of pG-1647(Bam) versus pG-1647((Bam) -350Elk-) versus pG-1647(Bam Delta -424/-273) versus pG basic in U937 cell line. Extracts were prepared from each sample and luciferase, growth hormone, and protein levels assayed. Luciferase activity was normalized by both growth hormone (transfection efficiency control) and protein levels (cell number control). Normalized activities are expressed relative to the pGbasic sample, which was arbitrarily assigned the value 1.0. Plotted values represent the average of at least three independent experiments for each construct.

Electrophoretic Mobility Shift Assays Using the Ets Consensus Site at -350-- The 151-base pair interval from -424 to -273 contains a consensus binding site for the Ets family of transcription factors located at -350. Oligonucleotides corresponding to FUTIV promoter region -350 and containing wild type or mutant Ets binding sites were used in EMSA to detect binding activity in U937 cells (Fig. 5). The Ets mutant oligonucleotide differs from wild type by a three-base substitution at the Ets consensus core sequence (see "Materials and Methods"). At least two binding activities (see s1 and s2 in Fig. 5) were detected with the wild type oligonucleotide. These activities were specific for the Ets consensus site since binding was competed with excess unlabeled wild type, but not mutant, oligonucleotide (Fig. 5, left panel, lanes 2 and 3). In addition, s1 and s2 bands were absent when the mutant oligonucleotide was used as probe (Fig. 5, left panel, lane 4).



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  		Fig. 5.   EMSA with probes from FUTIV -350 region encompassing the Ets/Elk-1 consensus binding site using extracts from FUTIV-expressing U937 or nonexpressing HepG2 cell lines. Left panel, wild type (W) or mutant (M) oligonucleotide probes were incubated without or with 100-fold molar excess of the indicated unlabeled oligonucleotide. Middle and right panels, EMSA after incubation in the absence (-) or presence of anti-Ets-1 (E), -Elk-1 (L), or -Pu.1 (P) antisera using extracts from U937 (middle panel) or HepG2 (right panel). s1 and s2 indicate specifically bound complex; ss indicates a "supershifted" complex.

The identity of the factor(s) bound to the FucTIV Ets consensus site was investigated using EMSA and antibodies to various Ets family members (Fig. 5, middle panel). The most abundant specific binding activity (s2) is shifted in mobility upon addition of anti-Elk-1 antibody to the binding reaction (middle panel, lane 3). Ets-1, pu.1 (middle panel, lanes 2 and 4, respectively), Ets-2, and SAP-1 antibodies failed to recognize U937 nuclear proteins bound to this site (data not shown). In addition, the s2 band is absent from FUTIV-nonexpressing HepG2 cells (Fig. 5, right panel).

Role of the Elk-1 (-350) Binding Site-- A promoter fusion construct (pG-1647Bam/-350(Elk-)) was made containing the same Ets-1 site mutation used above for EMSA. When compared with wild type (pG-1647Bam), promoter activity of the Elk-1 site mutant is significantly reduced in U937 cells (see Fig. 4).

The role of Elk-1 was further investigated by co-transfection experiments using the FucTIV promoter fusion construct pG-1647Bam, containing either the wild type or mutated Elk-1 -350 site, along with an expression vector encoding dominant negative Elk-1 protein (32). Results of these experiments are shown in Fig. 6. The data show a dramatic reduction (approximately 5-fold) in promoter activity of the pG-1647Bam construct when co-transfected with the dominant negative Elk-1-encoding, but not the empty vector plasmid. When the mutant Elk site construct was used as reporter, DN-Elk cotransfection reduced promoter activity to the same level seen for the wild type reporter plus DN-Elk cotransfection. The latter data suggest that Elk-1 binds to and regulates other sites in the FUTIV promoter, possibly a weaker consensus site at -610 base pairs (see Fig. 3). Collectively, these data show that Elk-1 binds specifically to the -350 site and regulates expression from the FUTIV promoter.



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  		Fig. 6.   U937 cotransfection with dominant negative Elk-1-expressing plasmid versus control cotransfections with pGL3 or pCMV5 vector plasmids. Reporter constructs were either wild type (pG-1647(Bam)) or the Elk site-mutated (pG-1647(Bam -350Elk-)) constructs. Extracts were prepared from each sample and luciferase, growth hormone, and protein levels assayed. Luciferase activity was normalized by both growth hormone (transfection efficiency control) and protein (cell number control) levels. Normalized activities are expressed relative to the pG-1647-Elk+ (cotransfected with pGbasic) sample, which was arbitrarily assigned the value of 100. Plotted values represent the average of three independent experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Six human alpha (1,3)-fucosyltransferase genes (FUTIII-FUTVII and FUTIX) have been described to date (28-30, 33-39). FUTIII, -V, and -VI have high sequence homology, whereas FUTIV, -VII, and -IX are less similar both to the former group and to each other. Each encoded enzyme displays a characteristic pattern of activity for potential glycolipid, protein and carbohydrate acceptors (8, 40-42). In addition, each FUT gene is expressed in a tissue and cell line characteristic pattern (15, 16, 20, 39, 43-45), suggesting unique regulation of individual gene expression. Functionally, the alpha (1,3)-FucTs fucosylate cell surface proteins and lipids that mediate cell adhesion, sorting, and activation (5, 46-49).

We have localized the predominant promoter for the FUTIV gene to just upstream from the translational start site. The FUTIV promoter lacks a TATA box and fits the criteria of a CpG island (50). Typical of such promoters, multiple, scattered transcriptional start sites were identified. In addition, a key regulator of the FUTIV promoter, Elk-1, was identified by DNA binding and cotransfection experiments.

Elk-1 was originally found by homology with the oncogene for Ets-1 (51) and is a member of the large family of Ets-related transcription factors. The Ets family regulates expression of other glycosyltransferases such as beta 4-galactosyltransferase-I by TASS-I (52), beta -1,2-N-acetylglucosaminyltransferase II and N-acetylglucosaminyltransferase-V by Ets-1 and Ets-2 (53-56), as well as members of the metalloproteinase gene family (57-61). Activity of N-acetylglucosaminyltransferase-V and metalloproteinases contributes to the metastatic capacity of tumors.

Ets family proteins bind to DNA either autonomously or as a member (called the ternary complex factor) of a complex with the dimeric serum response factor. Three Ets family members are capable of acting as ternary complex factors (Elk-1, SAP-1, and SAP-2/Erp/Net; reviewed in Ref. 62). Ets family proteins bind DNA sites containing a core GGA consensus sequence via a winged helix-turn-helix DNA binding motif (reviewed in Ref. 63). Binding site specificity for members of the Ets family is partially determined by DNA sequence flanking the core site. The FUTIV Elk-1 binding site at -350 (GCCCGGAAGCC) matches the 7 central bases of the 11-base consensus sequence (AACCGGAAGTG/a) to which Elk-1 binds autonomously (64). The latter sequence was also selected from randomized oligonucleotides by the Elk-1 DNA binding domain (65). SAP-1 is theoretically capable of autonomous binding to the FUTIV -350 site (65) but does not occur in U937 extracts (data not shown), so the identity of the s1 binding activity seen in Fig. 4 is unknown. Since a serum response element is absent in the surrounding FUTIV promoter region, Elk-1 may bind to this site autonomously rather than as a component of serum response factor. If so, this is the only functional gene promoter site yet described capable of autonomous regulation by Elk-1.

Elk-1 is a target of central importance in both Ras-dependent and -independent mitogen-activated protein kinase signaling pathways in numerous cell types (32, 66-69). Both growth factor-regulated kinases and two groups of stress-activated kinases (c-Jun N-terminal kinases and p38 kinase) have been found to activate Elk-1 (32, 70-72). Phosphorylation of Elk-1 induces a conformational change that increases its DNA binding, transcriptional activating and serum response factor binding activities, thus activating both the ternary complex and autonomous transcriptional regulatory functions of this protein (32, 66, 73, 74). The multiplicity of signaling pathways that activate Elk-1, and their deranged regulation in tumors, may explain the common expression of an autonomous Elk-1 target such as FUTIV in many tumors and tumor cell lines of various derivation (38, 43). In contrast, adults express the myeloid-type fucosyltransferase activity attributed to FucTIV only in adult human leukocytes and brain tissue (20).

Whether Elk-1 plays a role in FUTIV expression during normal development is not known. It is of interest, however, that a fucosyltransferase activity with acceptor and inhibitor specificity characteristic of FucTIV is expressed in the early stages of development of many human tissues. The embryonic fucosyltransferase activity gives way to a distinct tissue characteristic activity in the adult organ (20). This expression pattern is mirrored in the developing myeloid lineage, where immature promyelocytes express predominantly FUTIV while mature granulocytes express mostly FUTVII but very low levels of FUTIV (15). The developing human lung also shows stage specific patterns of Lex, Ley, and SLex expression. Lex is abundantly expressed in the early developmental stages, in cells of the presumptive bronchiolus, but declines in abundance after overt development of this tissue (19). Finally, down-regulation of FUTIV mRNA levels is seen upon Me2SO-induced differentiation of the promyelocytic HL60 cell line (16). Recently, a sixth alpha 1,3-fucosyltransferase (FucTIX) (39) has been described, which apparently shares acceptor specificity and NEM insensitivity (see analysis in Ref. 40) with FucTIV. Although some of the above studies specifically assay FUTIV mRNA expression (by RT-PCR), caution is warranted in attributing enzyme activity or end product (Lex) expression to FucTIV. Given all this information, it is likely that Elk-1 plays a role in establishing or maintaining expression of FUTIV in embryonic tissues or pluripotent lineages, but our data do not support a role for Elk-1 in FUTIV down-regulation during differentiation (data not shown).

Cell surface, stage-specific embryonic expression of Lex, determined by FucTIV activity, may mediate cell adhesive interactions during tissue morphogenesis. Evidence in support of this comes from studies of mouse preimplantation embryos, the blastomeres of which normally have a tightly compact form, but undergo decompaction when incubated with multivalent Lex in solution (75, 76). Blastomere compaction is known from gene knock-out experiments to be mediated by E-cadherin (77). Using mutant embryocarcinoma (EC) cell lines, other molecules involved in this process have been identified. Cells from two independent mutant lines, deficient either for embryoglycan (the major carrier of Lex in EC cells (Ref. 78)) or for alpha (1,3)-fucosyltransferase activity, could not aggregate with wild type cells (48, 79), despite a normal capacity for homotypic aggregation. These studies suggest that cell surface Lex plays a role in the initial stages of E-cadherin-mediated cell adhesion.

Enhanced FUTIV expression may also play a role in tumor progression, since the myeloglycan-type structure produced by FUTIV accumulates in metastatic colonic adenocarcinoma as well as lung and gastric tumor cell lines, but not in normal colonic mucosa or normal fibroblasts (9). Tumor cells expressing cell-surface myeloglycan structures might be better able to metastasize to tissues containing counter-receptors such as E-selectin or Lex.


    ACKNOWLEDGEMENTS

We thank Dr. Goran Larson and Dr. Atsushi Masamune for recovery of FUTIV genomic clone, Dr. Stephen Anderson for scientific editing and figure preparation, Wendy Smith for technical assistance, and Dr. Roger Davis (University of Massachusetts Medical Center) for the kind gift of dominant negative Elk-1-encoding plasmid.


    FOOTNOTES

* This work was supported in part by Grant OIG CA42505 from the NCI, National Institutes of Health, and by a grant from the Biomembrane Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF305082 and AF305083.

§ To whom correspondence should be addressed: Div. of Biomembrane Research, Pacific Northwest Research Inst., 720 Broadway, Seattle, WA 98122-4237. Tel.: 206-726-1242; Fax: 206-860-6751; E-mail: dwithers@pnri.org.

Published, JBC Papers in Press, September 26, 2000, DOI 10.1074/jbc.M007262200

2 FUTIV promoter deletion analysis identified a highly active region containing an Ets transcription factor consensus binding site, as reported at the First International Symposium on Glycosyltransferases and Cellular Communications, Mar 26-28, 1997, Osaka, Japan (21).


    ABBREVIATIONS

The abbreviations used are: FucT, fucosyltransferase; RT, reverse transcription; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; kb, kilobase pair(s); RACE, rapid amplification of cDNA ends.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Ozawa, M., and Muramatsu, T. (1996) J. Biochem. (Tokyo) 119, 302-308
2. Eggens, I., Fenderson, B. A., Toyokuni, T., Dean, B., Stroud, M. R., and Hakomori, S. (1989) J. Biol. Chem. 264, 9476-9484
3. Kojima, N., Fenderson, B. A., Stroud, M. R., Goldberg, R. I., Habermann, R., Toyokuni, T., and Hakomori, S. (1994) Glycoconj. J. 11, 238-248
4. Takeichi, M. (1991) Science 251, 1451-1455
5. Boubelík, M., Floryk, D., Bohata, J., Dráberová, L., Macák, J., Smíd, F., and Dráber, P. (1998) Glycobiology 8, 139-146
6. Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., and Reinhold, V. N. (1996) Biochemistry 35, 770-778
7. Handa, K., Stroud, M. R., and Hakomori, S. (1997) Biochemistry 36, 12412-12420
8. Handa, K., Withers, D. A., and Hakomori, S. (1998) Biochem. Biophys. Res Commun. 243, 199-204
9. Fukushi, Y., Nudelman, E. D., Levery, S. B., Rauvala, H., and Hakomori, S. (1984) J. Biol. Chem. 259, 10511-10517
10. Hakomori, S. (1996) Cancer Res. 56, 5309-5318
11. Ogawa, J., Inoue, H., and Koide, S. (1996) Cancer Res. 56, 325-329
12. Ito, H., Hiraiwa, N., Sawada-Kasugai, M., Akamatsu, S., Tachikawa, T., Kasai, Y., Akiyama, S., Ito, K., Takagi, H., and Kannagi, R. (1997) Int. J. Cancer 71, 556-564
13. Kudo, T., Ikehara, Y., Togayachi, A., Morozumi, K., Watanabe, M., Nakamura, M., Nishihara, S., and Narimatsu, H. (1998) Lab. Invest. 78, 797-811
14. Petretti, T., Schulze, B., Schlag, P. M., and Kemmner, W. (1999) Biochim. Biophys. Acta 1428, 209-218
15. Clarke, J. L., and Watkins, W. M. (1996) J. Biol. Chem. 271, 10317-10328
16. LeMarer, N., Palcic, M. M., Clarke, J. L., Davies, D., and Skacel, P. O. (1997) Glycobiology 7, 357-365
17. Cullen, P., Mohr, S., Brennhausen, B., Cignarella, A., and Assmann, G. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 1591-1598
18. Akamatsu, S., Yazawa, S., Zenita, K., Matsumoto, H., Tachikawa, T., and Kannagi, R. (1996) Glycoconj. J. 13, 1021-1029
19. Miyake, M., Zenita, K., Tanaka, O., Okada, Y., and Kannagi, R. (1988) Cancer Res. 48, 7150-7158
20. Mollicone, R., Candelier, J.-J., Mennesson, B., Couillin, P., Venot, A. P., and Oriol, R. (1992) Carbohydr. Res. 228, 265-276
21. Withers, D. A., and Hakomori, S. (1997) Glycoconj. J. 14, 764
22. Higuchi, R. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. , Gelfand, D. , Sninsky, J. , and White, T., eds) , pp. 177-183, Academic Press, New York
23. Schug, J., and Overton, G. C. (1997) TESS: Transcription Element Search Software on the WWW; Technical Report CBIL-TR-1997-1001-v0.0 , Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, Philadelphia, PA, http://www.cbil.upenn.edu/tess/index.html
24. Frohman, M. A. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. , Gelfand, D. , Sninsky, J. , and White, T., eds) , pp. 28-38, Academic Press, New York
25. Weissensteiner, T., and Lanchbury, J. S. (1996) BioTechniques 21, 1102-1108
26. Dent, C. L., and Latchman, D. S. (1993) in Transcription Factors: A Practical Approach (Rickwood, D. , and Hames, B., eds) , pp. 1-26, Oxford University Press, Oxford, United Kingdom
27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
28. Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349-1356
29. Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M., Macher, B. A., Kelly, R. J., and Ernst, L. K. (1991) J. Biol. Chem. 266, 17467-17477
30. Kumar, R., Potvin, B., Muller, W. A., and Stanley, P. (1991) J. Biol. Chem. 266, 21777-21783
31. McCurley, R. S., Recinos, A., Olsen, A. S., Gingrich, J. C., Szczepaniak, D., Cameron, H. S., Krauss, R., and Weston, B. W. (1995) Genomics 26, 142-146
32. Whitmarsh, A. J., Shore, P., Sharrocks, A. D., and Davis, R. J. (1995) Science 269, 403-407
33. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990) Genes Dev. 4, 1288-1303
34. Weston, B. W., Nair, R. P., Larsen, R. D., and Lowe, J. B. (1992) J. Biol. Chem. 267, 4152-4160
35. Weston, B. W., Smith, P. L., Kelly, R. J., and Lowe, J. B. (1992) J. Biol. Chem. 267, 24575-24584
36. Koszdin, K. L., and Bowen, B. R. (1992) Biochem. Biophys. Res. Commun. 187, 152-157
37. Natsuka, S., Gersten, K. M., Zenita, K., Kannagi, R., and Lowe, J. B. (1994) J. Biol. Chem. 269, 16789-16794
38. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269, 14730-14737
39. Kaneko, M., Kudo, T., Iwasaki, H., Ikehara, Y., Nishihara, S., Nakagawa, S., Sasaki, K., Shiina, T., Inoko, H., Saitou, N., and Narimatsu, H. (1999) FEBS Lett. 452, 237-242
40. Holmes, E. H., Xu, Z., Sherwood, A. L., and Macher, B. A. (1995) J. Biol. Chem. 270, 8145-8151
41. Grabenhorst, E., Nimtz, M., Costa, J., and Conrad, H. S. (1998) J. Biol. Chem. 273, 30985-30994
42. Niemela, R., Natunen, J., Majuri, M., Maaheimo, H., Helin, J., Lowe, J. B., Renkonen, O., and Renkonen, R. (1998) J. Biol. Chem. 273, 4021-4026
43. Yago, K., Zenita, K., Ginya, H., Sawada, M., Ohmori, K., Okuma, M., Kannagi, R., and Lowe, J. B. (1993) Cancer Res. 53, 5559-5565
44. Robinson, N. E., deVries, T., Davis, R. E., Stults, C. L. M., Watson, S. R., van den Eijnden, D. H., and Macher, B. A. (1994) Glycobiology 4, 317-326
45. Cameron, H. S., Szczepaniak, D., and Weston, B. W. (1995) J. Biol. Chem. 270, 20112-20122
46. Fenderson, B. A., Andrews, P. W., Nudelman, E. D., Clausen, H., and Hakomori, S. (1987) Dev. Biol. 122, 21-34
47. Eggens, I., Fenderson, B. A., Toyokuni, T., and Hakomori, S. (1989) Biochem. Biophys. Res. Commun. 158, 913-920
48. Boubelík, M., Dráberová, L., and Dráber, P. (1996) Biochem. Biophys. Res. Commun. 224, 283-288
49. Lo, S. K., Golenbock, D. T., Sass, P. M., Maskati, A., Xu, H., and Silverstein, R. L. (1997) Blood 89, 307-314
50. Bird, A. P. (1986) Nature 321, 209-213
51. Rao, V. N., Huebner, K., Isobe, M., Ar-Rushdi, A., Croce, C., and Reddy, E. S. P. (1989) Science 24, 66-70
52. Charron, M., Shaper, N. L., Rajput, B., and Shaper, J. H. (1999) Mol. Cell. Biol. 19, 5823-5832
53. Kang, R., Saito, H., Ihara, Y., Miyoshi, E., Koyama, N., Sheng, Y., and Taniguchi, N. (1996) J. Biol. Chem. 271, 26706-26712
54. Ko, J. H., Miyoshi, E., Noda, K., Ekuni, A., Kang, R., Ikeda, Y., and Taniguchi, N. (1999) J. Biol. Chem. 274, 22941-22948
55. Buckhaults, P., Chen, L., Freigen, N., and Pierce, M. (1997) J. Biol. Chem. 271, 19575-19581
56. Zhang, W., Revers, L., Pierce, M., and Schachter, H. (2000) Biochem. J. 347, 511-518
57. Gutman, A., and Wasylyk, B. (1990) EMBO J. 9, 2241-2246
58. Wasylyk, C., Gutman, A., Nicholson, R., and Wasylyk, B. (1991) EMBO J. 10, 1127-1134
59. Trimble, M. S., Xin, J., Guy, C. T., Muller, W. J., and Hassell, J. A. (1993) Oncogene 8, 3037-3042
60. Wernert, N., Gilles, F., Fafeur, V., Bouali, F., Raes, M. B., Pyke, C., Dupressoir, T., Seitz, G., Vandenbunder, B., and Stehelin, D. (1994) Cancer Res. 54, 5683-5688
61. Higashino, F., Yoshida, K., Noumi, T., Seiki, M., and Fujinaga, K. (1995) Oncogene 10, 1461-1463
62. Treisman, R. (1994) Curr. Opin. Genet. Devel. 4, 96-101
63. Macleod, K., Leprince, D., and Stehelin, D. (1992) Trends Biochem. Sci. 17, 251-256
64. Shore, P., and Sharrocks, A. D. (1995) Nucleic Acids Res. 23, 4698-4706
65. Shore, P., Whitmarsh, A. J., Bhaskaran, R., Davis, R. J., Waltho, J. P., and Sharrocks, A. D. (1996) Mol. Cell. Biol. 16, 3338-3349
66. Janknecht, R., Ernst, W. H., Pingoud, V., and Nordheim, A. (1993) EMBO J. 12, 5097-5104
67. Vossler, M. R., Yao, H., York, R. D., Pan, M.-G., Rim, C. S., and Stork, P. J. S. (1997) Cell 89, 73-82
68. Turner, H., and D., A., C. (1997) J. Exp. Med. 185, 45-53
69. Yang, S. H., Whitmarsh, A. J., Davis, R. J., and Sharrocks, A. D. (1998) EMBO J. 17, 1740-1749
70. Cuenda, A., Cohen, P., Buee-Scherrer, V., and Goedert, M. (1997) EMBO J. 16, 295-305
71. Whitmarsh, A. J., Yang, S. H., Su, M. S.-S., Sharrocks, A. D., and Davis, R. J. (1997) Mol. Cell. Biol. 17, 2360-2371
72. Yang, S. H., Yates, P. R., Whitmarsh, A. J., Davis, R. J., and Sharrocks, A. D. (1998) Mol. Cell. Biol. 18, 710-720
73. Janknecht, R., and Nordheim, A. (1992) Nucleic Acids Res. 20, 3317-3324
74. Yang, S. H., Shore, P., Willingham, N., Lakey, J. H., and Sharrocks, A. D. (1999) EMBO J. 18, 5666-5674
75. Fenderson, B. A., Zehavi, U., and Hakomori, S. (1984) J. Exp. Med. 160, 1591-1596
76. Bird, J. M., and Kimber, S. J. (1984) Dev. Biol. 104, 449-460
77. Larue, L., Ohsugi, M., Hirchenhain, J., and Kemler, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8263-8267
78. Childs, R. A., Pennington, J., Uemura, K., Scudder, P., Goodfellow, P. N., Evans, M. J., and Feizi, T. (1983) Biochem. J. 215, 491-503
79. Draber, P., and Maly, P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5798-5802

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