Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, Boston, Massachusetts 02215
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ABSTRACT |
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We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5'-GACTGGGCGGGGTCAAGATGGA-3'. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.
phosphatase; sodium butyrate; small intestine; transcription factor
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INTRODUCTION |
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THE MAMMALIAN SMALL intestine is lined by an epithelium in which undifferentiated, pluripotent crypt stem cells give rise to differentiated villus cells, including enterocytes and goblet, enteroendocrine, and Paneth cells. Enterocytes comprise approximately 95% of all villus cells and are the cell type responsible for the digestion and absorption of dietary nutrients. The transition from the undifferentiated, renewable crypt cell to the terminally differentiated villus enterocyte is thought to occur via the transcriptional activation of a number of cell type-specific genes, including the enzymes, transporters, and structural proteins that reside within the apical microvilli. The expression of these cell-specific genes is also regulated along craniocaudal and developmental axes. The molecular mechanisms responsible for the complex temporal and spatial patterns of intestinal gene expression are poorly understood (11, 21).
HT-29 cells are a human colon cancer-derived cell line that most closely resembles small intestinal crypt cells (20). Long-term incubation with the short-chain fatty acid sodium butyrate (NaBu) has been shown to induce biochemical and morphological changes characteristic of well-differentiated enterocytes (1). We and others have shown that even short-term NaBu treatment induces both growth inhibition and differentiation in HT-29 cells (2, 15). In addition, NaBu induces a shift from a secretory to absorptive phenotype, a transition that is characteristic of crypt-villus differentiation (23).
Among the differentiation markers induced in NaBu-treated HT-29 cells is intestinal alkaline phosphatase (IAP), a brush-border protein thought to play a role in fat absorption (33). IAP is part of a family of structurally related alkaline phosphatase (AP) proteins, including the liver/bone/kidney, placental, and germ cell (GCAP) forms. These alkaline phosphatase proteins are expressed in tissue-specific patterns, and each is encoded by a separate gene (17, 18, 30). Multiple IAP mRNA species have been described in both rat and human gut and are thought to derive from separate genes and different polyadenylation signals, respectively (8, 14).
In previous studies we have shown that IAP induction in butyrate-treated HT-29 cells occurs at the level of gene transcription and is entirely dependent on the synthesis of one or more new proteins (15, 16). The present work was undertaken to more precisely define the regulatory mechanisms underlying IAP gene activation in the context of crypt-villus differentiation. We show that IAP gene transactivation in HT-29 cells is largely dependent on a 28-bp Sp1-related DNA cis element (called IF-III) located within the 5' flanking region of the human IAP gene. Through electrophoretic mobility shift assays (EMSA) we have begun to characterize this novel cis element and its associated DNA binding proteins. These studies provide insight into the regulatory mechanisms underlying IAP transactivation in the context of enterocyte differentiation.
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MATERIALS AND METHODS |
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Cell culture. HT-29 and Hep G2 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD) and grown in 160-cm2 plastic flasks at 37°C, 5% CO2 in DMEM (GIBCO) supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine, and penicillin-streptomycin (100 U/ml). The medium was changed every 3 days, and the cells were separated via trypsinization when they reached confluence. Experiments were performed on cells at 80% confluence with differentiation induced by treatment with 5 mM NaBu, as previously described (15).
Northern blot analyses. Total RNA was extracted using the guanidinium thiocyanate method (6). Northern analyses were performed by loading 20 µg RNA in each lane of an agarose-formaldehyde gel, separating by electrophoresis, transferring to nitrocellulose membranes, and baking for 2 h at 80°C. An IAP-specific 32P-radiolabeled cDNA probe was made by the random primer method (9), as previously described (15). Conditions for hybridization were 5× standard saline citrate (SSC), 50% (vol/vol) formamide, and 1% (wt/vol) SDS at 42°C. Washing conditions were 2× SSC, 0.1% SDS at 50°C.
Transient transfections. Cells were transferred to 100-mm2 dishes and grown to 80% confluence. DNA (10-15 µg) was transfected in each experiment, along with 2 µg of a TK-GH plasmid, which was used to control for transfection efficiency. The DNA to be transfected was ethanol precipitated and resuspended in CaCl2-HEPES and then added directly to the culture dish. After 24 h the medium was changed, and the cells were incubated for 24 h ±5 mM NaBu. The cells were then washed with PBS, and extracts were prepared using a freeze-thaw method. Protein assays were done by the Bradford method (5), and CAT assays were performed using the FLASH CAT kit (Stratagene, La Jolla, CA).
Reporter plasmids.
The IAP2.4CAT (2400 to
49, relative to the start of translation),
IAP125CAT (
178 to
49), O-CAT (promoterless), and SV-CAT (SV40 early region,
including promoter and enhancer) plasmids were kindly provided by Dr.
P. Henthorn (14). IAPsty1CAT
(internal deletion of
254 through
1344) and
IAPsma1CAT (contains nucleotides 0 through
521) were constructed by restriction digestion of the IAP2.4CAT plasmid, followed by
ligation. The IAP75CAT plasmid (
128 to
49) was constructed by obtaining the DNA fragment
through the use of the PCR, adding
Hind III linkers, and then subcloning into the Hind III site of the parent
O-CAT plasmid. To ensure that differences between the
IAP125CAT and
IAP75CAT could be due only to the
5' deletion, a second reporter plasmid containing 125 bp
(
178 to
49) of the IAP 5' flanking region was made
using the identical 3' PCR primer as was used in the
IAP75CAT construction. No
differences were seen in activity between the two
IAP125CAT plasmids. The
IF-IIIHSV-CAT and
IF-IIIMSV-CAT plasmids were
constructed by using synthetic DNA oligomers containing one copy of the
human or mouse IF-III elements, flanked by
BamH I sites, and subcloning into the
parent SV-CAT plasmid. The
IF-IIIHX3SV-CAT contains three copies of the human IF-III element with
BamH I linkers in the SV-CAT plasmid.
The IF-IIIdelCAT and
IF-III
MCAT plasmids were
constructed using a site-directed mutagenesis kit (Stratagene) and the
appropriate PCR primers, to either delete IF-III or replace it with the
mouse element, respectively. All plasmids were sequenced through the
junction with the CAT gene, including the region of DNA manipulation,
to verify the integrity of the constructs.
DNase I footprinting.
Plasmid DNA containing the IAP 5' flanking region was cleaved
with Sty I to obtain an approximate 250-bp fragment, leaving a
3'-recessed end on either the sense or antisense strands. The ends were then filled in using the Klenow fragment of DNA polymerase I
in the presence of
[-32P]dCTP and the
probe purified through a Sephadex column (Pharmacia). DNA-protein
binding reactions were performed (~40,000 counts per min/reaction)
for 45 min in a buffer containing (in mM) 10 HEPES (pH 7.9), 2.5 MgCl2, 50 KCl, 0.1 EDTA, and 1 dithiothreitol, and 10% glycerol. Various amounts of
HT-29 cell nuclear extract were added along with 1 µg poly(dI/dC).
DNase I digestion was then carried out for 1 min, and the
phenol-chloroform extracted samples were run through a 6%
polyacrylamide DNA sequencing gel followed by autoradiography. G + A
Maxim-Gilbert sequencing reactions were run in each gel (not shown) to
determine the exact sites of DNA-protein interaction.
EMSA. Annealed, synthetic oligomers were radiolabeled using the T4 kinase in the presence of [32P]ATP and gel purified. The DNA sequences are depicted in Fig. 6. Approximately 0.5-1.0 ng (10,000 counts per min) of radiolabeled DNA (~1 × 108 counts per min/µg) was used in each binding reaction along with 2 µg bulk carrier DNA poly(dI/dC) and 5-20 µg nuclear extract. In some cases, purified Sp1 protein (Promega) was used or Sp1- or Sp3-specific antibodies (Santa Cruz Biotechnology) were added for supershift analyses. Binding reactions were performed for 40 min at 4°C in the identical buffer as was used for the DNase I footprinting. Excess cold competitor DNA (×100) was used to verify binding specificity. The reactions were then electrophoresed at room temperature on 4% polyacrylamide gels in 0.5× Tris-borate-EDTA and the gels were dried and then subjected to autoradiography.
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RESULTS |
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Localization of IAP cis-regulatory
element.
Transient transfections were performed to identify one or more
functionally important regions within the IAP gene. Figure 1A
depicts an experiment in which the
IAP125CAT reporter gene activity
in HT-29 cells was compared with the control plasmids. O-CAT lacks a
promoter and, as expected, is not expressed under either basal or
NaBu-treated conditions. The SV-CAT plasmid contains the SV40 early
region, including both the promoter and enhancer. Its expression is
quite low under basal conditions and is minimally increased by NaBu
(~2-fold). In contrast, the low basal expression of the
IAP125CAT reporter is dramatically
increased after 24 h of NaBu treatment (~17-fold).
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DNase I footprinting.
An approximate 250-bp segment of the IAP gene was used in footprinting
analyses to identify sites of interaction with HT-29 cell nuclear
proteins. As shown in Fig. 2, five
different footprints are seen and labeled as IAP footprint
(IF)-I-V. The footprinting pattern was identical when either the
sense (Fig. 2A) or
antisense (Fig. 2B) DNA strands were
radiolabeled. All five of the footprints appeared equally under control
and NaBu-treated conditions (not shown). It should be noted that IF-III
is the only footprint that resides within the 50-bp region identified
by the transfection studies and appears to span an approximate 35-bp
segment of DNA (nucleotides 165 through
130).
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IF-III is important for transactivation in HT-29 cells.
The IF-III element (135 through
163) was deleted from the
IAP125CAT plasmid by site-directed
mutagenesis (creating
IF-IIIdelCAT) and transient
transfections performed in NaBu-treated HT-29 cells. Figure
3A
demonstrates the dramatic decrease in reporter gene activity that
occurs on deletion of the IF-III element (compare IAP125CAT with
IF-IIIdelCAT activity). The
functional importance of the IF-III element was further assessed by
replacing the human IF-III element with the corresponding element from
the mouse IAP gene
(IF-III
MCAT). The mouse IF-III
element largely restored functional activity to the IAP promoter,
although the expression was slightly less than that seen with the
wild-type human IAP promoter.
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Cell type-specific pattern of IAP transactivation.
IAP is known to be expressed specifically within the mammalian small
intestine. Figure 4,
left, confirms the tissue-specific pattern of IAP expression using Northern analyses with RNA derived from
HT-29 and Hep G2 cells. As we have previously shown (6), HT-29 cells do
not express IAP under basal conditions, but the mRNA levels are quite
high under NaBu-treated conditions. In contrast, the IAP mRNA is not
detected in the nonintestinal Hep G2 cells, even in response to NaBu
treatment.
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EMSA.
Detailed analyses of IF-III were performed to characterize this DNA
cis element and its cognate binding
protein(s). Within IF-III is a guanine-cytosine (GC)-rich region, which
is a consensus binding site for the well-known transcription factor
Sp1. EMSA were therefore done to test whether Sp1
comprised the HT-29 cell nuclear protein(s) that bind to IF-III. Figure
5A shows
that four shifted bands, IB-1 through IB-4, are seen when the IF-III
fragment is incubated with HT-29 cell nuclear extract. Detailed
analyses with varying amounts of nuclear extract or DNA revealed no
differences between control and NaBu-treated cells, nor between HT-29
and Hep G2 cells (data not shown). All four bands (IB-1 through IB-4) are specific, as indicated by their absence when excess cold IF-III DNA
is added. In lane 4, coincubation with
an Sp1-specific antibody shows that the top band (IB-1) is partially
supershifted, whereas the other bands are unaffected. In
lane 6, the IF-III fragment is
incubated with purified Sp1 protein and a single-shifted band is seen,
all of which is supershifted by the Sp1 antibody (lane 7). The Sp1-DNA complex is competed away completely
by unlabeled IF-III oligomer (lane
8). Taken together, these data indicate that Sp1 is
able to bind to IF-III, comprising a portion of the IB-1 complex
created by HT-29 cell nuclear proteins. In Fig.
5B an Sp3-specific antibody is used in
supershift analyses and demonstrates that this protein also binds to
IF-III and comprises the portion of IB-1 other than that which contains
Sp1, as well as the IB-2 complex. It is clear therefore that the IB-1
complex is actually composed of two distinct bands, containing the Sp1
and Sp3 proteins, and that IB-2 is composed of Sp3. However, the other
IF-III complexes (IB-3 and IB-4) appear to be composed of proteins
distinct from Sp1/Sp3.
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DISCUSSION |
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In the present work we have employed NaBu-treated HT-29 cells as an in vitro model for the crypt-villus axis of enterocyte differentiation. We and others have shown that NaBu causes early G1 cell-cycle withdrawal in HT-29 cells, as well as inducing various markers of differentiation, e.g., IAP and villin (2, 15). NaBu also induces the expression of the cell cycle inhibitor p21 (15), paralleling its pattern of expression along the crypt-villus axis in the intact animal (24). In addition, expression of the Na-K-2Cl cotransporter (NKCC-1) is downregulated by NaBu treatment (23), consistent with the switch from a "secretory" to "absorptive" phenotype, as occurs during enterocyte differentiation. The alterations in HT-29 cell growth and differentiation occur within a 48-h period, reminiscent of the 2- to 5-day life span of intestinal epithelial cells in vivo and in contradistinction to the prolonged time needed in other models of enterocyte differentiation, e.g., postconfluent Caco-2 cells (34). Based on these data we believe that the short-term NaBu-treated HT-29 cell is a superb in vitro model for crypt-villus enterocyte differentiation.
The present studies indicate that IAP reporter gene activation appears to largely depend on a short DNA segment (IF-III) within the human IAP gene. We saw no differences in reporter gene activity among the IAP2.4-, IAPsty1-, IAPsma1-, and IAP125CAT plasmids, suggesting that important regulatory information is absent from the more upstream region. It is possible, however, that both enhancer and repressor activities could reside within this segment, functionally canceling each other out in the case of large deletions. Whether other more remote areas of the IAP gene play a role in its transactivation during the differentiation process will require further study.
The DNase I footprinting experiment revealed at least five protein binding regions within the 250 nucleotides 5' to the translational start site. IF-V corresponds to the putative TATA box, containing the DNA sequence TTTAAA. IF-I and IF-II both reside 5' to the region shown to be functionally important by transfection assays, suggesting that these binding sites may not play a critical role in IAP transactivation. IF-IV is a G-rich region located 3' to IF-III and may or may not play a role in IAP expression.
The mechanism by which NaBu affects the growth and differentiation of a
number of cell types is not well understood. It has been suggested that
the NaBu effects are a result of nonspecific alterations in the state
of histone acetylation (19, 26); however, more specific interactions
with DNA cis elements have been
described in the cases of the -globin- (10) and placental-like alkaline phosphatase (GCAP) genes (7). Interestingly, the
butyrate-response region in the GCAP gene contains the corresponding
IF-III element described in the present work on the IAP gene. It is
clear from our studies, however, that at least under some circumstances
IF-III can function even in the absence of NaBu, e.g., augmentation of SV-CAT reporter gene activity.
Although our transfection data demonstrate an important functional role for the IF-III cis element in mediating IAP transcription, the DNase I footprinting and gel shift analyses do not reveal any differences in nuclear binding as a function of either NaBu treatment or cell type (HT-29 vs. Hep G2 cells). We hypothesize therefore the existence of a protein(s) that is induced by NaBu and that acts as a "coactivator" of IAP transcription, regulating IAP transcriptional activation through an interaction with the IF-III nuclear proteins but without affecting their in vitro binding to IF-III. Such a coactivator(s) may be expressed in a cell type-specific manner because IF-III functions in HT-29 cells to a greater extent than in Hep G2 cells. Given the fact that NaBu is an inhibitor of histone deactylase, this model for IAP gene regulation is consistent with recent studies demonstrating the fundamental link between histone hyperacetylation and transcriptional activation (12, 31).
The key transcription factors and/or cis-regulatory elements, which are important for enterocyte differentiation, are largely unknown. Transgenic models using the 5'-flanking regions of the intestinal and liver fatty acid binding proteins (25) and sucrase (22) genes have provided some data as to which DNA segments function in regulating expression along the crypt-villus, craniocaudal, and developmental axes. In regard to transcription factors, the hepatocyte nuclear factor (32) and a homeodomain protein (Cdx2) (27) have been shown to regulate sucrase gene transcription and a novel transfactor (NF-LPH1) (29) has been shown to interact with the pig lactase promoter. Cdx2 may be of particular interest because its ectopic expression in the rat crypt IEC-6 cell line was associated with decreased cell growth and slight induction of sucrase expression (28). Sequence comparisons and direct competition experiments (data not shown) indicate that the nuclear factors that bind the IAP cis element (IF-III) are distinct from the previously characterized sites in these other intestine-specific genes.
Because IF-III contains a consensus Sp1 binding site (3), we examined whether Sp1 was contained within the HT-29 cell nuclear proteins responsible for the shifted complexes on EMSA. Our studies clearly indicate that both Sp1 and Sp3 bind to this DNA segment and comprise the IB-1 (both Sp1 and Sp3) and IB-2 (Sp3) shifted complexes. However, the protein binding activities that comprise IB-3 and IB-4 appear to be distinct from Sp1/Sp3. Sp1 has been found to interact with other transcription factors in regulating several genes and, interestingly, may be important in the NaBu induction of the human immunodeficiency virus type 1 gene expression (4). In some cases, Sp3 may repress the transcriptional activation mediated by Sp1 (13). In the case of the IF-III element, the binding of the non-Sp1/Sp3 proteins is unaffected by the presence of the Sp1/Sp3 antibodies, suggesting that their binding is independent from these proteins.
The EMSA demonstrate that the mouse IF-III element does not bind Sp1 or Sp3 and yet the transfection data indicate that this element functions almost to the same extent as the human IF-III element (see Fig. 3). As such, although there may be some importance for Sp1 and/or Sp3 in regard to IAP expression, it appears that most of the functional activity of IF-III relates to the proteins that comprise the IB-3 and IB-4 complexes and have yet to be identified.
In summary, we have identified a cis-regulatory element (IF-III) in the human IAP gene that appears to play a critical role in mediating its transcriptional activation during enterocyte differentiation in vitro. This GC-rich cis element binds Sp1 and Sp3, as well as one or more other proteins. IF-III functions in a cell type-specific manner, further suggesting an important role in IAP gene activation. Identification of the other transcription factors that mediate their effects via IF-III should provide important insight into the molecular mechanisms underlying intestinal epithelial differentiation.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47186 and DK-50623 to R. A. Hodin, and DK-34854 to the Harvard Digestive Diseases Center.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. A. Hodin, Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: rhodin{at}caregroup.harvard.edu).
Received 4 August 1998; accepted in final form 29 October 1998.
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