(Received for publication, August 10, 1994; and in revised form, December 1, 1994)
From the
Human carcinoembryonic antigen (CEA) belongs to a family of membrane glycoproteins that are overexpressed in many carcinomas; CEA functions in vitro as a homotypic intercellular adhesion molecule and can inhibit differentiation when expressed ectopically in myoblasts. The regulation of expression of CEA is therefore of considerable interest. The CEA gene promoter region between -403 and -124 base pairs upstream of the translation initiation site directed high levels of expression in CEA-expressing SW403 cells and was 3 times more active in differentiated than in undifferentiated Caco-2 cells, correlating exactly with the 3-fold increase in CEA mRNA seen in differentiated Caco-2 cells. Inclusion of additional upstream sequences between -1098 and -403 base pairs repressed all activity. By in vitro footprinting and deletion analyses, four cis-acting elements were mapped within the positive regulatory region, and one element within the silencing region. Several nuclear factors binding to these domains were identified: USF, Sp1, and an Sp1-like factor. By co-transfection, USF directly activated the CEA gene promoter in vivo in both SW403 and Caco-2 cells. In addition, the levels of factors binding to each positively acting element increased dramatically with differentiation in Caco-2 cells. Thus the transcriptional control of the CEA gene depends on the interaction of several regulatory elements that bind multiple specific factors.
CEA, ()a membrane glycoprotein first observed in
human fetal colon and colorectal cancer(1) , is a widely used
clinical tumor marker. CEA has been shown to function in vitro as a homotypic intercellular adhesion molecule (2, 3) and could thus play an important role during
development. A model for a possible carcinogenic role of CEA
overproduction in the colon has been suggested(2, 4) .
In support of this model, we have recently shown that the ectopic
expression of CEA on the surface of rat L6 myoblasts can completely
block terminal differentiation and the normal loss of proliferative
capacity(5) . These attributes make CEA an important candidate
for studies on control of gene expression.
CEA is a member of the immunoglobulin supergene family and is the prototype for its own subfamily of closely related molecules that vary in domain composition and tissue distribution (for a review, see (6) ). This subfamily consists of 29 closely linked genes on chromosome 19, including those coding for CEA itself, nonspecific cross-reacting antigen (NCA), biliary glycoprotein (BGP), CEA gene family member 6 (CGM6), and a number of other genes with yet undetermined products (hsCGMs)(6, 7) . As with CEA, NCA and BGP have been shown to function in vitro as intercellular adhesion molecules(2, 3, 6) .
Cloned cDNAs for CEA, NCA, and BGP have been used as probes to study their expression in normal and tumor tissue(6) . Whereas CEA mRNA is present at low levels in normal adult colon and is usually overexpressed in malignant colon and other cancers of epithelial cell origin, NCA mRNA is found in normal colon, lung, and granulocytes and is elevated by a greater factor in tumors of the colon, breast, and lung(6) . Several forms of BGP have been isolated from bile ducts, gallbladder mucosa, and various tumors(6) . In contrast to CEA and NCA mRNAs, the expression of two BGP mRNAs have been shown to be down-regulated in colorectal carcinomas in comparison to normal adjacent mucosa(8) . The increased levels of CEA have been shown not to be due to gene rearrangements or amplification (9) but, instead, to hypomethylation of upstream regions (9, 10, 11) and/or factor changes leading to altered rates of transcription; post-transcriptional changes have also been implicated(9, 12) .
CEA and NCA mRNA levels have also been investigated in the differentiating Caco-2 cell system(12) . When cultured in vitro, between 4 and 11 days after reaching confluence, this human colon adenocarcinoma cell line differentiates and becomes highly polarized, with tight junctions between individual cells and a brush border membrane containing enzymes characteristic of a fully differentiated intestinal epithelium(13, 14) . We found that CEA transcript levels were 3-fold higher in fully differentiated Caco-2 cells than in undifferentiated monolayers(12) . In the present study, we have used the differentiation control of the CEA gene in Caco-2 cells to check the biological validity of the CEA promoter analysis.
To carry out this analysis, we characterized the upstream noncoding region of the CEA gene. A 424-bp 5` flanking sequence has been reported to confer cell-type specific expression on a reporter gene(15) . Numerous purine-rich sites were postulated to play a role in transcriptional control(11) , but the exact regulatory elements involved remained unknown. We now demonstrate that both positive and negative elements reside within 1098 bp upstream of the translational start site and that a 403-bp upstream sequence confers cell type-specific and differentiation-dependent expression on the luciferase reporter gene. We identify five nuclear factor binding sites and three of the multiple trans-acting factors (USF, Sp1, and Sp1-like) interacting with the stimulatory domain. In addition, we present direct evidence that USF activates CEA gene transcription in vivo.
5` deletion mutants of the CEA gene promoter lacked the CEA
translation initiation codon and were fused immediately 5` to the
firefly luciferase (LUC) reporter gene (17) at the SmaI site of the pXP2 vector(18) . The resulting
plasmids were named according to the sizes of their respective CEA
promoter restriction fragments as shown in Fig. 1. Internal
deletion mutants of the CEA gene promoter, p1098279LUC and
p1098a
279LUC, were constructed as follows: the AvrII
to AvaI
fragment was blunt-ended and inserted in the sense and antisense
(a) orientation into the blunt-ended AvrII site in p124LUC,
thus preserving the transcriptional initiation site at its proper
position. p1098+279LUC contains the 974-bp AvrII
fragment, spanning from -124 to -1098, inserted into the AvrII site of p403LUC. pRSVLUC(18) , containing the
RSV long terminal repeat fused to the LUC gene in pXP2, was obtained
from Dr. M. Featherstone (McGill Cancer Centre, Montreal);
pRSVZ
-gal was obtained from Dr. E. Shoubridge (Montreal
Neurological Institute, Montreal) and contains the
-galactosidase
gene driven by the RSV long terminal repeat.
Figure 1:
Localization of the elements
determining CEA gene promoter activity. CEA promoter activity in
various cell lines is shown. The activity of each construct in each of
the cell lines is presented relative to the activity of the
promoterless vector, pXP2. In all cases, CEA sequences from -2 to
-124, including the 5` untranslated region and transcription
initiation site, are present in plasmid constructs. The translational
start site is at position +1. Ovals represent regions
protected in DNase I footprinting assays. Names of constructs
correspond to the sizes of the fragments tested. Sites used to create
constructs are shown on the restriction map, with precise positions
shown in Table 1. The data represent the mean ± S.D. of
three to four independent experiments, each performed in duplicate, and
corrected for protein concentration and for transfection efficiency by
the activity of the internal RSVZ-gal control plasmid. N.D., not determined.
Cells were
plated at a density of 1 10
cells/100-mm plastic
Petri dish 24 h before transfection. 10 µg of pRSVLUC or equimolar
amounts of the promoter-LUC gene constructs were cotransfected with 5
µg of pRSVZ
-gal and l0 µg of calf thymus carrier DNA by
calcium phosphate-DNA coprecipitation as described
previously(22) . The precipitate was removed after 15 h, and
the cultures were incubated for another 72 h in normal growth medium.
Undifferentiated Caco-2 monolayers were transfected at 2 days before
confluence; fully differentiated Caco-2 cells were transfected at 11
days after confluence. Their state of differentiation was confirmed by
the presence of domes and the development of a brush border membrane (12) .
-Interferon (Collaborative Research Inc., Bedford,
MA) was applied to Caco-2 cells at 2000 units/ml as described
previously (12) immediately following transfection, with a
fresh medium change. LUC activity was measured as described by DeWet et al.(17) .
-Galactosidase activity (23) in these same extracts was measured to correct for
variations in transfection efficiency. Relative luciferase activity was
calculated as the ratio between LUC and
-galactosidase activities
for each transfection and then reported as -fold above background
(considered as activity of the parental promoterless vector, pXP2).
Each plasmid was tested in duplicate plates and in three to five
different transfection experiments.
To assess the effect of USF on CEA promoter activity in vivo, 10 µg of p403LUC and either 5 or 15 µg of pRSV.USF (as indicated in Table 2) were coprecipitated with calcium phosphate and transiently transfected into the SW403 or Caco-2 cell line. pRSV.USF was constructed by inserting human USF cDNA (24, 25) into a pUC18-derived expression vector driven by the RSV long terminal repeat (18) . Rat HNF-4 cDNA in the pSG5 expression vector (26) was generously provided by Dr. F. M. Sladek (University of California, Riverside, CA).
DNase I footprinting assays were performed essentially as described by LeFèvre et al.(28) with modifications as by Howell et al.(29) . Briefly, 1-2 fmol (10,000 cpm) of DNA probes, labeled at only one end, were incubated with 0-160 µg of nuclear extracts for 15 min on ice; restriction enzyme-grade bovine serum albumin (Boehringer Mannheim) was added so that equal total amounts (160 µg) of protein were present in each reaction. Freshly diluted DNase I (Life Technologies, Inc.) at 20 ng/30 µl reaction volume was added for 3 min on ice. Reactions were stopped, and the DNA was digested with proteinase K, phenolextracted, and precipitated. The dried DNAs were suspended in formamide loading dye, and equal counts/min were loaded on an 8% polyacrylamide, 8 M urea sequencing gel. Sequencing reactions (23) were run alongside as size markers. The dried gels were autoradiographed at -75 °C with Cronex Quanta III intensifying screens (E. I. du Pont de Nemours & Co.).
To determine equivalent amounts of nuclear extracts from Caco-2 cells in their undifferentiated and differentiated states, oligonucleotides binding to ubiquitous factors such as Sp1 (31) or AP1 (32) could not be used since the levels of these factors have been observed to change with differentiation ( (33) and data not shown). Thus equal concentrations of isolated nuclei were used to normalize the extracts. Using this approach, with the preparations shown in Fig. 6, 20 µg of undifferentiated Caco-2 nuclear extract was equivalent to 18 µg of differentiated Caco-2 nuclear extract. The data shown in Fig. 6are representative of several independent experiments using independent nuclear extracts.
Figure 6: Analysis of Caco-2 nuclear proteins binding to CEA regulatory elements versus differentiation. Oligonucleotides containing the DNA sequence of three of the CEA regulatory elements were used as probes to reveal Caco-2 nuclear proteins which specifically recognized them and to indicate the relative abundance of these factors in equivalent amounts of nuclear extracts prepared from undifferentiated (U) and differentiated (D) Caco-2 cells. By using equal concentrations of isolated nuclei before the elution of nuclear proteins, 20 µg of undifferentiated Caco-2 nuclear extract was found to be equivalent to 18 µg of differentiated Caco-2 nuclear extract in the experiment shown here. The indicated unlabeled oligonucleotides used as competing DNAs assessed the specificity of the complexes formed. Arrows point to the specific complexes.
Deletion of the
-403 to -124 sequence, leaving only the transcription
initiation site and 5` untranslated region intact (p124LUC construct),
abolished all promoter activity (Fig. 1). Thus regulatory
elements responsible for the control of cell-specific expression of the CEA gene reside within this 279-bp upstream region. Inversion
of the 403-bp minimal promoter in the p403aLUC antisense construct
strongly reduced but did not abolish activity, suggesting that the
elements within the minimal promoter may use cryptic signals on either
strand for transcriptional initiation (no TATA box can be found in the
upstream sequence of the CEA gene). Inclusion of further
upstream sequences (-1098 to -403) in conjunction with the
minimal promoter, as seen with the p1098LUC construct, repressed
promoter activity markedly in SW403 cells (Fig. 1). The
-1098 to -403 region alone, placed in either orientation
before the fragment containing the transcription initiation site,
showed no activity whatsoever (constructs p1098279LUC and
p1098a
279LUC). Hence, a silencer region, which can down-regulate CEA gene transcription, must lie within the -1098 to
-403 bp sequence. This silencer can nevertheless be completely
overcome by the upstream placement of a second 279-bp (from -403
to -124) region at -1098 bp (construct p279+1098LUC) (Fig. 1).
As seen with the SW403 cell line, the inclusion of additional upstream (-1098 to -403) sequence also repressed promoter activity in Caco-2 cells, and further deletions of the minimal promoter past position 403 generally decreased activity. Only in Caco-2 cells does the p300LUC construct give higher activity than the p403LUC construct, however. It is thus possible that a second silencing element, recognized by factors only in Caco-2 nuclear extracts, is present in the -403 to -300 region.
Since
-interferon increases levels of CEA mRNA quite dramatically in the
Caco-2 cell line(12) , this cytokine was applied for 3 days to
examine changes in promoter activity. However, no effects on promoter
activity were seen (data not shown), despite the presence of several
possible
-interferon activation sites and
-interferon-stimulated response elements in the upstream promoter
region (-835 to -1650).
Figure 2:
Localization of binding sites for nuclear
proteins within the CEA promoter using DNase I footprinting. A, restriction enzyme map of the CEA promoter showing sites
used to generate the probes used in the footprinting experiments.
Probes I-III were labeled as indicated by asterisks at
either the 5` or 3` end. The circlednumbers show the
positions of the footprints detected. B, localization of sites
within the -2 to -403 region. 5`-end-labeled probe I was
incubated as described under ``Materials and Methods,'' with
nuclear extracts from LR-73, SW403, and Caco-2 (undifferentiated
monolayers) as indicated. G refers to the G sequencing track; lanes0 contained 160 µg of bovine serum albumin
only, and lanes 10-160 show results for 10-160
µg of the indicated nuclear protein extracts. The positions of the
various footprints are indicated on the right by bars and labels; their precise sequences are listed in Table 3.
The positions of some guanine residues are shown on the left for orientation purposes. Footprints were numbered according to
their proximity to the transcription initiation site. C,
localization of binding sites with a 3`-end-labeled -403 to
-2 probe (Probe II). Footprints FP2 and FP3 are revealed using
HepG2 nuclear extract. This probe is also shown using SW403 nuclear
extract for comparison. DNase I hypersensitive sites () produced
by binding of nuclear proteins to the FP2 and FP3 elements are visible. D, localization of binding sites in the silencer region with a
3`-end-labeled -835 to -403 probe (Probe III). In addition
to SW403 nuclear extract, a nuclear extract prepared from HT29 cells
was assayed for comparison. Both extracts reveal DNase I hypersensitive
sites (
) flanking the FP5 element.
Two additional footprints, labeled FP2 and FP3, were revealed using nuclear extract from HepG2 cells and were also present, although less apparent, with extracts from SW403 cells (Fig. 2C). Two DNase I-hypersensitive sites are visible between FP2 and FP3.
In Fig. 2D, the -835 to -403 silencing region was used as an end-labeled probe in DNase I footprinting experiments; only one protected region could be detected in repeated experiments. This footprint, labeled FP5, was more apparent in HT29 nuclear extract and was flanked by two DNase I-hypersensitive sites. Deletion of the -1098 to -403 region containing this element led to an increase in promoter activity in all cell lines tested (Fig. 1, p1098LUC versus p403LUC constructs).
Figure 3: Gel mobility shift assay reveals that the USF transcription factor binds to the FP1 regulatory element. A, synthetic, double-stranded oligonucleotide representing FP1 (Table 1) was end-labeled, incubated with 60 µg of SW403 nuclear extract and electrophoresed, showing two complexes (C1 and C2). 5-, 10-, or 100-fold molar excesses of competing oligonucleotides were added as indicated. B, USF synthesized in vitro (see ``Materials and Methods'') was added to the FP1 probe in lanes 9-11; a 5-fold molar excess of unlabeled FP1 DNA was added in lane10; lanes11 and 12, USF specific antibody was first added to USF protein or SW403 nuclear extract, respectively, for 15 min at 0 °C, after which the FP1 probe was added and incubated for another 15 min on ice. The supershifted (s.s.) complex is indicated by an arrow on the right. C, for comparison, 40 µg of HepG2 nuclear extract was analyzed for binding under the same conditions as that with SW403 extract. 5- and 50-fold molar excesses of unlabeled, double-stranded FP1 oligonucleotide were used as competitor DNAs.
Since the GAL2 USF oligonucleotide competed effectively for complex formation, we tested whether purified USF transcription factor would recognize the FP1 element. The complex formed (Fig. 3B, lanes9 and 10) comigrated with C1 from SW403 extract (Fig. 3B, lane8). As expected, specific anti-USF antibody supershifted the DNA/USF complex (Fig. 3B, lane11) but also supershifted the C1 complex from SW403 nuclear extract (Fig. 3B, lane12). Thus colon carcinoma cell lines clearly contain USF, which can bind to the FP1 element in the CEA gene promoter. The presence of two complexes in HepG2 nuclear extracts comigrating with those obtained with SW403 extract (Fig. 3C) suggests that USF is also present in this hepatoma-derived cell line.
Figure 4: Element FP2 is recognized by Sp1 and another novel transcription factor. A, end-labeled, double-stranded FP2 oligonucleotide (Table 1) was incubated with 40 µg of SW403 nuclear extract and subjected to electrophoretic analysis. Three complexes (C1-C3) were revealed. Unlabeled, double-stranded FP2, FP3, Sp1, and AP1 oligonucleotides (Table 1; 10- and 100-fold molar excesses) were used as competitor DNAs. B, 3.5 ng of purified Sp1 protein was incubated with FP2 probe in lanes 12 and 13 under the same binding conditions. Antibody specific to Sp1 was preincubated with 14 ng of Sp1 protein (lane 14) and 40 µg of SW403 nuclear extract (lane15). The Sp1 supershifted (s.s.) complex is indicated by an arrow on the right.
Figure 5: Element FP3 is bound by Sp1. A, electrophoretic analysis of end-labeled, double-stranded FP3 oligonucleotide (Table 1) incubated with 40 µg SW403 nuclear extract shows two specific complexes (C1 and C2). Unlabeled, double-stranded FP3, FP2, Sp1, AP1, and PY oligonucleotides (Table 1; molar excesses as shown) were used as competitor DNAs. B, in lanes 15 and 16, FP3 probe and 3.5 ng of purified Sp1 protein were incubated as described in Fig. 4. Sp1-specific antibody was preincubated with 14 ng of Sp1 protein (lane17) and 40 µg of SW403 nuclear extract (lane18). The Sp1 supershifted (s.s.) complex is indicated by an arrow on the right. C, 20 µg of HepG2 nuclear extract was incubated with FP3 probe under the same binding conditions for comparison purposes.
Probes FP1, FP2, and FP3 each produced several complexes (Fig. 6, lanes 1-12), similar to those seen with SW403 extracts (Fig. 3Fig. 4Fig. 5). The levels of the complexes obtained were dramatically higher using differentiated (D) than undifferentiated (U) Caco-2 nuclear extracts (Fig. 6). Thus, the levels of USF, Sp1, the Sp1-like, and the unknown factor responsible for the C3 complex with the CEA FP2 element all appear to increase with differentiation in Caco-2 cells. The higher levels support our contention that an increase in the abundance of positive factors interacting with the regulatory elements in the minimal promoter are partially responsible for the rise in CEA transcription observed in this differentiating system.
In this study, we have delineated the basic organization of the CEA gene promoter (summarized in Fig. 7and Table 3), which is the first to be determined for genes of the CEA subgroup of this human tumor marker family. Functional assays using various 5` flanking sequences of the CEA promoter linked to the luciferase reporter gene transfected into CEA-producing cells coupled with DNase footprint assays revealed that the 5` upstream region contains four positive (FP1-FP4) and one negative regulatory element (FP5). The upstream stimulatory factor (USF)(34) , also known as the adenovirus major late transcription factor (MLTF), was shown to bind to the FP1 element; the positive control of CEA transcription by USF was confirmed directly by the demonstration of specific stimulation of the CEA promoter in vivo by a co-transfected USF-producing plasmid. The Sp1 (38) and an Sp1-like transcription factor were found to bind to both the FP2 and the FP3 element. Through computer sequence analyses, FP4 was found to resemble an AP-2 transcription factor site (30, 39) , and preliminary experiments (data not shown) confirmed this possibility; oligonucleotides containing AP-2 binding sites competed with the FP4 site for nuclear factor binding and purified AP-2 protein was capable of forming a complex with the FP4 element. Other factors binding to the FP4 site, a third factor binding to the FP2 site and factors recognizing the FP5 silencer element remain to be identified.
Figure 7: Schematic representation of the various nuclear proteins binding the regulatory elements of the CEA promoter. Those factors that have been identified are indicated. Factors binding to the BGP promoter are also shown to allow comparison of transcription factor complexes specific to the CEA gene promoter. The arrow signifies the major transcriptional start site. Sequences and positions are compared in Table 3.
The biological significance of these element and factor assignments was further tested using the Caco-2 colonocyte system, which shows an increase in CEA mRNA with differentiation into polarized epithelium. Since the positively acting factor levels increased dramatically, it is thus possible that the transcription factors identified here could also control the expression of CEA in normal colonic epithelial cells, which show an increase in CEA mRNA (40) and protein (41) production during their differentiation in transit from the bottom to the top of a crypt. The basis for transcriptional changes seen for CEA and other CEA family members in tumors remains to be investigated.
Sequence comparisons of the CEA gene regulatory elements with the upstream noncoding sequences of other CEA gene family members are shown in Table 3. The control of expression of this family is of particular interest because of the unusually close alignment of the nucleotide sequences of its members (often over 90%). PSG5 and PSG11 are members of the pregnancy-specific glycoprotein (PSG) subgroup of the CEA gene family whose upstream sequences showed homology to the CEA FP2 and FP3 elements only. We have also analyzed the control of transcription of a second CEA family member, BGP (42) which, unlike CEA(6) , can show decreased transcript levels in colon carcinomas relative to adjacent normal tissue(8) . Comparison of the CEA and BGP gene promoters (see Fig. 7for summary) revealed differences that could explain their differential regulation. Only two of the corresponding BGP gene elements could be shown to bind nuclear factors: thus the Sp1, the Sp1-like (recognizing FP2 and FP3 in CEA), and the silencer factors (recognizing FP5 in CEA) do not bind to the BGP promoter, while a second factor, HNF-4(26) , as well as USF, binds to the USF site(42) . Experiments are in progress to determine whether the different changes in transcription of the CEA and BGP genes seen with colon carcinogenesis can be rationalized by changes in these factors.
Although the Sp1 transcription factor has long been characterized(31) , only recently have dramatically increased levels of this ubiquitously expressed factor been correlated with differentiation(33) . Genes regulated by Sp1 include the following: fibronectin, which shows greatly inhibited expression upon neoplastic transformation(43) ; E-cadherin, which is generally down-regulated in tumors(44) ; and the human papillomavirus type 18 E6-E7 oncogene, which also has an unusual Sp1 site(45) . Specific recognition of the DNA sequence is provided for by the three zinc finger domains of Sp1(46) . Although the core sequence is a typical GC box, substitutions are tolerated as long as certain G residues are present for contact with the ``fingers''(46) . These contact points exist in the FP2 and FP3 elements of the CEA gene, within the aligned homologous sequences of the NCA gene, and within the CGM1 sequence aligned to the CEA FP2 element (see Table 3).
The USF gene has recently been cloned and is now known to code for a ubiquitous factor with a helix-loop-helix repeat domain and a leucine zipper(34) . Its protein-binding interface is similar to that of Myc and Max(47) . All Myc family members recognize an identical core DNA target sequence of CACGTG and appear to bind to DNA as homo- or heterodimers, dependent on specificities contained within the leucine zipper(34, 48, 49) . Since we have directly demonstrated that USF activates the CEA promoter in vivo, any factors interacting with and modulating this nuclear factor should also modulate CEA gene expression. Although purified Myc protein did not bind to the CEA FP1 site (data not shown), it remains possible that the heterodimer c-Myc/Max could bind to this element or to USF itself. Both Myc/Max and USF have also been shown to bend DNA toward the minor groove to the same angle and orientation(50) . Pognonec et al.(51) have also demonstrated that the DNA-binding activity of USF is regulated via a redox dependent mechanism. The activity of the CEA promoter could therefore be partially controlled by a complex balance between the binding of various other b-HLH proteins to form heterodimers with USF, the redox mechanism, and competition for DNA binding by other factors, such as HNF-4 as shown for the BGP promoter(42) . As previously mentioned, CEA mRNA levels are up-regulated in colon carcinomas. About 70% of colon carcinomas overexpress the c-Myc gene as well as other members of the Myc gene family(52, 53) , although the status of USF expression is presently unknown.
This study has identified many of the cis-acting elements involved in the transcriptional control of the CEA gene and some of the trans-acting factors which interact with them. USF, in particular, is involved in both CEA and BGP control and may play an important part in the overall control system of the CEA gene family. These assignments, coupled with further studies on other family members, should lead to the rationalization of the observed tissue-specific, differentiation-dependent expression of the family. The possible deregulation of these trans-acting transcriptional factors in colon carcinogenesis could represent the basis for changes in the expression of CEA and other family members seen in tumors, changes that could be instrumental in the carcinogenetic process(2) . Ectopic expression of CEA, for example, has been shown recently to block myogenic differentiation and leave cells with division potential(5) . It will now be of interest to determine whether the CEA regulatory elements identified here are targets for the action of oncogenes and tumor suppressor genes.