Transcriptional regulation of the hamster Muc1 gene: identification of a putative negative regulatory element

Insong James Lee, Sang Won Hyun, Asit Nandi, and K. Chul Kim

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mucin gene Muc1 is expressed in glandular epithelial cells and is involved in lubricative and protective functions. It is also overexpressed in many carcinomas including breast and lung cancer cells. To study the transcriptional regulation of Muc1, we cloned a 2.4-kb fragment containing the promoter region of the hamster Muc1 gene and analyzed it for its ability to mediate transcription. Transcriptional initiation was localized to 22 base pairs downstream of the TATA box. We performed functional analysis of the Muc1 promoter in hamster (HP-1 and Chinese hamster ovary) and human cells (MCF-7, A549, and BEAS-2B) using deletion/reporter constructs. A positive regulatory region between bases -555 and -252 and a putative negative regulatory element (P-NRE) between nucleotides -1,652 and -1,614 were found to be active in transfected cells. The P-NRE contains a yin yang 1 (YY1) transcription factor binding site, and electrophoretic mobility shift assays with HP-1 cell nuclear extract revealed the binding of YY1 to this site. Our data suggest that YY1 may play an inhibitory role in the transcription of the Muc1 gene.

promoter; expression; negative regulatory region; protein binding; yin yang 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE GENE ENCODING THE TRANSMEMBRANE glycoprotein MUC1 was first identified in human breast cancer cells (MUC1 in humans and Muc1 in nonhuman species) (44). MUC1 is normally expressed in secretory epithelial cells of the respiratory, gastrointestinal, and female reproductive tracts as well as in lymphoid cells (6, 12, 16, 17, 21, 33, 36) and is highly overexpressed in most carcinomas (12, 13, 40) including those of the breast, ovary, pancreas, stomach, colon, and lung. Tumors induced by polyoma virus middle T antigen in Muc1-null mice develop more slowly than in their wild-type counterparts (45). Furthermore, MUC1 has been shown to be capable of acting as an antiadhesion molecule (16, 19, 23) and may promote metastasis. Although the function of MUC1 is not completely understood, MUC1 has been shown to be involved in acquired immunity, cell adhesion, and maintenance of normal cellular function and development (12, 13, 19). We have recently reported that MUC1 may act as a receptor for Pseudomonas aeruginosa (27), and furthermore, it has been reported that MUC1 is upregulated in cystic fibrosis cells (14), suggesting that MUC1 might play a role in the pathogenesis of cystic fibrosis.

Expression of MUC1 has been shown to be influenced by a number of factors, including hormones and retinoids (5, 18, 20, 29, 33, 34, 47), cytokines (9, 11), tyrosine kinase inhibitors (14, 50), and possibly by gene modifications such as DNA methylation or DNA conformational status (4, 31, 32, 52). Although the molecular mechanisms of the regulation of MUC1 transcription by these factors are not well defined, cis-acting elements within the MUC1 promoter mediating the effects have been identified. (1, 11, 24, 25, 43). MUC1 promoter regions containing some of these elements have been cloned upstream of heterologous genes and have been shown to be able to drive their expression when transfected into MUC1-expressing cancer cell lines. These gene constructs are being considered for possible targeting of genes to carcinomas for gene therapy (7, 28, 39). We recently reported the synthesis of Muc1 mucin by primary hamster tracheal surface epithelial cells (33, 35) and that the expression of Muc1 was density dependent and closely associated with goblet cell differentiation (33). Muc1 transcript levels increased threefold in dense but preconfluent cells and sixfold in confluent cells when compared with sparsely plated, actively growing cells. In an attempt to understand the regulatory mechanisms responsible for these observations, we isolated the 5'-flanking region of the Muc1 gene and characterized the promoter. Here, we describe the identification of both a positive regulatory element located between bases -555 and -252 and a putative negative regulatory element (P-NRE) located between -1,652 and -1,615 relative to the transcriptional initiation site. We demonstrate, using electrophoretic mobility shift assays (EMSA), that the yin yang 1 (YY1) transcription factor binds to the P-NRE. Our results suggest that Muc1 expression is regulated through both positive and negative pathways and that YY1 may be involved in the transcriptional repression of the Muc1 gene.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Cell culture. Human MCF-7 breast cancer and hamster HP-1 pancreatic cancer cells were grown in minimum essential medium with 10% fetal bovine serum (FBS). Human A549 lung adenocarcinoma cells were grown in Dulbecco's modified Eagle's medium with 10% FBS. Human BEAS-2B bronchial epithelial and Chinese hamster ovary (CHO) cells were grown in F-12-Dulbecco's modified Eagle's medium with 5% FBS.

Isolation and sequence of the hamster Muc1 promoter region. A hamster genomic DNA library made from the liver of an 8-wk-old male golden hamster (Stratagene, La Jolla, CA) was screened according to the manufacturer's protocol using a 1.5-kb hamster Muc1 cDNA probe (33). Positive plaques were isolated, and the phage was purified to homogeneity by repeated screening. Inserts from clones that likely contained the Muc1 promoter, as suggested by partial restriction mapping, were isolated by EcoRI digestion and subcloned into pBluescript KS+ (Stratagene). A clone was shown to contain up to a 2.3-kb region upstream of the Muc1 gene and the 3'-end of the hamster thrombospondin 3 gene as well as the 5'-untranslated region of the Muc1 gene (pMuc1pro-pBS) by sequencing (Biopolymer Core Facility, University of Maryland at Baltimore). We identified potential transcription factor binding sites by searching the Transfec database (51) with the MatInspector program (37), available through the internet.

Primer extension. An oligonucleotide complementary to the sense strand overlapping the ATG translational start site was synthesized and designated PEP-1: 5'-GCCCGGATGCCTGGGGTCATGA-3'. This oligonucleotide was used in primer extension analysis as described with modifications (41). Total RNA was prepared from either hamster tracheal tissue or cultured hamster pancreatic cancer cells (HP-1) using Trizol (8) (Invitrogen, Carlsbad, CA). PEP-1 was end-labeled with [32P]ATP (3,000 Ci/mmol) (NEN, Boston, MA) using Polynucleotide kinase (New England Biolabs, Waverly, MA), and then the labeled PEP-1 [2.1 × 106 counts per min (cpm)] was mixed with 20 µg of total RNA. This mix was incubated at 65°C for 90 min and then slowly cooled to 40°C. Extension was carried out in a reaction containing 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 200 µM dNTPs, 1 mM DTT, 0.13 mg/ml actinomycin D (Sigma, St. Louis, MO), and reverse transcriptase (Superscript; Invitrogen) at 42°C for 1 h. Extension products were resolved on a 6% 8 M urea acrylamide gel.

Preparation of Muc1 promoter/luciferase constructs and luciferase assay. Promoter deletion/luciferase (luc) constructs were prepared by digesting pMuc1pro-pBS with BspHI and then treating with Klenow enzyme (New England Biolabs) to get blunt ends. Then the plasmid was digested with SacI, and the fragment was purified and cloned into the pGL2-Basic vector (Promega, Madison, WI) prepared as described below. The plasmid pGL2-Basic was digested with XhoI and then treated with Klenow enzyme to make blunt ends and then digested with SacI. The resulting construct was designated as pMuc1-2354-luc. Subsequent deletion constructs were synthesized by digestion of pMuc1-2354-luc with SacI and one of the following restriction enzymes: SmaI, DraI, NcoI, PstI, BstXI, or ApaI, and treatment with mung bean nuclease and then religation and subsequent transfection into competent STBL cells (Invitrogen).

The Muc1 promoter/luciferase construct lacking the P-NRE was constructed as follows: pMuc1-2354-luc was digested with NcoI and then treated with mung bean nuclease. This plasmid was then digested with SacI, and the large 7.8-kb fragment was isolated. Another sample of pMuc1-2354-luc was digested with SacI and HindIII, and the 2.4-kb fragment was isolated. This fragment was digested with DraI, and the 699-bp fragment was isolated. This fragment was ligated to the 7.8-kb fragment prepared as described above to get a plasmid identical to pMuc1-2354-luc with the exception of the absence of bases -1,651 to -1,615 and designated as pMuc1-2354Delta P-NRE-luc.

To determine the effect of the P-NRE on transcription from a heterologous promoter, we cloned one or two copies of the P-NRE upstream of the SV40 promoter connected to a luciferase reporter gene. The plasmid pGL2-Control (pGL2-C; Promega) was digested with BglII and treated with Klenow enzyme to make blunt ends. This plasmid was then digested with calf intestinal phosphatase (New England Biolabs) to remove the phosphate groups and prevent religation. Oligonucleotides representing sense and antisense strands of the 37-bp P-NRE region (sense strand), 5'-AAATACTCAGACTGAGAGGCGCGGAGCTATTGCCATG-3', or the scrambled version, P-NRE-mut (sense strand): 5'-TAACAGCTGCATGGATGCTGTCACGACCGATTCAAGG-3', were allowed to anneal and were ligated to each other to create multimers. The ligated oligonucleotides were purified and then annealed to pGL2-C prepared as described above. The plasmids were digested with HindIII and KpnI to test for P-NRE or P-NRE-mutant (mut) inserts. Clones containing inserts were sequenced to confirm proper sequence and orientation of the P-NRE and P-NRE-mut. The constructs synthesized as described above were transiently transfected into human and hamster cells with SuperFect (Qiagen, Valencia, CA) according to the manufacturer's protocol. Briefly, cells were incubated with a mixture of plasmid DNA and SuperFect for 3 h at 37°C in 5% CO2, after which the transfection mixture was removed and replaced with media. The cells were grown for an additional 48 h and then lysed in reporter lysis buffer (Promega). Cells were scraped off and transferred to microcentrifuge tubes and subjected to three freeze-thaw cycles, after which the tube was vortexed for 20 s and then centrifuged for 3 min at 13,200 relative centrifugal force (RCF). The supernatant was used to measure luciferase activity with luciferase assay system kits (Promega).

EMSA. Nuclear extracts were prepared from HP-1 cells by a modification of the procedure of Andrews and Faller (2). Briefly, cells were washed twice with ice-cold PBS and lysed by resuspension in buffer A [10 mM HEPES-KOH, pH 7.8, 1.5 mM MgCl2, 10 mM NaCl, 1 mM dithiothreitol (DTT), 0.25% Igepal CA-630, 0.2 mM phenylmethylsulfonyl fluoride (PMSF)] and letting them sit on ice for 10 min. Nuclei were pelleted by centrifugation (1 min at 1,250 RCF), and the supernatant was removed. Proteins from nuclei were extracted in buffer B (20 mM HEPES-KOH, pH 7.8, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and 0.2 mM PMSF) for 25 min on ice. This mix was then centrifuged at 13,200 RCF for 5 min. The supernatant was collected and dialyzed in 500× volume of buffer C (20 mM HEPES-KOH, pH 7.8, 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 1 mM DTT, and 0.2 mM PMSF) for ~2 h. Protein concentration was measured by the method of Bradford (Bio-Rad, Richmond, CA), and extracts were stored at -80°C until use. Twenty micrograms of nuclear extract were incubated with 32P end-labeled synthetic P-NRE-47 oligonucleotide (sense): 5'-AAATACTCAGACTGAGAGGCGCGGAGCTATTGCCATGGTGACTGGTA-3' in a reaction buffer containing 10 mM HEPES-KOH, pH 7.8, 2 mM MgCl2, 0.1 mM EDTA, pH 8.0, 60 mM KCl, 10% glycerol, and 3.0 µg poly(di-dC)-(di-dC) (Pharmacia, Piscataway, NJ) for 20 min at room temperature. The reaction was then put on ice for 5 min and then run on a 4.5% polyacrylamide gel at 4°C in 0.25× Tris-borate-EDTA buffer and 1% glycerol and analyzed by autoradiography. In some experiments, 37- or 74-fold molar excess of unlabeled oligonucleotide competitors (underlined letters indicate altered sites in mutated oligonucleotides) as listed below or antibodies to YY1 (Santa Cruz Biotechnology, Santa Cruz, CA) were added to verify binding of YY1 protein. Competitor oligonucleotides were (sense strand): P-NRE-47-YY1-mut, 5'-AAATACTCAGACTGAGAGGCGCGGAGCTATTGCGGTGGTGACTGG- TA-3'; YY1-con, 5'-ACGACGCCATTTTGAGTGTGCA-3'; YY1-mut, 5'-ACGACGCGGTTTTGAGTGTGCA-3'; p53-YY1, 5'-ACTTGTCATGGCGACTGTCCAGCTTTGTGC-3'.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Sequence of the hamster Muc1 gene 5'-flanking region. By screening a hamster genomic DNA library with a 1.5-kb Muc1 cDNA (33) we isolated a 2.4-kb fragment containing all of the 5'-flanking region of the Muc1 gene. This fragment (nucleotide -2,354 to +72, numbered relative to the transcriptional initiation site) begins 106 bp upstream of the polyA signal of the thrombospondin 3 gene and ends at the ATG initiation codon of the Muc1 gene (Fig. 1). When we compared the sequence of the hamster promoter region encompassing the transcriptional initiation site and 1,999 bp upstream with the corresponding region of the mouse Muc1 promoter (49) using the pairwise blast program (National Center for Biotechnology Information), an 81% identity was seen with the gap extension penalty set to 1, the gap open penalty set to 1, and mismatch penalty set to 1. When the same parameters were used to compare hamster Muc1 promoter to the human MUC1 promoter (26), an identity of 68% within the first 1,101 bp in human and 1,093 bp in hamster was seen. However, the next 294 bp upstream of the human MUC1 promoter ranging from -1,102 to -1,395 have no detectable similarity to the hamster or the mouse Muc1 promoters. This region, however, is present with high similarity (98%) in gibbon Muc1 promoter (46). Immediately upstream of this 294-bp region, the similarity between the hamster Muc1 and human MUC1 promoters continues at 61% identity for an additional 530 bp. Thus there seems to be an extra 294-bp region present in the human and gibbon promoters that is absent in both the hamster and mice Muc1 promoters. This extra sequence may have arisen out of an insertion event. Accordingly, the distance from the initiation site of the human MUC1 gene to the polyA signal of the thrombospondin 3 gene, which is located immediately upstream of the MUC1 gene, is 2,660 bp, whereas in the hamster, the distance is 2,244 bp and in the mouse, 2,229 bp. The extra 122 bp (compared with the distance in the hamster Muc1 5'-flanking region) of the distance between the transcriptional initiation site of the MUC1 and the polyA signal of the thrombospondin 3 gene not accounted for by the 292-bp region may arise from additional insertion or duplication events. The TATA boxes of the MUC1/Muc1 promoter from the four aforementioned species were identical (5'-TATAAA-3'). By primer extension, using RNA from hamster trachea tissue, we mapped the transcription initiation to a cytosine residue located 22 bp downstream from the most 3'-A of the TATA box (data not shown). This distance is identical to that reported for mouse (49) and one nucleotide shorter than that reported for human (26). However, when RNA from the hamster pancreatic tumor cell line HP-1 was used for primer extension, an alternative transcriptional start site 37 bp downstream from the TATA box was seen (Fig. 2). Because the distance between the TATA box and the transcriptional start site is 22 bp for the mouse and 23 bp for the human and because we believe the hamster tracheal tissue is more reflective of in vivo conditions, we have decided to use the initiation site determined in the hamster primary tracheal tissue to number nucleotides of the hamster Muc1 promoter. No other significant bands except for a weak band 41 bp downstream from the TATA box were observed in the primer extension assays, indicating that the sites 22 bp and 37 bp downstream from the TATA box are the major transcriptional start sites in hamster tissue cells and HP-1 cells, respectively. Sequence search revealed a 100% homology to three previously characterized transcriptional regulatory elements (Fig. 1) described in the human MUC1 gene promoter (1, 24, 43) as well as several potential transcription factor binding sites.


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Fig. 1.   Nucleotide sequence analysis of hamster Muc1 gene 5'-flanking region. A total of 2,424 bp of the 5'-flanking region and the translational initiation site codon ATG of the hamster Muc1 gene is illustrated. The polyadenylation signal (CATAAA) of the thrombospondin 3 gene and the TATA sequence (TATAAA) of the Muc1 gene are shown in boxes. The transcription initiation site is indicated with an arrowhead, and the ATG start codon at the 3'-end of the sequence is underlined. Several potential transcription factor binding sites are underlined and boldfaced. The starting nucleotide of each of the region of the promoter fused to the luciferase reporter cDNA to prepare the deletion constructs are indicated by broken arrows and marked as M, S, D, N, P, B, or A. The bases within the region responsible for negative regulation [putative negative regulatory region (P-NRE), from -1,651 to -1,615] is italicized and underlined. Underlined regions with #1, #2, and #3 written underneath represent regulatory elements reported by Shirotani et al. (43), Abe and Kufe (1), and Kovarik et al. (25), respectively. Nuclear factor (NF)-kappa B, activator protein (AP)-1, cAMP response element binding protein (CREB), growth hormone-thyroid responsive element (GH-TRE), and NF-1 binding sites were identified by database search.



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Fig. 2.   Identification of the transcription initiation site of the hamster Muc1 gene. RNA from hamster HP-1 cells was reverse transcribed using the labeled primer PEP-1 (sequence in METHODS) and resolved in a 6% acrylamide gel. Larger arrow at right, the position of a 75-nucleotides-long primer extension product; smaller arrow, a 71-nucleotide minor product. M13seq, M13 mp18 sequencing ladder; PE, primer extension product; m, size markers (10-bp ladder).

Functional promoter analysis. Functional analysis of the Muc1 gene 5'-flanking region was carried out by the construction of various deletion/luciferase reporter plasmids followed by transfection into a Muc1-expressing hamster cell line, HP-1 cells. Although HP-1 cells were originally reported as lacking Muc1 protein expression on the basis of Western blot analysis (3), we identified Muc1 mRNA in these cells by reverse transcriptase-PCR (data not shown). Seven deletion constructs containing varying regions of the Muc1 promoter as shown in Fig. 3 were tested. Two regions were identified as affecting the expression of the luciferase reporter gene. The first region is located between nucleotides -555 and -252 and contains a positive response region. Removal of this region resulted in a decrease in transcription by 50-70% in the human cells and 80% in the hamster cells. This region is highly conserved among the hamster, mouse, and human muc1 and MUC1 promoters and contains sequences identical to previously characterized positive cis-acting elements in the human MUC1 promoter. The second element is located in between -1,652 and -1,614, and the removal of this region resulted in a 1.8- to 2.7-fold increase in transcription in human cells and a 2.3-fold increase in HP-1 cells. This 37-bp region was active in hamster HP-1 but not in CHO cells (Fig. 3A), and it was active in all of the three human cells tested (Fig. 3B).


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Fig. 3.   Analysis of hamster Muc1 gene promoter activity. Hamster [HP-1, Chinese hamster ovary (CHO)] and human (MCF-7, BEAS-2B, A549) cells were transiently transfected with deletion constructs prepared by cloning various regions of the 5'-flanking region of the Muc1 gene to a luciferase cDNA (pGL2-Basic), and luciferase activity was determined 48 h after transfection. Top: schematic diagram of the full-length Muc1-promoter/reporter construct (M) and the regions contained in the 6 deletion constructs (S, D, N, P, B, A) with nucleotide sequences of the respective segments shown in Fig. 1. Bas, the pGL2-Basic plasmid with no insert. A and B: Muc1 promoter activity in hamster and human cells, respectively. Removal of 37 bp from construct D (-1,651) resulted in an increase in luciferase activity in HP-1 and all human cells (compare constructs N and D). Deletion of a 304-bp region from -555 to -252 (compare constructs A and B) uniformly led to decreased reporter activity. Each value represents the mean ± SE of 5 independent experiments. The difference in activity between constructs D and N in HP-1 cells and all the human cells tested is statistically significant (P < 0.01).

Identification of the P-NRE as a YY1 transcription factor binding site. Analysis of the DNA sequence of the P-NRE and the immediately surrounding areas indicated a potential YY1 binding site toward the 3'-end of the P-NRE-37 mer (5'-CCATG-3'). To determine whether YY1 can bind to this region, we synthesized and labeled an oligonucleotide spanning the 37 mer and an additional 10 bp downstream (designated P-NRE-47) and used it as a probe in EMSA experiments. Nuclear extract was prepared from hamster HP-1 cells. As shown in Fig. 4 (lane 2), migration of the probe was retarded after incubation with the HP-1 nuclear extract, indicating the binding of nuclear protein(s) to the labeled P-NRE-47. Addition of unlabeled oligonucleotide (P-NRE-47) as a competitor decreased the band intensity in a dose-dependent manner (lanes 3 and 4). To determine whether YY1 is binding to this region and causing the shift in migration of the probe, we used a competitor DNA identical to the P-NRE-47 with the exception that the CA bases at the 3'-end of the oligonucleotide were changed to a GG (P-NRE-47-YY1-mut). The CA bases have been shown to be critical for the binding of the YY1 protein (22). The P-NRE-47-YY1-mut oligonucleotide did not compete away the binding factor (lanes 5 and 6). When an oligonucleotide bearing a consensus YY1 binding site was used as a competitor (YY1-con) (22), the binding factor was competed away effectively (lanes 7 and 8). When the critical CA bases were changed to a GG in this consensus YY1 oligonucleotide (YY1-mut), this oligonucleotide could not compete away the binding factor (lanes 9 and 10). We also used an oligonucleotide bearing a region of the human p53 gene promoter previously shown to bind YY1 (10), and this oligonucleotide competed effectively (lanes 11 and 12). Finally, an antibody made against YY1 and known to recognize both rodent and human YY1 was added to the band shift reaction, and a "supershift" was clearly seen confirming that YY1 binds to the P-NRE region (lane 13).


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Fig. 4.   Electrophoretic mobility shift assay using the P-NRE-47 oligonucleotide as a probe. Nuclear extracts were prepared from HP-1 cells as described in METHODS. Nuclear extract (20 µg) was incubated with 32P-end labeled synthetic oligonucleotide (P-NRE-47) encompassing residues -1,651 to -1,605. The binding mixture was resolved on a 4.5% polyacrylamide gel and analyzed by autoradiography as described in METHODS. Addition of either 37- or 74-fold molar excess of unlabeled P-NRE-47 resulted in a decreased intensity of the band in a dose-dependent manner (lanes 3 and 4). However, when the crucial CA nucleotides of the putative YY1 binding site was changed to a GG, no competition was seen (lanes 5 and 6). Oligonucleotides bearing known yin yang 1 (YY1) binding sites competed effectively for the band (lanes 7, 8, 11, and 12), but mutated competitors (-mut) did not (lanes 9 and 10). The reaction run out in lane 13 contained YY1-specific antibody, and a supershift (arrow at right) was seen. YY1-con, YY1-consensus.

To determine the effect of the removal of the P-NRE region in the context of the full-length Muc1 promoter, we made a promoter/reporter construct identical to the full-length pMuc1-2354-luc with the exception of the absence of bases -1,651 to -1,615 (pMuc1-2354Delta P-NRE-luc). This construct was then transfected into HP-1 cells along with the wild-type pMuc1-2354-luc as well as the deletion constructs containing up to -1,698, -1,651, and -1,614 of the Muc1 promoter region, and then reporter activity was measured. The deletion of bases from -1,651 to -1,615 resulted in a twofold increase in transcription compared with the wild-type pMuc1-2,354-luc as well as deletion constructs containing the P-NRE (pMuc1-1,698-luc and pMuc1-1,651-luc). The level of transcription from the pMuc1-2,354Delta P-NRE-luc construct was similar to or slightly higher than the deletion construct lacking the P-NRE (Fig. 5). This result indicates that the region between -1,652 and -1,614 normally represses transcription of the Muc1 gene in HP-1 cells and that the repression can be relieved by the removal of this site.


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Fig. 5.   Effect of removal of the P-NRE domain of the hamster Muc1 promoter on Muc1 transcription. The Muc1 promoter/luciferase construct lacking the P-NRE (pMuc1-2354Delta P-NRE-luc, labeled as No-NRE) was constructed and transfected into HP-1 cells as described in METHODS. Its level of promoter activity was measured by luciferase assay and compared with the levels of other constructs as shown in Fig. 3 (top). Note that the removal of the P-NRE region (No-NRE) resulted in a twofold increase in transcription relative to the pMuc1-2354-luc construct (M) as well as constructs S (pMuc1-1698-luc) and D (pMuc1-1651-luc). The 37-bp P-NRE is located between construct D and construct N (pMuc1-1615-luc). *P < 0.01 (compared with construct M, S, or D).

To determine whether the P-NRE is capable of repressing transcription from a heterologous promoter, we placed the P-NRE upstream of the SV40 promoter connected to the luciferase reporter gene and transfected into HP-1 cells. As can be seen in Fig. 6, the presence of a single copy of the P-NRE in the correct orientation (same direction as in Muc1 promoter) upstream of the SV40 promoter resulted in a small decrease in transcription. However, when two copies of the P-NRE were attached in the correct orientation, a further decrease in transcription was seen. Interestingly, when two copies of the P-NRE were cloned in the opposite orientation, the level of inhibition of transcription was higher than when two copies were placed in the correct orientation. Cloning a copy of a scrambled P-NRE sequence (P-NRE-mut) resulted in no significant change in transcription from the SV40 promoter/reporter construct. These results indicate that the P-NRE is capable of inhibiting transcription from a heterologous promoter and in an orientation-independent manner. Furthermore, the repressive effect of P-NRE is additive; that is, two copies result in a greater level of inhibition of transcription than one copy.


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Fig. 6.   Effect of P-NRE on the transcriptional activity of a heterologous promoter. One or two copies of the P-NRE were cloned upstream of the SV40 promoter connected to a luciferase reporter gene, the resulting plasmid constructs were transfected into HP-1 cells, and luciferase activity was measured as described in METHODS. 1M, 1 copy of a scrambled P-NRE (P-NRE-mut in the text). Correct, same direction as in Muc1; rev, reverse direction. Note that the mutation of P-NRE resulted in a complete loss of the P-NRE activity, whereas the presence of the intact P-NRE upstream of the SV40 promoter resulted in a significant inhibition of the promoter activity in a copy number-dependent but orientation-independent manner. *P < 0.01 (compared with the 0 copy group).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results from the body of literature on the transcriptional regulation of the MUC1 gene suggest that the process is complex and multifactorial in nature. Although several cis-acting elements and trans-acting factors that influence the level of MUC1 expression have been identified, the molecular mechanism of the regulation of the MUC1 gene during epithelial differentiation and carcinogenesis has not been well elucidated. We have cloned and analyzed ~2.4 kb of the 5'-flanking region of the hamster Muc1 gene containing putative transcriptional regulatory elements. The region up to 1 kb upstream of the transcriptional initiation site shows relatively high sequence homology to the corresponding segment of the human MUC1 gene (68% identity) and a higher level of similarity (81%) to the mouse Muc1 promoter. Upstream of the 1-kb region, the human MUC1 promoter contains an ~295-bp region not found in either the hamster or mouse Muc1 promoters. The functional significance of this region in the regulation of the human MUC1 gene remains to be determined. Further upstream of this 295-bp region, the similarity between hamster and human promoters continues for ~530 bp at 61% identity.

Functional analysis of the hamster promoter in HP-1 cells revealed a positive regulatory region between nucleotides -555 and -252, since deletion of this region resulted in lower transcription levels in all of the cell lines tested regardless of MUC1/muc1 expression. Therefore, this region may not be responsible for the cell type-specific expression of MUC1/Muc1 in the cells tested. The presence of several positive regulatory regions in the human MUC1 gene promoter has been previously reported (1, 24, 43). Abe and Kufe (1) identified a 21-bp cis-acting element between nucleotides -505 and -485 that bound to a 45-kDa nuclear protein present in MUC1-positive MCF-7 cells but not in a MUC1-negative subclone of MCF-7. Shirotani et al. (43) reported a region between nucleotides -531 and -520, referred to as a responsive mucin element, that interacts with a 65- to 70-kDa nuclear protein and functioned as a positive regulatory element (PRE) for the MUC1 gene in a human colon cancer cell line. Because these two regions do not share sequence similarity, it is currently unclear whether they represent separate PREs or whether their differences in activity reflect differences in the cell types examined. A third study (24) identified two additional MUC1 regulatory elements (Sp1 and E-MUC1 binding sites) more proximal to the transcriptional initiation site relative to the two cis-acting elements described above. The authors of the study reported the importance of these two elements for the epithelium-specific expression of MUC1. The 304-bp region containing a putative PRE in the promoter region of the hamster Muc1 gene reported here has a 68% identity to the corresponding region in the human MUC1 gene. Moreover, the three cis-acting elements described above are 100% conserved between the human MUC1 and hamster Muc1 promoters, suggesting that the positive regulatory mechanisms used to regulate the MUC1 and Muc1 genes are similar.

Our functional studies in HP-1 cells also identified a P-NRE between nucleotides -1,652 and -1,614. The removal of the P-NRE resulted in higher transcriptional rates in all the MUC1/Muc1-expressing cells tested. However, no change was observed in CHO cells that do not express the Muc1 gene, suggesting the possible involvement of the P-NRE region in the regulation of MUC1/Muc1 transcription. There has been a previous report describing the negative regulation of the MUC1 gene. Kovarik et al. (25) identified a novel protein (SpA) capable of repressing MUC1 gene transcription by competing with Sp1 for binding to its consensus sequence between nucleotides -99 and -91. The recognition sequence of SpA overlapped with the Sp1 consensus motif but also required an additional adenine residue at the 5'-end for binding. Deletion of the 37-bp P-NRE within the context of the full Muc1 promoter resulted in a twofold increase in transcription, supporting the notion that the P-NRE can function to regulate the transcription of Muc1. Furthermore, our results demonstrate that the P-NRE can act as a transcriptional repressor in an orientation- as well as distance (from the transcriptional initiation site)- independent manner. Also, the P-NRE functions in an additive fashion; that is, two copies placed upstream of the SV40 promoter inhibited transcription to a greater extent than a single copy. The P-NRE is well conserved in the mice Muc1 promoter (30/37 bases) at the corresponding location as well as in the human MUC1 promoter (24/37 bases). The corresponding P-NRE in the human MUC1 promoter is found at nucleotides -2,041 to -2,005 due, in part, to the presence of the 295-bp region described earlier. It has been reported that the 1.4-kb upstream region from the transcriptional initiation site of the human MUC1 promoter is sufficient to confer epithelium-specific expression as well as induction during lactation and polyoma T protein-induced tumorigenesis in transgenic mice (15). The corresponding human MUC1 P-NRE site that we describe in this report is located upstream of the 1.4-kb region, and thus the P-NRE may be involved in the modulation of the correct MUC1/Muc1 regulation during epithelium-specific expression or induction during lactation or tumorigenesis. Alternatively, the putative function of the P-NRE in the transcriptional regulation of MUC1 may be compensated for in the MUC1 transgenic mice by the presence of the appropriate chromatin structure at the MUC1 gene integration site. It should be noted that only 2 out of 10 founder mice carrying the MUC1 promoter demonstrated correct tissue-specific expression and induction during lactogenesis and tumorigenesis and that the level of induction was relatively low (15). It is also possible that the P-NRE is involved in the regulation of Muc1 gene expression during states not described above or in a species-specific manner.

As seen in Fig. 4, our detailed EMSA analysis clearly demonstrates that YY1 can bind to the Muc1 P-NRE. These results indicate that YY1 binds to the P-NRE and may have a repressive role in the transcription of the Muc1 gene, although we cannot rule out that other factors undetectable by EMSA also bind to the P-NRE and affect the transcription of Muc1. YY1 is a C2H2 zinc finger protein that is ubiquitously expressed and can act as both a transcriptional repressor and activator, depending on the promoter context (48). It has been shown to be involved in the repression of the beta -casein gene in mammary epithelial cells (30). YY1 has been shown to be bound to Sp1 transcription factor (42) as well as both histone deacetylases (HDAC1 and HDAC2) and histone acetyltransferase (HAT) (48). Thus it may possible that YY1 represses the activation of Muc1 transcription by interactions with Sp1 or HDAC1/2 or HAT. Our computer searches indicate the presence of four other potential YY1 binding sites within the Muc1 promoter. It would be important to determine whether YY1 also binds to these sites and whether the binding of YY1 affects the transcriptional level from the Muc1 promoter.

In conclusion, the molecular mechanisms involved in the regulation of MUC1 transcription seem to be rather complex, but the details are beginning to emerge. We report here a PRE in the hamster Muc1 promoter that seems homologous both in sequence and function to the PREs described in the human MUC1 gene promoter. A novel P-NRE located ~1.6 kb upstream from the transcriptional initiation site, which is a binding site for the YY1 transcription factor, seems to be important for the transcriptional regulation of the Muc1 gene on the basis of the series of experiments described here. We are currently investigating the transcriptional regulation of the Muc1 gene based on the YY1 binding to the P-NRE site as well as other potential binding sites on the Muc1 promoter.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Steve Georas (Johns Hopkins University) and Erik Lillehoj (University of Maryland, Baltimore) for valuable comments. HP-1 cells were kindly provided by Dr. M. A. Hollingsworth, University of Nebraska Medical Center, Omaha, NE.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-63742.

Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 N. Pine St., Rm. 446, Baltimore, MD 21201.

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.

September 27, 2002;10.1152/ajplung.00342.2000

Received 27 September 2000; accepted in final form 11 September 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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