transcriptional activation of human leukosialin (cd43) gene by sp1 through binding to a gggtgg motif

Eur. J. Biochem. 223, 319-327 (1994) 0 FEBS 1994

Transcriptional activation of human leukosialin (CD43) gene by Spl through binding to a GGGTGG motif Shinichi KUDO and Minoru FUKUDA La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, USA

(Received April 5, 1994) - EJB 94 0456/2

Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell-type specific and differentiation-stage-specific manners. Previously we found that the sequence from - 53 to -40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483 - 84891. A transient-expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin-producing and non-producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel-mobility-shift assays. The nuclear extracts from both leukosialin-producing Jurkat cells and non-producing Hela cells showed a footprint on the 5‘ flanking region from -58 to -34. Gel-mobility-shift assays revealed that DNA- protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Spl -binding consensus sequence. Prior incubation of anti-Spl antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA-protein complex. In addition, the footprint produced by the purified Spl transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Spl to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophilu Schneider cells demonstrated that cotransfection with Spl expression plasmid increased the transcriptional activity. These results establish that Spl can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non-producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue-specific expression is probably achieved by suppression of the basal transcriptional activity in non-producing cells.

Human leukosialin (CD43) makes up the heavily glycosylated membrane protein of leukocytes and platelets [ l - 31. Although its function is unknown, data have accumulated to suggest that leukosialin is involved in signal transduction. It was demonstrated that the addition of monoclonal antibodies specific to leukosialin leads to the activation of leukocytes. Upon addition of these specific antibodies, T-cell prolifera- tion [4] and the activation of NK cells [5] and monocytes [6] were observed. One of the signals may be mediated by protein hnase C and the down-regulation of the phosphorylation was found to be associated with T-cell activation [7, 81. It is known that oligosaccharides attached to leukosialin are al- tered in patients with the Wiskoff-Aldrich syndrome, which is an X-linked recessive disorder and associated with throm- bocytopenia, eczema and severe immunodeficiency [4, 91. Recently, it was demonstrated that leukosialin on T-cells in- teracts with antigen-presenting B-cells [lo]. This interaction

Correspondence to S . Kudo or M. Fukuda, La Jolla Cancer Research Foundation, 10901 North Torrey Pines Roads, La Jolla, CA 92037, USA

Fax: +I619 450 2101. Abbreviations. CAT, chloramphenicol acetyltransferase;

DMEM, Dulbecco’s modified Eagle’s medium; PGS, promoter genomic sequence.

was found to be mediated through the binding to ICAMl expressed on B-cells [ l l ] . These observations have led to the speculation that communication between B-cells and T-cells can be mediated by ICAM1-leukosialin interaction.

Leukosialin is expressed on T-lymphocytes, granulocytes, monocytes, platelets and hematopoietic stem cells, but absent from erythrocytes [3,5, 12, 131. In the erythroid cell lineage, leukosialin is produced during an early stage of differentiation then decreases during its maturation [14]. While resting B-lymphocytes do not express leukosialin, antibody-producing B-lymphocytes and some myeloma cells do [13]. Leu- kosialin is expressed in cell-type-specific and differentiation- stage-specific manners, and its appropriate expression in hematopoietic cell lineages seems to be essential for its function. In this regard, it is noteworthy that the amplification or rearrangement of the leukosialin gene took place in some Friend leukemia cells, suggesting that the aberrant expression of leukosialin might be one mechanism of leukemogenesis

In an attempt to understand the mechanism of the leukosialin gene expression, we have investigated the transcription-regulatory region of the leukosialin gene. We found that the region immediately upstream from the transcription-start site does not possess a typical TATA box or CAAT box, but

1151.

contains a guanine-rich region in the sense strand [16]. The transient-expression assay with the leukosialin-producing Jurkat cells revealed that the 14-nucleotide sequence posi- tioned at -40 relative to the transcription-start site was critical to the promoter function [16]. In an effort to further delin- eate the transcriptional regulation of the leukosialin gene, we characterized the promoter and its binding protein. We found that the Spl transcription factor could interact with the promoter sequence and positively regulate the leukosialin gene expression. Based on these observations, the regulatory mechanism of the leukosialin gene expression is discussed.

MATERIALS AND METHODS Cell culture

Human leukemia Jurkat (T-lymphocytic), Raji (B-lymphocytic), and K562 (erythroid) cells were grown in RPMI 1640 medium supplemented with 10% or 15% fetal calf serum, 2 mM glutamine, penicillin (500 U/ml), streptomycin (100 pg/ml). Hela (epithelial) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum and the same other supplements. The Drosophila cell line Schneider 2, obtained by the courtesy of Dr R. M. Evans, was cultured in Schneider’s Drosophila medium (GIBCO BRL) with 10% fetal calf serum and 2 mM glutamine.

Chloramphenicol acetyltransferase (CAT) assay The various 5’ regions of the leukosialin gene were gen-

erated by means of the PCR [17] and were cloned into the promoterless pCAT-Basic vector (Promega) as described previously [16].

For the transient-expression assay with human cell lines, cells were plated at a density of approximately 1 .OX lo6 cells/ 100 mm-diameter dish. Equimolar amounts of the plasmids, equivalent to 10 pg pCAT-Basic (Promega), were transfected. To obtain sufficient expression of the reporter gene, the following transfection methods were utilized. Jurkat and K562 cells were transfected by the calcium phosphate method [18]. Raji cells were transfected by the DEAE- dextran method as described [19]. Hela cells were transfected by the lipofectin method [20]. As a positive-control plasmid for the transient-expression assay, pcDNACAT was constructed by cloning the CAT gene block (Pharmacia) into pcDNAI (InVitrogen), which contains a cytomegalovirus enhancer and promoter. The transfected cells were harvested after 48 h, and the CAT assay was performed as described by Gorman et al. [21].

For the transient-expression assay with Drosophila cells (Schneider line 2), 5 pg CAT constructs were cotransfected with 1 pg Spl expression plasmid, pPacSpl (kindly provided by Dr J. T. Kadonaga) or 1 pg A5C vector (obtained by the courtesy of Dr R. M. Evans). 1 .OX lo6 cells were transfected by the calcium phosphate method [18] and after 2 days, CAT activities were determined.

For a stable expression assay, equimolar amounts of the CAT constructs, equivalent to 10 yg pCAT-Basic were cotransfected into Hela cells with 2 pg pcDNANeo (InVitro- gen) by the calcium phosphate method [18]. After 2 days, the medium was changed to DMEM containing 400 yg/ml G418 (GIBCO BRL) and the cultivation was continued for 3 weeks. Around 60 visible colonies in each plate were trypsin- ized and pooled for replating on 100-mm-diameter dish. Af-

ter the cultivation, 5x10‘ cells were subjected to the CAT assay as described above. The erythroid-specific expression plasmid, pHEGCAT, which has the human P-globin enhancer and promoter, was constructed as will be described else- where.

DNase I footprinting DNA probes were obtained as follows. A DNA fragment

from -130 to t-37 nucleotide position, with respect to the transcription-initiation site of the leukosialin gene [ 161, was synthesized by PCR using synthetic oligonucleotides. The 5‘- and 3’- primers contained a-specific 24-nucleotide sequence followed by HindIII and SulI recognition sequence, respec- tively. The PCR product was cloned once into a Bluescript plasmid vector (Stratagene) and the nucleotide sequence was confirmed by dideoxynucleotide sequencing [22]. The 5’ end of the insert was cut with HindIII and labeled with [;1- ’*P]ATP and T4 kinase [23]. The insert was then excised from the plasmid by SalI digestion and used as a probe for the coding strand footprinting. The insert labeled at the SalI site as described above, was excised from the plasmid by HindIII digestion and was used as a probe for the non-coding footprinting. The nuclear extracts from Jurkat and Hela cells were prepared as described [24]. Binding reactions were car- ried out on ice for 15 min in SO 1-11 10 mM Tris/HCl pH 7.5, SO mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 2 pg herring sperm DNA, 2% poly(viny1 alcohol), 0.5-1 ng end- labeled DNA probe (around 20000cpm), and nuclear extracts or purified Spl transcription factor (Promega). MgCI, was then added to a final concentration of 2.5 mM, followed by the addition of freshly prepared DNase I (0.02-1.5 U). After incubation at room temperature for 2 min, the resulting DNA fragments were extracted with phenol, precipitated with ethanol and separated by 8% sequencing gels together with a Maxam-Gilbert sequencing [25] ladder of the same DNA probe.

Gel-mobility-shift assay Gel-mobility-shift assays were performed essentially as

described by Singh et al. [26]. A promoter genomic sequence (PGS)DNA TGGGTGGGGTGGGTGGAGCCAGGGCCC- ACT that corresponds to the sequence from -59 to -30 of the 5’ flanking region of the leukosialin gene was prepared by annealing complementary synthetic oligonucleotides and labeled with [y3*P]ATP and T4 kinase [23]. Approximately 20000 cpm of this ’2P-end-labeled probe was incubated with nuclear extracts or purified transcription factors in a 20-pl reaction of 10 mM Tris/HCl, pH7.5, 50 mM NaCI, 5 mM MgC12, 1 mM dithiothreitol, 1 mM EDTA, 2 pg herring sperm DNA and 5 % glycerol for 20 min at room temperature. The double-stranded oligonucleotides which had a transcription-factor-binding motif were used for the competition analysis. The sequences of these oligonucleotides obtained from Promega were as follows : Spl, 5’-ATTCGATC- GGGGCGGGGCGAGC-3’; NFl, 5’-CCTTTGGCATGCT- GCCAATATG-3’ ; AP1, 5’-CGCTTGATGAGTCAGCCG- GAA-3‘; AP2, 5’-GATCGAACTGACCGCCCGCGGCC- CGT-3’; AP3, 5’-CTACTGGGACTTTCCACACATC-3’. The mutated DNA fragments in the promoter were prepared from the inserts of the CAT constructs utilized in the previous study [16]. These unlabeled competitors were used in an approximately 100-fold excess against the probe for the competition assays. Purified Spl or AP2 transcription factor ob-

321

tained from Promega, was added to the reaction together with 3 pg bovine serum albumin as a carrier. The resultant reactions were loaded onto 4% polyacrylamide gel and run in OSXTBE buffer (TBE; 89 mM Tris, pH 8.3, 89 mM boric acid, 0.2 mM EDTA). Gels were fixed in 10% methanol/lO% acetic acid and dried for autoradiography.

The affinity-purified polyclonal Spl antibody purchased from Santa Cruz Biotechnology Inc. and the affinity-purified lamp-2 antibody prepared in our laboratory [27] were incubated with nuclear extracts or purified Spl transcription factor in gel-supershift assay as described [28].

RESULTS

High basal transcriptional activity of leukosialin promoter in various human cell lines

Previously we studied the transcription-regulatory region of the leukosialin gene by the transient-expression assay using the leukosialin-producing Jurkat cell line [16]. The maximum promoter activity was observed in the sequence from -91 to f 9 0 , relative to the transcription-start site. Mu- tational analyses showed that the sequence 5’-GGGTGGGT- GGAGCC-3’ from -53 to -40 was critical to the promoter function [16]. To investigate whether this leukosialin promoter can convey the cell type-specific expression in the transient-expression system, we introduced a series of CAT constructs into other cell lines (Fig. 1). K562 cell line, from which leukosialin was originally isolated, can produce a comparable amount of leukosialin [3]. In Raji B lymphocytic cells and Hela cells, expression of leukosialin was not detected by immunostaining analyses or by Northern blot hy- bridization (unpublished results). To obtain sufficient transfection efficiency, suitable transfection methods were employed for each cell line, as described in Materials and Meth- ods. Significant promoter activities were also obtained in the series of CAT constructs in those non-producing cell lines (Fig. 1). The maximum CAT activity was obtained in LS5CAT (-91 to +90), which demonstrated considerable transcriptional activity compared to that of the cytomegarovirus enhancer and promoter in pcDNACAT. The CAT constructs having further proximal regions showed barely detectable CAT activities, as previously shown in Jurkat cells (Fig. 1, [16]). The addition of a further distal region to the sequence from -91 to $90 resulted in a gradual decrease in the CAT activity in all of the cell lines analyzed (see PSCAT to LS5CAT in Fig. 1). These data showed that the leukosialin promoter confers the high basal levels of transcription in both leukosialin-producing and non-producing cells.

We extended the analysis of the other leukosialin genomic region in transient-expression system by cloning the DNA fragments in the PSCAT (-1793 to +90). Each DNA fragment of about 1 kb was synthesized by PCR, and cloned into a Hind111 or BarnHI site of this CAT construct and tested for its effect on the CAT expression in both Hela and Jurkat cells. These results showed that the region from -5.9 kb to + 5.1 kb relative to the transcription-start site, did not con- tain a regulatory element that could significantly differentiate the transcriptional level between these two cell lines (data not shown). Whereas, inclusion of the intron (+71 to +448) in the CAT construct led to a twofold increase in the CAT activity in all of the cell types studied here (data not shown, and [16]), indicating a constant enhancer function of this region.

The transcription-factor-binding site is consistent with the promoter functional region

The transcription factor(s) interacting with the leukosialin promoter was analyzed by DNase I footprinting. At first, the nuclear extract from Jurkat cells was tested with the 32P- labeled AN7 probe containing the sequence - 130 to + 37 of the leukosialin promoter. A single footprinting protected the region from -55 to -34 in the coding strand (Fig. 2A). This region is almost the same as the functional promoter sequence obtained by the transient-expression assay [16]. No other obvious protection site was detected in this experiment. A consistent result was obtained in the non-coding strand with a single footprint from -58 to -36 (Fig. 2B). In this case, a hypersensitive band was observed at the distal ends of the protection. Very similar footprinting results were obtained when the nuclear extract from Hela cells was used (Fig. 2C). Taken together with the results obtained in the transient-expression assay, it appeared that the transcription factor(s) interacting with this promoter can play a dominant role in providing high basal transcriptional activity of the leukosialin gene.

Spl transcription factor can bind to the promoter of the leukosialin gene

To characterize the transcription factor interacting with the leukosialin promoter, gel-mobility-shift assays were performed (Fig. 3). When the nuclear extract from Jurkat cells was incubated with the PGS DNA probe containing the sequence from -59 to -30 of the leukosialin gene (Fig. 5B), two shifted bands were observed (Fig. 3A). These two complex formations were abolished by the addition of an excess amount of unlabeled PGS. To determine a possible transcription factor interacting with the promoter, several oligonucleotides having different transcription-factor-binding motifs were tested for competition in this gel-mobility-shift assay. Among these oligomers, the oligonucleotide with the Spl consensus sequence significantly competed in formation of both of the complexes, and its effect was almost equivalent to that of the same unlabeled DNA fragment PGS (Fig. 3A). This competitor contained the consensus Spl-binding GGG- CGG motif [29], while the promoter had the GGGTGG- sequence in the core region. A slight reduction of complex formation was detected by means of the oligonucleotides containing the binding motif for the AP2 transcription factor, which can also recognize the G+C-rich sequences [30]. None of the other oligonucleotides having NF1-, AP1- and AP3-binding motifs showed inhibitory effects for complex formation. When the nuclear extract from Hela cells was used, a similar result was obtained (Fig. 3B), except that the AP2 consensus oligomer did not show a competitive inhibition of the binding. The transcription factor responsible for the complex formation was then compared with purified Spl and AP2 transcription factors in the gel-mobility-shift experiment. Interaction of the Spl transcription factor with the PGS probe produced a single shifted band, which migrated with the upper shifted band in the assay with Jurkat and Hela nuclear extracts (Fig. 4A). However, no obvious binding to this probe was detected by the AP2 transcription factor (Fig. 4A) in spite of the above marginal competition effect of the AP2 consensus oligonucleotide on the complex formation with Jurkat nuclear extract.

To better understand the mode of binding of the transcription factor, the footprint with Jurkat nuclear extract was com-

322

Construct Jurkat K562 Hela Raji

PSCAT (-17931+90)

LSlCAT (-952/+90)

LS2CAT (-614/+90)

LS3CAT (-2l7/+90)

Ls4CAT (-130/+00)

-AT (-91/+W)

LS7CAT (+ll+W)

Vector

pcDNACAT

u u u u u u u u CM A- CM ACCM CM AcCM CM ACCM

Fig.1. Transient expression of the leukosialin promoter in various human cell lines. A series of CAT constructs was generated by employing the PCR technique described previously [16]. Equimolar amounts of the plasmid DNAs, which were equivalent to 10 pg of promoterless pCAT-Basic (Vector), were introduced into each cell line, as described in Materials and Methods. pcDNACAT, in which the CAT gene was under the control of the cytomegarovirus enhancer and promoter, was used to trace the transfection efficiency in each cell line. After 48 h, cells were harvested and their extracts were assayed for CAT activity by separation of non-acetylated (CM) and acetylated forms (AcCM) of chloramphenicol with thin-layer chromatography. The results obtained here were reproduced in repeated experiments.

pared to that of purified Spl transcription factor. As shown in Fig. 4B, Spl and Jurkat nuclear extracts protected the identical sequence, nucleotides -58 to -36, and also exhibited the hypersensitivity site at nucleotide-58, showing indis- tinguishable binding patterns at the promoter.

To confirm the binding of Spl to the leukosialin promoter sequence, an antibody-specific to Spl was tested in the mobility-shift experiments. This affinity-purified antibody was raised against the amino acid residues 520-538 of Spl, which are adjacent to its DNA-binding domain. After the nuclear extracts or purified Spl transcription factor were incubated with this antibody preparation, they were mixed with the PGS probe and analyzed in the gel-shift assay. As shown in Fig. 4C, the addition of Spl-specific antibodies reduced the mobility of the upper band, indicating with the formation of the triple complex with DNA-Spl and Spl antibody. In contrast, the affinity-purfied antibody against human lamp-2, which is a lysosomal membrane protein, did not show any effects in the mobility of complexes. Thus, Spl was thought to be a major transcription factor interacting with the leukosialin promoter sequence.

The effect of Spl on the leukosialin promoter To investigate the relationship between the binding-speci-

ficity of Spl to the promoter and the transcription-regulatory effect, the affinity of Spl transcription factor to the mutated promoter sequence was examined by gel-mobility-shift assay (Fig. 5A). The mutated DNA fragments having dinucleotide substitutions were prepared from the CAT constructs, which were previously tested by the transient-expression assay [ 161.

The positions of these mutations are indicated in Fig. SB. In our previous study, it was shown that promoter activity gradually decreased when the mutant had substitutions to- ward the transcription-start site. The mutant RE4 (mutated at -47 and -46) gave only 12% of wild-type MT2 promoter activity. Mutants having further proximal substitutions reco- vered the activity, and RE6 (mutated at -37 and -36) and RE7 (mutated at -35 and -34) restored almost equal activity to that of wild-type MT2 [16]. In this study, 100-fold excess amounts of these DNA fragments were used as competitors for the complex formation between the purified Spl and the PGS probe. Consistent with the result of the transient-expression assay, the mutants possessing less promoter activity showed less competitive effect on binding between Spl and the PGS probe (Fig. SA). In particular, the least inhibition was observed in the mutant RE4, which had the substitution from GT to AC in the GGGTGG motif, indicating that this motif is important for Spl recognition (Fig. SB). Almost the same results were obtained when instead of Spl, nuclear extracts from Jurkat and Hela cells were used (data not shown). These results indicate that the extent of Spl- binding affinity in different oligonucleotide sequences is pro- portional to the extent of promoter activity. To further exam- ine the effect of Spl transcription factor on the transcriptional activity of the leukosialin promoter, Drosophila cells (Schneider cell line 2), known to lack endogeneous Spl [31], were used as recipient cells for the transient-expression assay. LSSCAT (-91 to +90), which provided the highest transcriptional activity in the transient assay with human cell lines (Fig. l) , was utilized. When this construct was cotransfected with Spl expression plasmid, pPacSpl , significantly

323

A. Coding

A

1 2 3 4 5 6

B. Non-Coding

A

G k 1 2 3 4 5 6

-34

1 -55

-58

1 -36

C. Non-Coding

-56

1 -36

Fig.2. Footprinting for the nuclear factor binding site on the leukosialin promoter sequence. (A) The AN7 DNA (-30 to +37), labeled at the 5’ end of the coding strand, was allowed to react with Jurkat nuclear extracts (lane 1, none; lane 2, 18 pg; lane 3, 24 pg; lane 4, 30 pg; lanes 5 and 6, 36 pg) and treated with DNase I (lane 1, 0.02 U; lane 2, 0.3 U; lane 3, 0.5 U; lane 4, 0.8 U; lane 5 U; lane 6, 1.5 U). The unlabeled AN7 DNA (X100 molar ratio to the probe) was used as a competitor for the binding (lane 6). Maxam-Gilbert sequencing ladders (G and A+G) of the labeled DNA were used for comparison. The protected region is indicated by a bracket, and the nucleotide positions are numbered from the transcription-start site. (B) The same analyses were performed on the non-coding strand of the AN7 DNA. (C) The AN7 DNA labeled at the 5‘-end of the non-coding strand was reacted with Jurkat nuclear extract (32 pg), Hela nuclear extracts (24 pg or 32 pg), and without nuclear extract (control).

higher CAT activity was observed compared with the cotransfection with only the A5C expression vector (Fig. 6). Taken together, these results strongly indicate that Spl transcription factor can positively regulate the expression of the leukosialin gene.

Regulation of the leukosialin promoter in a stable expression system

The transient-expression system is sometimes difficult to use as a representation of the natural state of a gene’s expres-

sion mainly because of the high copy number of introduced genekell. Therefore we employed the stable expression system to produce the situation close to a real gene expression. We transfected a series of the CAT constructs into Hela cells and obtained the transformants by the neomycin (G418) selection. These stably transfected cells were once pooled and subjected to the CAT assay. As shown in Fig. 7, the region from -91 to +90 possessed considerable promoter activity, and the further proximal region (-46 to +90) significantly reduced the activity as observed in the transient assay

3 24

A B

Jurkat Hela - + + + + + + + Nuclear Extract: - + + + + + + + -

Nuclear Extract - Competitor: - - Self Spl NF1 APl APZ AP3 Competitor: - - Sell slrl NF1 APl APZ AP3

Fig. 3. Complex formation with nuclear extracts and leukosialin promoter DNA, and its competition analyses. (A) Gel-mobility-shift assay with the Jurkat nuclear extract. The PGS probe (encompassing the sequence from -59 to -30 relative to the transcription-start site) was incubated with 3 pg Jurkat nuclear extract. Competition of the binding was performed with a 100-fold excess of the non-labeled fragment or oligonucleotides containing the binding motif of the indicated transcription factor. Shifted bands are indicated by arrows. (B) Gel-mobility-shift assay with the Hela nuclear extract. The same analysis was performed with 3 pg Hela nuclear extract.

(Fig. 1). Addition of further distal regions produced larger variety in the activity due to several factors, including the efficiency of integration and copy number. The results demonstrated that the region up to -1.7 kb apparently possessed the high transcriptional activity in this leukosialin non-producing cells. In this system, pHEGCAT containing the P-globin enhancer and promoter, which is functional in erythroid cells, exhibited a low level of CAT activity, This result further confirmed that this 5' flanking region did not bear a regulatory element involved in the cell-type-specific gene expression. Therefore, the leukosialin gene is probably regulated by the repression of a high basal transcription level mediated by the Spl transcription factor.

DISCUSSION In this study, we characterized the leukosialin promoter

and the transcription factor interacting with this regulatory squence. The 5' region from -91 to + 90 of the leukosialin gene could provide a high transcriptional level in both leukosialin producing and non-producing cells. In particular, the transcriptional activities in Jurkat and Raji cells in the transient-expression system were higher than that driven by the cytomegalovirus enhancer and promoter (Fig. l) , which is known as a strong regulatory segment in mammalian expression systems [32]. Here, we obtained evidence that Spl is a major transcription factor interacting with this functional regulatory sequence of the leukosialin gene.

The DNA-protein complexes formed with Jurkat and Hela nuclear extracts were effectively inhibited by oligonucleotides containing the Spl -binding motif. Purified Spl transcription factor binds to the promoter sequence in an almost identical manner as these nuclear extracts, as shown by DNase I footprinting. Mutational analyses of the promoter sequence demonstrated that there is a strong positive correla- tion between the Spl-binding affinity and the promoter activity. Cotransfection of the CAT construct with Spl expression plasmid into Drosophila Schneider cells exhibited the increase of the transcriptional activity. These results establish that Spl can bind to the promoter and positively regulates the transcription of the leukosialin gene.

During these studies, the antibody-specific to Spl was utilized to further confirm whether or not formed complexes are actually due to Spl (Fig. 4C). Although the mobility of the upper band of the complexes in the gel was clearly reduced after treatment with the antibodies, it was not apparent if the lower band migrated less after the same treatment, partly because it might migrate with the remaining upper band. To further resolve this, the nuclear extracts of Jurkat and Hela cells were tested with the antibodies by Western blotting. It was found that the antibodies reacted with two proteins, the protein with the higher molecular mass migrated to the same position as that of the purified Spl (data not shown). These results indicate that Spl proteins might have two different molecular-mass forms in the nuclear extracts utilized here. The reason for these two forms could be due to either a product of proteolytic digestion of the native mol- ecules, or to the different degree of post-transcriptional modi- fication. It was shown that Spl can be modified by O-glycosylation [33] or phosphorylation [34], both of which are thought to be involved in the regulation of Spl function.

The present study revealed that GGGTGG in the leukosialin gene promoter is the critical sequence recognized by Spl transcription factor. This core sequence is different from the consensus Spl-binding motif, GGGCGG [29]. The GGG- TGG motif was, however, reported for the Spl-binding site in the promoters of several other genes [3S-391, including the retinoblastoma control element of insulin-like growth factor I1 gene [38]. Thiesen and Bach studied the Spl-binding sequences using PCR, by amplifying the oligonucleotides after the affinity selection with the recombinant Spl transcription factor [40]. Their results showed that two out of the eleven oligonucleotides identified contained the GGGTGG motif. It has been reported that replacement of cytosine in the GGGCGG motif weakens the binding of Spl [41]. How- ever, the GGGTGG motif is repeated four times in the leukosialin promoter (Fig. 5B) and such repeats, likely to increase the affinity to Spl, could result in high transcriptional potential of this sequence.

In this study, we demonstrated that Spl can bind to the promoter and provide a high basal transcription level of the leukosialin gene. The question thus remains as to how the

325

A

Jurkat Hela spl AP2 NE NE

Protein: - = I + + u + + I n Self Competitor: - - + - + - + - +

C

SPl Jurkat NE Hela NE

B

68 I, Protein: - I + + + u + + + I I + + + I

Antlbody: - - SD1 Lamp:! - Spl Lamp2 - Spl Lamp:!

Fig. 4. Binding of Spl transcription factor to the leukosialin promoter. (A) The same DNA probe as used for Fig. 3 was used for the gel-shift assay with purified Spl transcription factor (IS ng), purified AP2 transcription factor (15 ng), Jurkat (3 pg) and Hela (3 pg) nuclear extracts. Inhibition of the binding was performed with the same unlabeled DNA (100-fold molar excess). Shifted bands are indicated by arrows. (B) The same DNA fragment in Fig. 2B and C was used for the footprinting analysis. The DNA probe was reacted with purified Spl transcription factor ( S O ng or 1.50 ng), or Jurkat nuclear extract (36 pg). The competition of the binding was performed with 100-fold molar excess of the same, unlabeled DNA fragment. (C) Reaction of Spl antibody with DNA-protein complexes, Purified Spl transcription factor (50 ng), Jurkat nuclear extract (3 pg) and Hela nuclear extract (3 pg) were incubated with an affinity-purified Spl antibody, or an affinity-purified lamp-2 antibody, before the binding reaction with the PGS probe. The samples were run as described in Materials and Methods. The open arrow indicates the band which was shifted to the band (indicated by the filled arrow) by an affinity-purified Spl antibody.

leukosialin gene is expressed in a cell-type-specific manner. Transient-expression assay with the CAT reporter gene did not reveal a regulatory element responsible for cell-type-specific expression in the region from -5.9 kb to +5.1 kb. It was conceivable that the promoter sequence is a binding site for other factors which modulate the binding of Spl. It is possible that such a factor functions as a repressor and is

present in low abundance in a non-leukosialin-producing cell line of Raji or Hela. If this is the case, the effect of a repressor might be detected in the system that provides only few copy number of an introduced genekell. To test this possi- bility, we examined the CAT constructs in the stable expression system using Hela cells (Fig. 7). This result was largely consistent with the result obtained by the transient-expres-

326

A SPl

Purified protein: - ' + + + + + + + + +

Competitor: - - MT2 RE1 RE2 RE3 RE4 RE5 RE6 RE7

4-

6

-30 EXON 1 -60 -50 -40 5'---- C--- ---TCCC .---I

I ~~ RE1 RE2 RE3 RE4 RE5 RE6 RE7

CA AC CA CA TA TA AT GT TG GT GT AT AT TA

Fig. 5. Affinity of Spl transcription factor for mutated promoter sequences. (A) The complex formation with purified Spl transcription factor and the PGS probe was competed with the 100-fold excess amount of wild (MT2) or mutated (RE1 to RE7) promoter sequences, which were excised from the CAT constructs utilized in the previous study [16]. Positions and substituted sequences of mutants are shown in Fig. 5B. The shifted band is indicated by an arrow. (B) Schematic representation around the leukosialin promoter. The sequence of the PGS (-59 to -30) used for the gel shift assay is indicated by the bracket. The mutated sites within the wild- type promoter sequence (MT2; -63 to +90) are underlined with the name of the mutant (RE1 -RE7) and substituted sequences. The protected regions in the footprinting analyses are shaded.

.I

1 CM

pPaCsp1 - + - + A!X + - f -

Fig. 6. Effect of Spl on the transcriptional activity of leukosialin promoter in Drosophila cells. CAT activity of LSSCAT (-91 to +90) and pCAT-Basic vector in Drosophila cells (Schneider cell line 2) were examined by cotransfection with Spl expression plamid, pPacSpl, or Drosophila A X expression vector. The assay was performed as described in Materials and Methods.

600 -

500 - - 8 Y

5 400 > m

.- .- I

t- 300 U 0 al .- zoo - 0 a

100

0 I

Fig. 7. Stable expression experiment in Hela cells. The CAT constructs utilized in the transient-expression assay (Fig. 1 ) were stably expressed in non-leukosialin-producing Hela cells and CAT activities were determined as described in Materials and Methods. LSSIN- CAT includes the first intron sequence at the SalI site of LSSCAT (-91 to +90), and pHEGCAT is a erythroid expression plasmid containing human p-globin enhancer and promoter. CAT activities are expressed relative to that of pcDNACAT. Each value is presented as the mean and standard deviation of three independent experiments.

sion assay. The region up to -1.7 kb apparently possessed the high transcriptional activity in this cell line. Thus, the promoter function appeared to be barely affected by such a repressor. If the chromatin structure could play a role in the- specific expression pattern of the gene, the regulatory region might be fully functional only in a native state. Further studies including in vivo footprinting experiments are re- quired to reveal the configuration of this regulatory region.

The authors wish to thank Dr J. T. Kadonaga for providing the Spl expression plasmid, Dr R. M. Evans for providing Drosophila cells and the A5C vector, Mr J. Knight for oligonucleotide synthesis, Dr Y. Kohwi, Dr T. Shigematsu-Kohwi and Dr R. Maki for helpful discussions, and Ms. Leslie Depry for typing and editing the manu- script. This work was supported by grant R01 CA33895 from the National Cancer Institute.

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transcriptional activation of human leukosialin (cd43) gene by sp1 through binding to a gggtgg motif

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