Background
The MUC1 gene belongs to a family of genes encoding mucin glycoproteins [
1]. MUC1 is normally expressed on the apical surface of mammary epithelial cells. However, in breast adenocarcinoma and a number of epithelial tumors, MUC1 is upregulated with aberrant expression over the entire cell surface [
1‐
4]. This characteristic makes the MUC1 protein valuable as a marker in breast cancer diagnostics and prognosis [
2]. In addition to epithelial cells, expression of MUC1 glycoprotein has been observed also in hematopoetic cells, T- and B-lymphocytes, hepatocytes, myocytes and nerve cells [
5,
6].
Molecular studies have revealed several MUC1 protein isoforms: MUC1/TM, MUC1/SEC, MUC1/Y, MUC1/X and MUC1/Z [
4,
7,
8]. The MUC1 isoforms demonstrate a diversity of properties and functions which may explain the crucial role of the MUC1 protein in many physiological and pathological processes. It participates in signal transduction, blastocyst implantation, epithelial cell morphogenesis, cellular adhesion, T-cell immunosuppression and matastases progression [
9]. Expression of the MUC1/TM, MUC1/X, MUC1/Y and MUC1/Z is associated with the presence of malignancy, whereas expression of MUC1/SEC is observed mostly in non-malignant tissue [
10].
The coordinated expression of multiple MUC1 isoforms in a wide spectrum of cells suggests a fine-tuning of mechanisms regulating MUC1 transcription in a cell- and tissue-specific manner. One of the essential steps of transcriptional regulation is binding of transcription factors to transcription cis-elements in promoter DNA. The combinatorial arrangement of cis-elements in a gene promoter is one of the important factors that determine gene transcription. For this reason, identification of cis-elements and their arrangement in a given promoter is of great importance and may provide clues to unravel potential mechanisms regulating transcription of a given gene.
The computer analysis of promoter DNA sequences is an appropriate method for identification of transcription
cis-elements. Previously, we analyzed the content and composition of transcription
cis-elements in the 3'-end 725 bp fragment of the MUC1 promoter [
11]. In this relatively short fragment we found more than one hundred
cis-elements demonstrating the high level of MUC1 promoter complexity both structurally and functionally. Nonetheless, these findings represented only a small portion of the
cis- elements operating in the full length MUC1 promoter. One of the goals of this study was to further analyze the content and arrangement of transcription factor binding sites in the full 2872 bp MUC1 promoter and to construct a MUC1
cis- element map.
Many studies of transcriptional regulation have been performed by transient transfection. Usually, in such experiments, a recombinant plasmid containing a promoter (or part of it) obtained from a gene of a given type (human, for example) and coding region (test-gene) from a gene of another type (bacterial CAT-gene, luciferase gene) is used. This artificial construct is transfected into cells (mouse, for example) that naturally do not express the gene of interest. Analysis of endogenous genes in homologous systems is more biologically relevant than these artificial set-ups for several reasons. It is known that introns play important role in transcriptional regulation [
12], however, in artificial systems they are excluded from this process. The results of transcription, as evaluated by RT-PCR, depend on the stability of nuclear RNA-transcripts that, in turn, is determined by the gene's 3'UTR [
13]. It should be underscored that usage of transfection assays with a promoter separated from its exon-intron sequences and 3'UTR, may distort the natural mechanisms of transcriptional regulation of the given gene. The inadequacy of the described system for analysis of natural regulation of transcription becomes even more obvious when properties of transcription factors of different origins are taken into account. For instance, in certain conditions the affinity and binding specificity of mouse transcription factors for ligands and
cis-elements may be different from those of corresponding human transcription factors or transcription factors of other non-relative species [
14,
15]. This may lead to aberrant transcription of a human gene in mouse cells upon transient transfection. Therefore, for obtaining more objective information on the promoter activity and transcriptional regulation, it is important to study the expression of a gene both in transiently transfected heterologous cells and in cells in which the gene of interest is expressed endogenously. In this study, we investigated the roles of different regions of the MUC1 promoter in expression of human MUC1 isoforms using transient transfection. However, in addition to ectopic transfection assays we also investigated expression of the human MUC1 gene in human epithelial cells.
MUC1 expression is predominantly hormonally regulated [
16‐
20]. Although intensively studied [
16‐
18], the mechanisms of steroid regulation of MUC1 transcription are still obscure. Estrogen increases MUC1 expression [
17], whereas the antiestrogen ICI 164,387 inhibits estradiol-stimulated MUC1 synthesis [
19]. These results imply a specific effect of estrogen and suggest that nuclear estrogen receptor(s) are likely to be involved in MUC1 transcription [
19]. However, in the mouse MUC1 promoter the estrogen receptor (ER) does not bind directly to estrogen receptor
cis-element, ERE [
18]. Among the
cis- elements found in the 725 bp fragment of the human MUC1 promoter, we have detected several sites homologous to ERE [
11]. In this study of the full MUC1 promoter, we identified an additional ERE. We analyzed the role of estrogen in regulation of the MUC1 transcription in human epithelial cells
in vivo and have investigated the potential of ER to bind
cis-ERE of the human MUC1 promoter
in vitro. We showed for the first time that estrogen differentially regulates expression of the MUC1 isoform specific mRNAs in human breast cancer cells: it activates expression of the MUC1/SEC isoform but does not affect expression of the MUC1/TM mRNA. By electrophoresis mobility shift assay (EMSA) we were able to demonstrate that ERα present in T47D cell lysate could directly bind to MUC1 promoter EREs, thus supporting the hypothesis that regulation of the MUC1 expression by estrogen may be realized by ER binding to EREs present in the MUC1 promoter.
Discussion
The expression of multiple isoforms of the MUC1 gene [
2,
4,
7,
9] in a variety of cell types [
2‐
6] and the active role of the gene in different physiological processes [
9] suggest a fine-tuned transcriptional regulation. Some specific questions that we have addressed in this study include: 1) Does the MUC1 promoter regulate differential expression of isoform specific mRNAs? 2) Is the expression of human MUC1 gene transfected into heterologous (mouse) cells relevant to its endogenous expression in homologous (human) cells? 3) How does estrogen regulate MUC1 transcription? Some of these questions have been answered in this study, others are still issues for our ongoing investigations.
Numerous overlapping and densely distributed cis-elements that potentially could bind a multitude of transcription factors demonstrate the structural and functional complexity of the MUC1 promoter. Further evidence of the MUC1 promoter complexity is the presence of several elements that participate in the development of RNA-Pol II initiation complexes (TATA-boxes, GC-boxes and initiators) and multiple transcription start sites (CAP-sites).
Usually, eukaryotic promoters that regulate transcription by RNA polymerase II belong to one of four types: 1) TATA-box-containing promoter (facultative genes) [
21]; 2) GC-box-containing promoter (housekeeping genes) [
22]; 3) Initiator-containing promoter (first described in genes expressed in B-cells) [
23]; 4) "Dual" promoter that have both TATA- and GC-elements and drive both TATA- and GC-boxes specific transcription (found in cathepsin D and methallotheonin genes) [
27]. The MUC1 promoter demonstrates properties characteristic for promoters of different types. It contains several TATA- and GC-boxes and a number of initiators (see "Additional files
1,
2,
3"). It contains multiple
cis-elements that potentially transcribe the MUC1 gene in many different cell types and tissues and
cis-elements specific for viral promoters [
2,
3,
5,
28‐
30]. These peculiarities of the MUC1 promoter allow us to classify it as a "mixed polypotent promoter". We believe that studies of eukaryotic transcription will reveal increasing number of genes with this type of promoter.
Our bioinformatic study of the MUC1 promoter resulted in construction of the MUC1
cis-element map. This map has several advantages. Using the map, one may design a functional analysis of transcription factors potentially competing for overlapping
cis-elements in the MUC1 promoter. It also allows prediction of cells in which the MUC1 gene might be expressed. Based on the content of the MUC1
cis-elements, we predicted expression of the MUC1 gene in lymphoid and muscle cells that had been thought to be MUC1 non-producing cells. Subsequently, the MUC1 expression in these cells was confirmed experimentally [
5]. Obviously, in a single study it is impossible to evaluate functional activities of all
cis-elements detected in the MUC1 promoter. Nevertheless, several
cis-elements that were identified by our bioinformatic approach (MZF-1, Sp1 and STAT) have been already found active in transcriptional regulation of the MUC1 gene
in vivo [
28‐
30].
Transient transfection of plasmids that have different deletions within the promoter DNA can identify promoter regions necessary for the expression of specific mRNA isoforms. We combined this approach with our bioinformatics information to experimentally characterize the MUC1 promoter activity. In our study, the Dpr plasmid that contained a full-length MUC1 promoter supported expression only of the MUC1/SEC isoform (Fig.
1B, lane – 1). In contrast, the plasmid DprΔ2154 lacking a ~2 kb fragment deleted from the 5'-end of the promoter demonstrated higher activity and supported expression of both MUC1/SEC and MUC1/TM mRNA (Fig.
1B, lanes – 4 and 5, respectively), indicating that within the deleted fragment are transcriptional repressor binding sites. Our
cis-element map supports this hypothesis, since the deleted fragment of the MUC1 promoter (-2872/-718) contains multiple binding sites for transcriptional repressors: 9 sites for δEF1-repressor (-2180/-2170, -1546/-1536, -1522/-1512, -1486/-1476, -1462/-1452, -1418/-1408, -1175/-1165, -1029/-1019 and -1008/-998), 7 sites specific for the Gfi-1 repressor (-2627/-2515, -2589/-2574, -2530/-2515, -1738/-1723, -1435/-1420, -1306/-1291 and -1141/-1126), 5 sites specific for YY1-repressor (-2786/-2776, -2772/-2762, -1428/-1418, -1395/-1385 and 1203-1193) and a single ELP repressor binding site (-1172/-1165) (see "Additional files
1,
2").
Plasmid DprΔ2446, lacking an additional 292 bp, elevates MUC1/TM expression but leads to a decrease in the expression of the MUC1/SEC mRNA compared to DprΔ2154 (Fig
1B, lanes – 7 and 8). These observations suggest that the deleted 292 bp fragment might contain both additional sites for MUC1/TM mRNA repressors as well as sites important for MUC1/SEC expression. Indeed, as the map shows, the deleted fragment contains
cis- elements specific for two repressors, YY1- (-670/-660) and δEF1- (-472/-462), in addition to several
cis-elements specific for mammary epithelial cell activators: CTF/NF, ESE1, MAF, MGF, MP4 and RME [
31‐
34] (see "
Additional file 3").
According to results obtained with the DprΔ2839 plasmid, a minimal set of transcription elements (one TATA-box, one CAP-site and several
cis-elements specific for transcriptional activators RCE, SRE and ETF-RE) (see "
Additional file 3") is sufficient for expression of MUC1/SEC isoform (Fig.
1B, lane – 10). Transcriptional activity of the minimal MUC1 promoter has been observed in transfection studies [
35,
36]. Notably, two very different plasmids, one of which contains the full MUC1 promoter (Dpr) and the other which contains only a minimal promoter sequence (DprΔ2839), drive similar expression of the MUC1/SEC isoform. It appears that in our experimental conditions, the positive and negative effects of the
cis-elements in the full promoter seem to balance-out each other resulting in expression of MUC1/SEC similar to that of the minimal promoter.
The results demonstrating higher activity of the truncated promoter (DprΔ2154, DprΔ2446) compared to the full length MUC1 promoter (Dpr) are not surprising. Earlier, Kovaric et al [
36] showed that expression of the CAT-test gene driven by the full length MUC1 promoter was almost half than that directed by 743 bp promoter fragment. Abe and Kufe [
35] also observed higher CAT activity when the test-gene was driven by a smaller promoter fragment (-686/+33) than by a larger one (-1656/+33). Moreover, according to these authors, the 114 bp fragment of the MUC1 promoter located between -598 and -485 bp demonstrated the highest CAT gene expression. Superimposition of the promoter fragments analyzed in these studies and our MUC1
cis- element map leads to similar schematics of activating and repressing regions.
One of the issues addressed in our research was whether the expression of the human MUC1 gene transfected into heterologous (mouse) cells is relevant to its endogenous expression in homologous (human) cells. The results described in this study showed that in our experimental conditions, the patterns of the MUC1 gene expression in both cell systems were different. In mouse cells, human MUC1 gene (Dpr plasmid) directed expression of the MUC1/SEC isoform, whereas in human cells, the predominant mRNA was MUC1/TM. In transfected mouse DA3 cells, we could not detect human MUC1/Y isoform, whereas in human T47D and MCF7 cells this isoform was observed. The basis for such differential expression is presently being studied.
One of the important issues in regulation of MUC1 expression is the role of estrogen and estrogen receptors. Several studies have showed that the MUC1 gene positively responds to estrogen [
16‐
19]. However, in our study, we observed that not all MUC1 isoforms responded to estrogen in the same manner. The MUC1/SEC mRNA was expressed in T47D cells (ER+ clone 10) only after treatment with estrogen. In contrast to T47D cells, in ER-positive MCF7 cells, expression of the MUC1/SEC mRNA was observed in the absence of estrogen, although, similarly to T47D cells, estrogen increased and 4-OHT decreased its expression. The MUC1/TM mRNA could be expressed both in T47D estrogen receptor positive cells (clone 10) and estrogen receptor negative cells (clone 8) as well as in MCF7 cells. Moreover, this expression was not affected by 4-OHT. The dependence of the MUC1/Y isoform expression on estrogen and ER was not clear cut. On one hand, we observed some increase in MUC1/Y expression in T47D and MCF7 cells after estrogen treatment but on the other hand, 4-OHT did not inhibit its expression. Further experiments will be needed to clarify this matter.
The above data suggest that the expression of MUC1 isoforms in MCF7 cells somehow differs from their expression in T47D cells. Perhaps relevant to our observations is that MCF7 and T47D cells express different levels of steroid receptors. In MCF7 cells, ER is expressed at much higher levels than progesterone receptors (PR), whereas in T47D cells, the expression of PR is higher than that of ER [
37,
38]. Since estrogen regulates the transcription of the ER gene [
39], it appears that T47D cells may require exogenous supplements of estrogen to activate expression of the ER gene. In contrast, the endogenous expression levels of estrogen receptors in MCF7 cells might be high enough to support expression of the MUC1/SEC isoform. In accordance with our results, Hurd et al [
40] observed expression of hyperphosphorylated retinoblastoma protein (ppRB) in MCF7 but not in T47D cells when cells incubated without estrogen. A gradual increase of its expression after treatment with estrogen was observed in MCF7 cells in time-dependent manner. In T47D cells, longer estrogen treatment was necessary to detect ppRB than in MCF7 cells.
Our data demonstrating the responsiveness of the MUC1/SEC isoform expression in human epithelial cells to inhibitory effects of 4-OHT are in agreement with observations that the expression of MUC1 gene in human adenocarcinoma cells is also sensitive to antiestrogens [
41,
42]. These data suggest that, in human cells, estrogen may regulate the MUC1 gene transcription by interaction with ER directing them to
cis- ERE in MUC1 promoter. The results obtained by us with EMSA using T47D cell lysates support this hypothesis. However, they are in contradiction with the observations made in a mouse system. Studying the role of ER in transcriptional regulation of the mouse Muc1 gene, Zhou et al [
18] concluded that ER did not directly bind to the
cis- ERE of the murine Muc1 promoter. We found that all
cis-ERE detected in the human MUC1 promoter could form complexes with human ERα
in vitro. Several factors may explain this discrepancy. First, different
cis-EREs were used in both studies. Although human and mouse MUC1 promoters have high degree of homology, their
cis-EREs demonstrate pronounced diversity. We have analyzed the mouse Muc1 and human MUC1 promoter sequences and found that each promoter contains six
cis-EREs. Comparison of these elements revealed both homology and differences in their sequences. Second, Zhou et al [
18] analyzed binding of estrogen receptors that have been
in vitro translated, whereas in our binding assays we used ERα endogenously synthesized in T47D cells that express human MUC1 gene.
It should be noted, that, although all MUC1
cis-EREs bound ERα, the properties of the complexes developed by different
cis-EREs were different. Several
cis-EREs, (ERE1, ERE3, ERE4 and ERE5) containing only half of the classical palindrome sequence produced a weak, fast migrating complex with ERα. Interestingly, ERE2, which also contains only half of the ERE palindrome sequence, developed two complexes that at least partially correspond to those observed with classical ERE from the vitellogenin gene and with "
putative" ERE-6 of the MUC1 gene. The different electrophoretic mobility of the complexes might be explained by content of ER-cofactors in the complexes. It is not clear why oligonucleotides that have identical or very similar ERE core sequences recruit different cofactors to ER-containing complexes, however, the flanking sequences may play a crucial role in this process [
43]. Importantly, all complexes developed with tested
cis-ERE contained ERα since the binding of ERα was specific and could be inhibited by antibodies developed against the DNA-binding domain of human ERα.
Whereas our
in vitro studies clearly showed that physical binding of ERα with
cis- EREs of the MUC1 promoter occurs, definitive
in vivo binding still remains to be proven. Orientation of ERE within the synthetic oligonucleotides appears not to be important for
in vitro binding, but orientation and distribution of EREs among
cis-elements within promoter, are presumably crucial for proper
in vivo binding [
44‐
46]. A special approach will be needed to study this issue. In the MUC1 promoter, two estrogen responsive elements (ERE-1 and the "putative" ERE-6), have direct sequence orientation while others (ERE-2, ERE-3, ERE-4 and ERE-5) have opposite orientation. Three elements (ERE-1, ERE-2 and ERE-3) are located relatively distant from the active TATA-box and from each other. Three others (ERE-4, ERE-5 and the "putative" ERE-6) are located in close proximity to each other between nucleotides -389 and -337 (see "Additional files
1,
2,
3") and may function as an estrogen responsive unit [
47,
48].
Very few mRNAs that are known to be directly regulated by estrogen in mammary epithelial cells are actually induced via canonical EREs [
49]. In fact, most estrogen-responsive genes identified to date contain one or more imperfect EREs or multiple copies of an ERE half-site rather than the classical ERE [
50,
51]. The MUC1
cis-EREs also are not absolutely identical to the consensus sequence. Although the affinity of the estrogen receptor for the classical ERE is higher than for imperfect ERE-like sequences, most of the imperfect EREs bind ER [
52]. The MUC1
cis-EREs, although imperfect, also bound ER
in vitro. It is becoming clear that EREs function as allosteric modulators of ER conformation [
53,
54]. The conformational changes in ER induced by individual ERE sequences lead to specific association of the receptor with other transcription factors and assist in differential transcription of estrogen-responsive genes [
54,
55]. In light of these data, the imperfect MUC1
cis-EREs might be significant, perhaps by differential usage of ERE in diverse cells recruiting distinct cofactors for the MUC1 expression.
We have discussed the possible involvement of ER and MUC1 EREs in regulation of the MUC1 gene transcription. However, it is known that ER may regulate gene transcription also by interaction with other transcription factors (STAT, AP1, EGFR or NFkB) without direct binding to ERE [
56]. Further studies are needed to understand the process of MUC1 transcription
in vivo and to elucidate the mechanism by which estrogen activates transcription of the MUC1 gene.
Although our study revealed some new and important features of the MUC1 promoter
cis-element content and structure, the precise mechanisms by which these
cis-sites are involved in the regulation of MUC1 expression have not been fully elucidated. On one hand, elements within the promoter could determine usage of different transcription start sites specific for individual MUC1 isoforms. The presence of multiple CAP-sites in the MUC1 promoter together with previously documented multiple transcription start sites of the MUC1 gene in T47D cells [
11] support this hypothesis. On the other hand, the promoter
cis-elements might be involved in regulating of alternative splicing of a single pre-mRNA common to all MUC1 isoforms. A growing body of evidence suggests that transcription and splicing are highly coordinated processes both at the structural and functional levels [
57‐
61]. For instance, it has been shown that mutations introduced into promoter
cis-elements could change the alternative splicing patterns [
58]. Moreover, the RNA pol II large subunit physically associates with spliceosomes and the SR proteins that regulate alternative splicing act through specific promoter occupation [
62].
In light of these data, we suggest that cis-elements of the MUC1 promoter may be involved in mechanisms that regulate both transcription and splicing of the MUC1 pre-mRNAs. In this study, we used total RNA extracted from transfected cells. However, for a better understanding of the role of the MUC1 promoter in transcription and splicing of MUC1 isoforms, an additional study of the 5'-ends of nuclear pre-mRNA is needed. Additionally, the effect of different mutations within cis-sites of the MUC1 promoter on isoform expression could be more thoroughly dissected. These experiments are currently in progress.