Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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 basesEMSA.
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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RESULTS |
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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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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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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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DISCUSSION |
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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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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