Many reports have implicated cell surface sialic acid moieties in the regulation of the immune response, as mediators of disorders, and in alteration of the metastatic potential of tumorigenic cells (for examples, see Dennis and Laferte, 1987; Piller et al., 1991; Pilatte et al., 1993; Takada et al., 1993; Umansky et al., 1996; Thomas, 1996). Of particular significance is that the B cell-restricted molecule, CD22, has a carbohydrate-binding domain that specifically recognizes [alpha]2,6-linked sialic acid residues (Bast et al., 1992; Keppler et al., 1992; Munro et al., 1992; Powell and Varki, 1994). During B-cell activation, CD22 associates with the BCR complex that includes protein tyrosine phosphatase 1C, Syk, and phospholipase C-[gamma]1 via their Src-homology 2 (SH2) domains. Together, the BCR complex triggers elevation of intracellular Ca2+ and subsequent downstream signaling events (Lankester et al., 1995; Law et al., 1996); for review see (Law et al., 1994). It is proposed that interaction of CD22 with [alpha]2,6-sialic acid ligands is a modulatory mechanism for B-cell activation and proliferation (Stamenkovic et al., 1991; Aruffo et al., 1992; Schulte et al., 1992; Tuscano et al., 1996; Cyster and Goodnow, 1997). Recognition of CD22 to [alpha]2,6-linked sialyl ligands may also participate in adhesion of B-cells to activated endothelial cells (Hanasaki et al., 1994; Hanasaki et al., 1995). B-Cell maturation is also accompanied by appearance of cell-surface [alpha]2,6-sialic acids (Erikstein et al., 1992) and induction of the cognate sialyltransferase, ST6Gal I ([beta]-galactoside [alpha]2,6-sialyltransferase, SiaT-1, ST6N, EC# 2.6.99.1). Mice unable to express ST6Gal I exhibit severely compromised B-cell proliferative response, retarded T-cell dependent and T-cell independent activation, and dramatically depressed IgM (Hennet et al., 1998).
While SIAT1, the gene encoding the human ST6Gal I, is expressed in most tissues, its level of expression varies dramatically from tissue to tissue (O'Hanlon and Lau, 1989; Kitagawa and Paulson, 1994). Differential SIAT1 expression among tissues is due largely to the existence of multiple promoters (Wang et al., 1990, 1993). Transcription from the different promoter regions results in a family of mRNAs sharing the same protein coding domains and differing among each other only at their 5[prime]-untranslated regions. In human, at least three physically distinct promoter regions exist (see Figure
Figure 1. Schematic of ST6Gal I mRNA isoforms. The exon organization of the human ST6Gal I gene, SIAT1, is shown. Shaded regions represent protein coding domains; unshaded regions represent transcribed but untranslated regions. P1, P2, and P3 are transcriptional regulatory regions for the three known SIAT1 mRNA isoforms, Form 1, Form 2, and Form 3, respectively. For example, transcription initiation from P2 yields an mRNA containing Exons X, I, II, III, etc., whereas transcription initiation from P1 yields an mRNA containing only Exons I, II, III, etc.
To understand the regulatory pathways controlling appropriate SIAT1 expression in maturing B-cells and other cell types, we have previously mapped the transcription initiation sites for Form 1 and Form 2 mRNAs (Lo and Lau, 1996). We demonstrated appropriate cellular specificity in the transient expression of reporter genes under the control of SIAT1 sequences flanking these initiation sites. Here, we have extended this study by demonstrating that multiple regulatory elements reside in both the 5[prime]-flanking sequence and in the divergent first transcribed exon unique to Form 2 mRNA, Exon X. Together, these elements confer high level expression in Louckes (a lymphoblastoid cell line of the mature B phenotype), while repressing expression in Reh (a T-null/B-null precursor cell phenotype), and HepG2 (a cell line with hepatic origins). Appropriate regulated expression of a reporter gene requires only a 495 bp SIAT1 P2 segment, comprised of 370 bp of 5[prime]-flanking sequence and the initial 125 bp of the first transcribed exon of Form 2 SIAT1 mRNA.
Specificity of the SIAT1 P2 promoter in transient transfections
The three known isoforms of human SIAT1 mRNA,Form 1, Form 2, and Form 3 are diagrammatically represented in Figure
Figure 2. Progressive truncation of the 5[prime]-flanking segment of P2 region. Schematic diagram of CAT constructs regulated by progressively 5[prime]-truncated fragments of the P2 region. Position +1 corresponds to the previously defined transcriptional initiation site (Wang et al., 1993). Shaded balloons represent putative cis-acting elements and TATA box. X represents Exon X. In pSR[alpha]CAT, P+E is the SV40 early promoter and enhancer. LTR is the human T-cell leukemia virus type-1 long terminal repeat. The expression plasmids as diagrammed were transiently introduced into Louckes cells, Reh cells, and HepG2 cells. The results are shown in the bar graph with each data set corresponding to the plasmid schematically diagrammed directly to the right. Relative CAT activity is normalized with 100% arbitrarily assigned to the CAT expression in Louckes cells from pCAT-P2(-784). This experiment was performed four times independently. The data as shown are the mean of the four experimental values. Error bar indicates the range of the experimental values. Contribution of the 5[prime]-flanking region to transcriptional regulation
The genomic segment 5[prime]-adjacent to the transcription initiation point contains regions with consensus to the TATA box (position -29), two potential NF-[kappa]B/Rel binding sites (position -703 and -296), and an AP2 site (position -357). To examine further the contribution of this 5[prime]-flanking region to P2 activity, a series of CAT expression vectors that are identical except for the progressive truncation of the 5[prime]-P2 sequence were constructed. As indicated in Figure Transcribed Exon X region is sufficient for expression in Louckes but not Reh or HepG2
pCAT-P2(-36), containing only 36 bp of 5[prime] flanking information, was further manipulated to remove the remaining 5[prime]-flanking region, the putative transcription initiation site, and the first 32 bp of transcribed Exon X sequence. The resulting construct, pCAT-P2(+32), contains only the +32 to +125 region of Exon X (Figure
Figure 3. Regulated CAT expression from Exon X sequences. X denotes sequence of Exon X from nucleotide +1 to +125 [pCAT-P2(-36)] or from nucleotide +32 to +125 [pCAT-P2(+32)] in sense-orientation. Shown at the bottom is the CAT activity in Louckes, Reh, and HepG2 cells transiently transfected with pCAT-P2(-36) (lane 1), pCAT-P2(+32) (lane 2), and pSR[alpha]CAT (lane 3).
To examine the molecular basis for pCAT-P2(+32) activity in Louckes, the +32 to +125 segment was cloned in the reverse orientation (Figure
Figure 4. Truncated 5[prime]-untranslated region encoded on Exon X displays promoter-like activity. (A) CAT fusion constructs containing truncated fragment (from position +32 to +125 of Exon X with either sense (X) or anti-sense (-X) orientations, pCAT-P2(+32) and pCAT-P2(-X), respectively, were constructed. Also constructed is a pCAT-P2(+32) derivative, pZCAT-P2(+32) with a triple polyadenylation signal (shaded arrows in diagram) inserted immediately 5[prime] of the Exon X segment. (B) CAT activity in Louckes cells transiently transfected with pCAT-P2(+32), pCAT-P2(-X), and pZCAT-P2(+32) (lane 1, 2, and 3, respectively). (C) Northern analysis of Louckes cells transiently transfected with pCAT-P2(+32) and probed for Exon X + CAT coding sequences. Mobilities of ribosomal RNAs are marked with 28S and 18S on the left. The native SIAT1 mRNA signal migrates immediately below the 28S rRNA {1655}. Arrow points to the expected mobility for CAT fusion mRNAs.
The inability of the +32 to +125 Exon X segment, when cloned in the reverse orientation, to mediate CAT expression (see Figure Contribution of sequences 3[prime] of transcription initiation site to P2 activity
To explore further the role of Exon X, the P2 region is arbitrarily divided into three segments; (1) the genomic region 5[prime] of position -36; (2) the 68 bp region (position -36 to +32) containing the TATA box, the transcriptional initiation site, and the first 32 bp of the transcribed Exon X sequence; and (3) the 94 bp Exon X region between +32 and +125. Constructs variously deleted of one or two of these segments were transiently introduced into Louckes, Reh, HepG2, and CHO cells (Figure
Figure 5. Differential contribution of the 5[prime]-flanking and Exon X segments in P2 promoter specificity. The P2 promoter region is divided into three segments: the 5[prime]-flanking region, the -36 to +32 segment containing the TATA box and transcription start site, and the +32 to +125 portion of Exon X. Shown on the left is a schematic of P2-CAT fusion constructs deleting one or two of these three segments. The expression plasmids as diagrammed were transiently introduced into Louckes cells, Reh cells, HepG2 cells, and CHO cells. The results are shown in the bar graph with each data set corresponding to the plasmid schematically diagrammed directly to the right. Each data point is the mean of four independent transfections for CHO cells and two independent transfections for Reh, Louckes, and HepG2 cells. Error bar indicates the range of the experimental values; 100% CAT relative activity is that expressed by pCAT-P2(-784) in Louckes cells (see Figure 2).
In the absence of both the -36 to +32 segment and the +32 to +125 segment [pCAT-P2(-415dTA/X)], P2-mediated CAT expression was below detectable levels in all cell lines examined, including CHO cells. This demonstrates that the 5[prime]-flanking region from position -415 to -36 alone is insufficient for regulated expression of a heterologous gene. As expected from data shown earlier (Figure
It is clear that the -36 to +32 region of P2 contains the minimal information necessary for transcription. In CHO cells, CAT expression from pCAT-P2(-36dX) was clearly detectable and only reduced by 50% compared to other constructs that contain either of the 5[prime]-flanking sequence and the +32-+125 Exon X segment or both. Nevertheless, CAT expression from pCAT-P2(-36dX) was negligible in Louckes, Reh, and HepG2 cells.
Together, the above data indicate that the +32-+125 Exon X region is necessary not only for high level expression in Louckes cells, but also contributes to cellular specificity of expression. Because this region is also the transcribed leader sequence of the Form 2 mRNA, there is the possibility that some of the observed properties may be due to posttranscriptional mechanisms. To address this issue, CAT expression in Louckes cells and Reh cells was monitored on both RNA and protein levels by Northern analyses and CAT assays, respectively. As shown in Figure
Figure 6. Contribution of the Exon X segment on the transcriptional and posttranscriptional effects. Shown on the left is a schematic of the P2-CAT fusion constructs deleting one or two of the three segments. Louckes cells and Reh cells were transiently transfected with these CAT-fusion constructs and then analyzed for their CAT expression on RNA and protein levels by Northern analyses and CAT enzyme assays, respectively; 100% CAT expression is assigned to the RNA and protein levels in Louckes cells by pCAT-P2(-370).
Past work on the mechanism of SIAT1 expression has been focused on liver where SIAT1 transcription is regulated by P1 (Wang et al., 1990; Svensson et al., 1992). The P1 promoter is utilized only in cells of hepatic and intestinal epithelial origins (Wang et al., 1990a,b; Vertino-Bell et al., 1994). Little is known about P2, the promoter utilized for the stage-specific induction of ST6Gal I and the consequent display of cell surface [alpha]2,6-linked sialic acids on maturing B-lymphocytes. Our data indicate that a 68bp segment containing the TATA box/transcriptional start site (as in pCAT-P2(-36dX)) is sufficient as a minimal promoter. however sequences necessary for lymphocyte lineage-specific expression reside 5[prime] and 3[prime] of this core 68 bp segment.
Either the 5[prime] or 3[prime] segment alone can confer expression in B-lineage cells, but not in the hepatic line HepG2. However, the highest level of expression in Louckes, a cell with the mature B-cell phenotype, can be achieved only with the presence of both 5[prime] and 3[prime] segments.
The 94 bp region (+32 to +125) of the transcribed 5[prime]-UT region encoded in Exon X was also sufficient for expression in Louckes, albeit at a substantially reduced level from constructs including sequence both 5[prime] and 3[prime] of the transcriptional start site. CAT expression under the control of the +32 to +125 P2 segment remained negligible in the precursor cell type, Reh, indicating the presence of element or elements specifying stage-specific expression in this region. The elements residing in the +32 to +125 region are not enhancer-like elements, since they did not exhibit position and orientation independence either alone or in conjunction with an SV40 promoter. At least two transcriptional regulatory properties are conferred by this 94 bp Exon X segment, an activator for expression in mature B-lymphoid cells and a repressor in immature or precursor cells of the lymphocytic lineage. This was evident by deletion of this Exon X segment which resulted in a 3-fold reduction of CAT expression in Louckes cells, but also a 3-fold activation in Reh (see pCAT-P2(-415dX) in Figure
On the RNA level, suppressed CAT expression in Reh was not alleviated upon removal of either the 5[prime] or the 3[prime] segments. This suggests that elements conferring B-developmentally specific expression reside in the 5[prime]-flanking region as well as in the transcribed Exon X segment. Quite interestingly, removal of the Exon X segment (+32 to +125) resulted in enhanced CAT enzymatic expression in both Reh and Louckes without a concomitant elevation in RNA levels. The latter is consistent with an earlier report that the divergent 5[prime]-UT regions of ST6Gal I mRNAs endow different translational efficiencies to the transcripts (Asheim et al., 1993).
Together, the data suggest that the +32 to +125 segment contributes multiple regulatory properties to the expression of ST6Gal I in B lineage cells. First, in additional to transcriptional regulation, presence of Exon X region may confer altered stability of mRNA in different cell types. Our present data cannot distinguish between altered transcription and mRNA stability as the mechanistic basis of the cellular specificity provided by Exon X. Finally, our data is also consistent with the ability of the Exon X region to alter the efficiency of the mRNA as templates for protein synthesis.
Presence of transcriptional modulatory elements in transcribed sequences is not frequently considered, although examples of this exist in the literature. For example, sequences downstream of the HIV-1 and HIV-2 promoters have been implicated in the control of basal transcription (Emerman et al., 1987). The cyanobacterial photosensibility gene, psbA, is transcriptionally regulated by a small enhancer region between the cap site and nucleotide +41 (Li and Golden, 1993). Among eukaryotic genes, silencer type elements are reported to exist within the 5[prime]-UT region of the gene for the neuron-glia cell adhesion molecule (Ng-CAM) (Kallunki et al., 1995). In the estrogen receptor gene (ER), a 75-bp region in the 5[prime]UT sequence augments expression from the ER promoter (DeConinck et al., 1995).
Tissue- and cell-specific expression of SIAT1 gene is the result of differential usage of a number of physically distinct promoter regions. Consequently, distinct mRNA isoforms, differing in their 5[prime]-UT domains, are generated. An earlier report implicated these 5[prime]-UT regions in posttranscriptional regulation (Asheim et al., 1993). Different translational efficiency was observed when the different isoforms were synthesized by in vitro translation, with Form 1 sequence being the most efficiently translated and Form 2 being less efficiently utilized. Here, we provide evidence that the Exon X region that contributes to the 5[prime]-UT sequence of Form 2 mRNA participates in specifying cellular specificity by modulating the level of mRNA. In keeping with the view that multiple and complex pathways of regulation control the expression of SIAT1 in different tissues and cell types, we provide characterization of a promoter region that confers specific regulated expression in mature B cells. Construction of plasmids
For progressively 5[prime]-truncated constructs, pSK-del22.5a, containing 784 bp sequence upstream from transcriptional initiation site and 125 bp 5[prime]-untranslated sequence (Exon X) of P2 promoter (Wang et al., 1993), was used as starting material. This plasmid was first double-digested at contiguous restriction sites ApaI and XhoI followed by the Exonuclease III/mung bean nuclease treatment with various time points to generate serial 5[prime] deletions. The deleted inserts were then released by single digestion of PstI, separated from their vector by electrophoresis, eluted and re-inserted into upstream of the CAT coding region of pCATenhancer, a promoterless CAT vector (Promega). The positive clones were screened for Exon X and sequence analyzed to determine their precise degree of deletions (see Figure Cell culture and Northern analysis
The human hepatoma cell line HepG2 and the human lymphoblastoid lines Louckes and Reh were obtained and maintained as previously described (Wang et al., 1993). The Chinese hamster ovary (CHO) cells were grown in minimum essential medium (GIBCO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µl/ml streptomycin. Total cellular RNA was prepared from cells using the guanidine isothiocyanate method (Chirgwin et al., 1979); 10 µg of each total RNA was separated on formaldehyde agarose gel (Boedtker, 1971) and electrotransferred onto nylon filter (Zetabind, Cuno Inc., Meriden, CT). For probe, a 457 bp insert fragment of pCAT-P2(+32) was generated by PCR (see Figure Transient cell transfection and transfection analysis
Plasmid DNA was prepared over double cesium chloride gradients. Transient transfection of HepG2 cells and CHO cells was performed using the calcium phosphate transfection technique; transient transfection of Louckes cells and Reh cells was accomplished by electroporation as previously described (Wang et al., 1993). Preparation of cell lysates and subsequent CAT assays were performed within 24-48 h after transfection according to Ausubel et al. (Ausubel et al., 1989) with some modifications. In brief, transfected cells were washed twice with phosphate-buffered saline (PBS), once with TEN (40 mM Tris-Cl, pH 7.5, 1 mM EDTA, pH 8.0, 150 mM NaCl), and resuspended in 100 µl of 0.25 M Tris-Cl, pH 8.0. Cells were sonicated briefly, incubated at 65°C for 10 min, and subjected to freeze-and-thaw once. The lysates were then clarified by quick centrifugation. Protein concentration in each cellular extract was determined by the Bradford reagent (Bio-Rad, Richmond, CA) using bovine serum albumin as a reference standard. Unless stated elsewhere, 20 µg of cell extract were used for each measurement for CAT activity. Each CAT assay, containing 0.25 µCi of [14C]-chloramphenicol (Amersham) and 1 µg of acetyl CoA (Sigma), was assayed at 37°C for 1 h. CAT activity was quantified by thin layer chromatography.
This work is supported by Grant GM38193 from the National Institutes of Health to J.T.Y.L. and by institute core grant CA16056-21 to Roswell Park Cancer Institute.
Discussion
Materials and methods
Acknowledgments
References
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