INTRODUCTION

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THE SMALL INTESTINAL epithelium is composed of a self-renewing monolayer of cells. As cells migrate from the crypts to the villus tips, they cease to proliferate and terminally differentiate before apoptotically dying and exfoliating. A single stem cell in each crypt gives rise to four distinct epithelial lineages, which include Paneth cells, goblet cells, enteroendocrine cells, and absorptive enterocytes (11). Each lineage of cells expresses a distinct repertoire of molecules that are required for physiological functions of the intestine.

To assimilate lactose, differentiated absorptive enterocytes express lactase-phlorizin hydrolase (LPH), an enzyme that hydrolyzes the -1,4-galactoside bond of the disaccharide into its monosaccharide components. Similar to other glycohydrolases, whose expression is restricted to absorptive enterocytes, LPH transcription is regulated during development (29). LPH expression on the surface of apical membrane microvilli, which commences at the crypt-villus junction, is programmed in enterocytes both during maturation and development and is unaffected by the presence of substrate in the lumen (20). In the human LPH promoter, DNA sequences upstream of a single TATA box possess binding sites for transcription factors that are expressed in a variety of cell lineages, including Sp1, SRF AP-2, CTF/nuclear factor-1, cyclic adenosine monophosphate response element binding protein, and Oct1/Oct2 (4). In addition, there is a GATA motif, ~60 bp upstream of the TATA box. This motif is conserved in the LPH promoters of all the mammalian species that have so far been analyzed (4, 5, 27).

Members of the family of GATA binding proteins all contain a segment with two conserved zinc finger DNA binding domains. These target the consensus sequence (A/T)GATA(A/G). GATA-1, GATA-2, and GATA-3 play a critical role in the regulation of hematopoiesis and are also expressed in nonhematopoietic tissues, such as Sertoli cells (GATA-1), endothelial cells (GATA-2), and nerve tissue (GATA-2 and GATA-3) (30).

Recently, the cDNAs of three new GATA binding proteins have been cloned (1, 17, 21). The mRNAs of GATA-4, GATA-5, and GATA-6 are expressed primarily in the heart and gut. Each of these transcripts encodes amino acid sequences in the zinc finger domains, which are conserved across species (1, 17, 26, 33). Both GATA-4 and GATA-6 are expressed in mammalian enterocytes. Although overlapping, the developmental and tissue-specific patterns of their expression differ significantly. In vitro, studies have indicated that GATA-4 contributes to the regulation of genes crucial for myocardial function and for extracellular matrix formation in the yolk sac (3, 13, 23). Targeted disruption of GATA-4 in mouse embryonic stem cells resulted in the loss of visceral endoderm formation (23). The rat homologue of GATA-6 has been shown to bind to and activate the promoters of both the - and -subunit genes of the H⁺-K⁺-adenosinetriphosphatase enzyme in the gastric parietal cells of rats and humans (26). These findings, along with the presence of conserved GATA elements in the promoters of intestinal fatty acid-binding protein and sucrase-isomaltase, suggest that GATA binding proteins may play a significant role in the regulation of some gut-specific genes. In this report, we show evidence that GATA-6 may play an important role in stimulating cell type-specific activation of the human LPH promoter.

	MATERIALS AND METHODS

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Cell lines, plasmids, RNA purification, and DNA sequencing. Caco-2 and HT-29 human colon adenocarcinoma cells, SW-13 human adrenal carcinoma cells, and cervical carcinoma HeLa cells (purchased from the American Type Culture Collection) and human jejunal HIE-7 cells (24) were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum. A 5.5-kb segment of the human LPH gene that contains 3.5 kb of the promoter region, exon 1 and part of intron 1, was excised from LPH 7 (4) (generously provided by N. Mantei, Swiss Federal Institute of Technology, Zurich, Switzerland), using Sac I and Sal I, and cloned into pUC 19, to produce p19/5.5.

Transfections. Transient transfections of Caco-2 cells were performed by electroporation, using a Bio-Rad gene pulser system and capacitance extender set at 250 V and 960 µF. Transfections were performed with 2 × 10⁷ cells, 40 µg of a selected LXP reporter construct, and 2 µg of pOBCAT0 (2). Cells grown to 80% confluence were trypsinized, thoroughly suspended in 0.5 ml of cell culture media, chilled to 4°C, and aliquoted in 0.4-cm electroporation cuvettes (Bio-Rad). After electroporation, the cells were cultured for 42 h in petri dishes 100 mm in diameter. To prepare cellular extracts from transfected Caco-2 cells, the adherent and nonadherent cells were harvested together. At the time of harvest for analysis of reporter expression, the adherent cells were 60-80% confluent and growing in clusters of 10-20 cells. In some experiments, 10 µg of an RSV/chicken GATA-6 expression vector (9) (generously provided by J. B. E. Burch, Fox Chase Cancer Center, Philadelphia, PA) was cotransfected with 35 µg of an LXP construct and 2 µg of pOBCAT0 into cells. Identical amounts of DNA were also transiently transfected into Cos-1 cells, by calcium phosphate transfection. Each culture of transfected cells was lysed in 100 µl of buffer containing 1% Triton X-100 and 0.1 M potassium phosphate (pH 7.8) and stored at 70°C. To measure chloramphenicol acetyltransferase (CAT) expression, we mixed 10 µl of the cleared supernatants in scintillation vials with 200 µl CAT reaction buffer containing 10 µl [³H]acetyl-CoA (Dupont NEN) diluted 1:35 with 2.5 mM acetyl-CoA, 50 µl 5 mM chloramphenicol, and 140 µl 0.1 M tris(hydroxymethyl)aminomethane (Tris) · Cl (pH 7.7). The assay mix was gently overlaid with 2 ml of Econofluor scintillation fluid and immediately placed in a 1209 Rack Beta liquid scintillation counter. The samples were then counted at 1-h intervals. Sample readings of reaction rates were used to establish transfection efficiencies. Based on transfection efficiencies, standardized volumes of each extract (range 4-20 µl) were then analyzed for luciferase content, using a Monolight 2010 luminometer (Analytic Bioluminescence Laboratories). For each cell line tested, differences in the volumes of extracts that were tested were less than twofold.

Ribonuclease protection, reverse transcription-PCR, and Northern blot analysis. To detect LPH mRNA from human enterocytes and Caco-2 cells ³²P radiolabeled riboprobes were made from a 578-bp LPH segment (bases 198 to +380) inserted into Bluescript SK and transcribed from the T3 and T7 promoters to produce sense and antisense probes, respectively. To detect reporter mRNA from Caco-2 cells transiently transfected with LXP plasmids, riboprobes were made from a Bluescript SK plasmid containing a 208-bp segment between bases 112 of LPH and +95 of the luciferase reporter. After linearization of the plasmid, the probes were transcribed from the T7 and T3 promoters to produce sense and anti-sense probes, respectively. After hybridization, the mRNA-riboprobe duplexes were treated with ribonucleases (RNases) A and T1, denatured, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then dried and autoradiographed. To determine the presence of LPH mRNA in cell lines, RNA samples were reverse transcribed with random hexamer primers and reverse transcriptase (RT). The RT products were then amplified with LPH primers to generate a 225-bp segment between +15 and +237 (5' primer: 5'-GAGCTGTCTTGGCATGTAGT-3'; 3' primer: 5'-GGAGACTGCTGAAGTACTCTGGC-3'), which was detected by agarose gel electrophoresis. For Northern blot analysis of GATA-6 mRNA, electrophoretically separated total RNA was transferred to a nylon membrane. GATA-4 mRNA was detected with a 44-base oligonucleotide probe (AGGCTGTGCAGGACCGGGCTGTCGAAGGGGCCGGCG- GAGGCGGC), which is complementary to both human and rodent GATA-4 forms, but not to other GATA-binding protein family members (1, 21, 26, 33), after ³²P end-labeling with T4 polynucleotide kinase. A human GATA-6 cDNA probe (kindly provided by T. Evans, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY), consisting of a 1.5-kb EcoR I fragment not overlapping with the conserved zinc finger DNA binding domain, was ³²P radiolabeled with random hexamers and the Klenow fragment of Escherichia coli DNA polymerase, and hybridized to membrane-bound mRNA. After washing, the membranes were dried and autoradiographed.

Nuclear extract preparation and analysis of protein-DNA binding. Nuclear extracts of cell lines were prepared according to a modified protocol of Dignam et al. (10), and extracts were stored at 70°C. Briefly, after being rinsed and scraped in cold phosphate-buffered saline (PBS), the cells were centrifuged. The cell pellet was then resuspended in an equal volume of cold 2× buffer A [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.25 mM dithiothreitol (DTT), 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin]. After the cells were swollen on ice and disrupted by passage through a 26-gauge needle, the mixture was layered over a sucrose cushion (0.8 M sucrose, 10 mM Tris · Cl, pH 7.5, 80 mM KCl, 5 mM MgCl₂, 0.25 mM DTT, and the protease inhibitors listed above) and centrifuged at 2,000 revolutions/min (rpm) for 15 min. The nuclear pellet was resuspended in an equal volume of 2× buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl₂, 200 mM EDTA, 0.25 mM DTT, and the protease inhibitors listed above in 20% glycerol). The mixture was then centrifuged at 37,500 rpm for 30 min, and the supernatant was stored at 70°C. Protein quantitation was determined by the Bradford protein assay (Bio-Rad) (7). EMSAs were performed using plasmid restriction endonuclease fragments or double-stranded DNA oligonucleotides that were ³²P end-labeled with T4 polynucleotide kinase. The radiolabeled double-stranded oligonucleotides or restriction fragment probes (0.2-0.4 ng) were added to 8 µl of EMSA binding buffer [25 mM Tris · Cl, 200 mM glycine, 1 mM EDTA, 10% glycerol, 1 mM 2-mercaptoethanol, 50 mM NaCl, 0.025% bromophenol blue, and 1 µg poly(dI-dC)] before the addition of 1 µl nuclear extract (5 µg/sample). The mixtures were incubated at room temperature for 30 min and then loaded onto 5% polyacrylamide gels. After separation of the protein-DNA complexes, the gels were dried and autoradiographed.

	RESULTS

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The LPH promoter is only activated in Caco-2 cells. To identify a human cell line that endogenously expresses LPH mRNA, PCR amplification was performed on the RT products of total RNA isolated from intestinal and nonintestinal cell lines using primers from LPH exon 1. Of the five cell lines tested, only Caco-2 cells exhibited expression of the LPH transcript (Fig. 1A). This human colon carcinoma cell line forms a monolayer of polarized cells, after growing to confluence, and expresses a number of proteins associated with the brush borders of small intestinal enterocytes (14, 15). LPH mRNA expression is low in preconfluent Caco-2 cells and increases significantly in postconfluent culture, as the cells become more differentiated (Fig. 1B). Although the electrophoretically separated RT-PCR product of preconfluent cell LPH mRNA was not detected by ethidium bromide staining, after Southern blotting it was detected by hybridization with a radiolabeled LPH cDNA probe (data not shown). A series of LPH promoter segments of increasing length were fused with luciferase (Fig. 2). To determine whether LPH promoter activity is restricted in a cell line-specific manner, the LXP fusion constructs were transiently transfected into Caco-2 cells as well as the other four cell lines tested above (Table 1). Luciferase expression was then analyzed after standardization to CAT levels generated by a cotransfected plasmid containing the CAT gene linked to the SV40 early promoter. All the cell lines tested were efficiently transfected with this CAT reporter plasmid and exhibited high levels of CAT expression. In sharp contrast, transcription from the LPH promoter was only measurable in Caco-2 cells. In these LPH-producing cells LPH promoter activity of the LXP constructs was high, whereas the promoterless construct was transcriptionally silent (Table 1). Initial promoter mapping, using the LXP constructs with increasingly extended LPH promoter segments, indicated that the region required for transcriptional activation in Caco-2 cells is between the LPH TATA box (LXP/46) and a site 66 bp further upstream (LXP/112). Consistent with this finding, a construct with an internal deletion between the TATA box and 112 (LXP/315) was transcriptionally inactive. These experiments revealed the unique ability of Caco-2 cells to activate transcription from the LPH promoter.

View larger version (109K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   Fig. 1.   A: lactase-phlorizin hydrolase (LPH) mRNA expression is detectable in postconfluent Caco-2 cells but not in other intestinal and nonintestinal cell lines. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from each cell line. Primers encompassing a region in the first exon of LPH from +15 to +237 were used to amplify a 225-bp segment (arrow indicates PCR product). Control lanes include primers without reverse-transcribed products (Primers only) and Caco-2 cell reverse-transcribed mRNA without primers (Template only). With control primers encompassing a segment in the 2-microglobulin cDNA, PCR of the RT products from each tested cell line produced equivalent levels of electrophoretically detectable products (not shown). B: LPH mRNA expression is induced after confluence in Caco-2 cell cultures; d, day; 2-microglob, 2-microglobulin control primers.

View larger version (6K): [in this window] [in a new window]   Fig. 2.   Schematic representation of LPH promoter segments of increasing length fused with luciferase (LXP). The 5' end of each promoter segment is numbered relative to the LPH translation start site. TATA represents the LPH TATA box, which is retained in all fusion constructs. ATG indicates the translation start site in the luciferase gene, and the filled arrowhead indicates the promoter transcription start site. Experimental results involving the use of these constructs are seen in Table 1 and Figs. 4 and 5.

Analysis of transcription start sites of the endogenous and transfected LPH promoters. To determine if the 5' end of LPH mRNA in Caco-2 cells is consistent with the transcription start site in human primary enterocytes, RNase protection analysis was performed. We hybridized 10 µg of total RNA from both human enterocytes and Caco-2 cells to a 578-bp riboprobe made from the LPH bases 198 to +380. Nuclease digestion resulted in a 399-bp protected fragment from both samples, suggesting that identical LPH transcription start sites are utilized 15 bp upstream of the translation start site (Fig. 3). This site is within a 5-bp segment that was previously found to encompass the transcription start site (4). To determine whether the appropriate LPH promoter sequence was utilized by the transfected LXP constructs, mRNA from Caco-2 cells transiently transfected with LXP/315 was analyzed by RNase protection. For this purpose, a 207-bp riboprobe, which consisted of 112 bp of the LPH promoter segment fused with a 95-bp segment of the reporter plasmid, was used. RNase protection analysis of mRNA from transfected cells demonstrated a 114-bp protected fragment, which was absent when mRNA from nontransfected cells was analyzed (Fig. 4). The size of the protected fragment is consistent with appropriate usage of the LPH transcription start site in the transfected LPH-luciferase gene.

View larger version (69K): [in this window] [in a new window]   Fig. 3.   RNase protection analysis demonstrates identical transcription start sites of the LPH gene in human enterocytes and Caco-2 cells. Arrow indicates a 399-bp protected fragment after nuclease digestion of LPH mRNA-probe hybrid products from human enterocytes and Caco-2 cells.

View larger version (84K): [in this window] [in a new window]   Fig. 4.   RNase protection analysis shows identity of the transcription start sites in a transfected LXP plasmid and the endogenous LPH gene. Arrow indicates a 115-bp protected fragment after nuclease digestion of the LXP/315 mRNA-probe hybrid product from transfected Caco-2 cells that is absent in nontransfected cells. The size of this product corresponds to the predicted transcription start site of the LPH gene.

Identification of an 18-bp LPH upstream segment that is required for LPH transcription and that binds a Caco-2 cell line-specific nuclear protein(s). To locate a regulatory element within the 66-bp region required for the activation of the LPH promoter in Caco-2 cells, LXP constructs were made that contained segments of the LPH promoter 39 and 49 bp upstream of the TATA box (LXP/85 and LXP/95, respectively). When transiently transfected into Caco-2 cells, these constructs exhibited no significantly greater activity than the TATA box construct LXP/46 (Fig. 5). This result indicated that the crucial area for LPH transcription activation is in an 18-bp segment that is located between 95 and 112 bp upstream of the LPH translation start site. This segment has been designated the LUE.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Mapping of an LPH upstream element (LUE) that is required for promoter transcription activation in Caco-2 cells. Transfections and measurements were performed as in Table 1. The values of luciferase expression represent fold increases over pXP1 background activity. Error bars indicate the standard errors of the mean (SEM).

View larger version (26K): [in this window] [in a new window]   View larger version (118K): [in this window] [in a new window]   View larger version (73K): [in this window] [in a new window]   Fig. 6.   A Caco-2 cell line-specific protein(s) (CCP) binds the LPH upstream element in a 10-bp stretch that contains a GATA consensus motif. A: wild-type (WT) and mutated sequences of a 22-bp segment containing the LUE (112 to 91) that were tested for binding to CCP. Bases that were mutated in tandem 5-bp segments are shown in lowercase letters. Bases that match the position of the GATA motif are underlined. Of the mutated LUE sequences, LUE/mutB and LUE/mutC contain substituted bases within the GATA consensus motif, whereas LUE/mutA and LUE/mutD contain substituted bases that flank this site. B: gel shift assay of the radiolabeled WT/LUE fragment bound by CCP. Where indicated, the binding reactions were performed with a molar excess of nonradiolabeled WT or mutated LUE competitors. Arrow indicates the site of migration of the CCP-LUE complex. C: formation of the CCP-LUE complex (arrow) is competed by excess molar amounts of a 119-bp plasmid restriction fragment encompassing LPH bases 112 to 1, but not of a control 125-bp restriction fragment. A series of more rapidly migrating complexes are not competed in a DNA sequence-specific fashion.

LUE bases within and 3' of the GATA motif are required for promoter activity. If CCP binding to the LUE is necessary to stimulate the LPH promoter, it is predicted that mutations that abolish the protein-DNA interaction also block transactivation. To test this prediction, four LXP constructs were made that incorporated each of the four tandem blocks of LUE mutations (LXP/MutA, LXP/MutB, LXP/MutC, and LXP/MutD), previously analyzed for binding in Fig. 6B. Expression by each of these altered LPH promoter-luciferase constructs in Caco-2 cells was compared with expression by the wild-type LUE reporter construct (LXP/112) (Fig. 7). Although none of the constructs containing mutated sequences demonstrated transcriptional activity as high as the wild-type control (LXP/112), the construct with mutations in the segment encompassing bases 112 to 107 (LXP/MutA) reduced transcriptional activity by only 1.5-fold (Fig. 7). In contrast, mutations that block CCP binding (LXP/MutB and LXP/MutC) reduced transcriptional activity more dramatically, consistent with a critical role for binding of the CCP in LPH promoter activation. Unexpectedly, LXP/MutD, which contains mutated bases located downstream of the GATA element that do not disrupt CCP binding, was also transcriptionally silent. Interestingly, the mutated bases in LXP/MutD are part of a DNA sequence in which 11 of 13 bases are identical with the reported consensus sequence for hepatocyte nuclear factor-1 (HNF-1) (28, 31). Although contiguous, the GATA element is not overlapping with this site.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Mutations that eliminate binding of the CCP to the LUE block transcriptional activation of the LPH promoter in Caco-2 cells. Transfections and measurements were performed as in Table 1.

GATA-6 stimulates the LPH promoter. GATA-4 and GATA-6 mRNA are both expressed in small intestinal epithelial cells. Compared with the rat heart mRNA, none of the cell lines tested by Northern analysis, including Caco-2 cells, expressed significant levels of GATA-4 mRNA (Fig. 8A and data not shown). In contrast, Caco-2 cells produce high levels of GATA-6 mRNA. Expression of GATA-6 transcripts at equivalent levels by the LPH nonproducer cell lines was not observed. The cell line-specific expression of high amounts of GATA-6 mRNA parallels the restricted expression of CCP detected by EMSA analysis. These data suggest that GATA-6 is a CCP that binds the LUE in vivo. Because of the difference between GATA-4 and GATA-6 mRNA levels, it is unlikely that GATA-4 is as abundant as GATA-6 in the CCP-LUE complex. This is supported by the observation that mobility of the CCP-LUE bound product, detected by EMSA analysis, is not affected by GATA-4 "supershifting" antibodies (data not shown). Unlike LPH expression, GATA-6 mRNA synthesis in Caco-2 cells is constitutive and is not influenced by confluence-induced differentiation (Fig. 8B).

View larger version (44K): [in this window] [in a new window]   View larger version (75K): [in this window] [in a new window]   Fig. 8.   Caco-2 cells express constitutively high levels of GATA-6 mRNA in a cell line-specific manner. A: postconfluent Caco-2 cells express negligible levels of GATA-4 mRNA but high levels of GATA-6 mRNA. Northern blot analysis of total RNA was performed with a GATA-4 probe complementary to both human and rodent species and a human GATA-6 probe. Rat heart mRNA served as a positive control for detection of GATA-4. Also shown is the ethidium bromide (EtBr) staining of 28S and 18S ribosomal RNA used to normalize the loading of samples. B: GATA-6 mRNA levels are the same in preconfluent and 7- and 10-day postconfluent Caco-2 cells.

View larger version (10K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Fig. 9.   Mutation of the GATA binding site eliminates LPH promoter stimulation by GATA-6. LXP plasmids were transiently transfected with (+) or without () a GATA-6 expression vector (pRSV/GATA-6). Measurements of luciferase expression were performed as in Table 1. The reporter activities (relative light units) are normalized to the activity of LXP/112 (WT) in the absence of pRSV/GATA-6. A: relative luciferase expression by Caco-2 cell transfectants. B: relative luciferase expression by Cos-1 cell transfectants.

	DISCUSSION

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The coincident expression of GATA-6 and LPH mRNA in Caco-2 cells and the ability of a GATA-6 expression vector to stimulate transcription from cotransfected LXP plasmids establish a connection between the GATA binding protein and LPH promoter activation. Consistent with an activating role for GATA-6, mutations that altered the GATA binding site also abrogated promoter activation in Caco-2 cells. The GATA sequence in the LUE, AGATAA, is identical to a sequence to which GATA-6 is known to bind preferentially, as measured by selected and PCR-amplified binding (J. B. E. Burch, personal communication). When linked to a minimal promoter, this sequence has been found to effectively mediate GATA-6-induced transactivation (9). Further evidence of the activation of the LPH promoter by GATA-6 was demonstrated by transiently cotransfecting the GATA-6 expression plasmid and LXP reporter constructs into Cos-1 cells, which ordinarily do not express significant amounts of GATA-6. In association with the absence of GATA-6 expression in these cells, the transfected LPH promoter was transcriptionally silent. However, when GATA-6 was produced by a transfected expression plasmid, LPH promoters containing wild-type sequences or a block of substitution mutations 5' to the GATA motif (LXP/MutA) in the LUE were strongly activated. In contrast, the promoters with mutations overlapping the GATA site (LXP/MutB and LXP/MutC) were not stimulated by GATA-6 to produce significant levels of luciferase.

In both Caco-2 cells and small intestinal enterocytes, the expression of GATA-6 alone is not sufficient to stimulate LPH transcription. This is supported by the observation that the level of GATA-6 mRNA expression is the same in preconfluent and confluent Caco-2 cells, whereas LPH mRNA expression is low in preconfluent cells and dramatically increases in 7- and 14-day postconfluent cells. There are also important differences in the time of onset of both GATA-6 and GATA-4 mRNA expression and LPH mRNA expression during development of the mouse. In the primitive gut, the transcripts that encode these GATA binding proteins are detectable as early as embryonic day 9.5 (21). In contrast, LPH mRNA only appears after the cytodifferentiation of endodermal cells into a monolayer of polarized epithelium, which occurs several days later (29). Thus, in addition to GATA binding protein expression, another differentiation-associated event(s) is required to stimulate transcription from the genomic LPH promoter, in both preconfluent Caco-2 cells and undifferentiated small intestinal epithelial cells. This may be an alteration(s) in chromatin structure or a modulation(s) of another nuclear protein(s) that activates or suppresses LPH transcription.

The inability of GATA-6 to activate the LPH promoter with mutations downstream of the GATA binding site (LXP/mutD) contrasts with binding studies that indicate that the GATA-binding CCP is able to form a stable complex with the LUE containing these mutations (LUE/mutD). There are two possible explanations that may account for this discrepancy. First, the binding affinity of a putative GATA-6/LUE complex in vivo might be modulated by bases 3' to the GATA motif. Previously, it has been observed that bases flanking the GATA motif affect the binding of GATA-1, GATA-2, and GATA-3 (16, 18). These proteins demonstrate base preferences that affect the binding affinities at bases +3 and +4 on the 3' end of the GAT core. Moreover, cooperative GATA protein binding to second contiguous sites composed of a variety of sequences that deviate from the consensus sequence has been demonstrated. However, it is unlikely that the base pair substitutions in LUE/mutD cause a significant change in the affinity of the GATA binding protein-DNA interaction, since the mutated bases 3' to the GATA motif, which disrupted LPH promoter transactivation, did not affect formation of the CCP-LUE complex in vitro. This was demonstrated by the identical efficiency of competition by titrated amounts of excess LUE/mutD and LUE/wild-type (LUE/WT) sequences for binding of the CCP with the radiolabeled LUE/WT.

A second explanation for the discrepancy between preservation of CCP binding to LUE/mutD and the loss of transactivation by LXP/MutD is that GATA-6 may require the presence of a downstream accessory factor to activate the LPH promoter. Analogously, GATA-1 has previously been shown to functionally interact with SP1 and erythroid Kruppel-like factor (12). It has recently been demonstrated that GATA-6 functionally cooperates with other transcription factors (9). In the vitellogenin II promoter, estrogen-induced transcription depends on the contribution of this GATA binding protein, which binds a site flanking the estrogen receptor element. In the case of the LUE, accessory factor binding may be a necessary step for LPH promoter transactivation. By EMSA analysis, we have found that a second Caco-2 cell protein binds a separate site that is immediately 3' of the GATA binding site (data not shown). This site (GTTAAATATTAAG) includes 11 of 13 bases that are identical with the reported consensus motif for HNF-1 (GTTAATNATTAAC) (28, 31). In vitro binding of the second Caco-2 cell nuclear protein at the HNF-1-like site in the LPH promoter occurs independently of binding at the GATA element by CCP and is selectively disrupted by the base substitutions identical with those in LUE/mutD. Both the related homeoproteins HNF-1 and HNF-1 have been identified in primary enterocytes and Caco-2 cells (31). These transcription factors, along with CDX homeodomain proteins, were shown to regulate the small intestine- specific product of the sucrase-isomaltase gene (SI) (31). Consistent with a role in the activation of LPH expression in the small intestine during murine development, HNF-1 mRNA is expressed by embryonic day 12.5 (19), which precedes LPH mRNA expression by a few days. The possibility that a GATA binding protein(s) cooperates with a member(s) of the HNF-1 family to stimulate the LPH promoter awaits further studies.

The region between the GATA motif in the LUE and the TATA box of LPH contains other elements known to bind nuclear transcription factors. A second GATA binding site located 77 to 72 bp upstream of the LPH translation start site, which has previously been designated as +1 and is ~62 to 57 bp upstream of the LPH transcription start site (4), does not stimulate transcription from the LPH promoter in Caco-2 cells, as measured in the transfection analysis. This element, whose orientation is opposite of the GATA element in the LUE, may not be positioned to effectively interact with other critical accessory protein binding sites and/or the RNA polymerase complex. The CE-LPH1 element is located 63 to 51 bp upstream of the LPH translation start site (27). This element has been shown to bind an intestinal nuclear protein (NF-LPH1), whose expression in pig enterocytes changes in parallel to lactase expression. Although it may also play a role in LPH activation, the range of NF-LPH1 expression does not parallel the cell type-restricted pattern of LPH mRNA synthesis (6).

Whether GATA-4, which similar to GATA-6 is expressed in the small intestine, stimulates or regulates LPH transcription has yet to be studied. Overlapping expression of GATA proteins in cells may lead to additive, synergistic, or inhibitory effects on LPH transactivation. There is precedent for such regulatory interactions between GATA factors. GATA-1 and GATA-2 show signs of mutual cooperativity. In addition, GATA-1 has been shown to repress GATA-2 expression (30). Studies of the ranges of expression and functional interactions of intestinal GATA binding proteins will lead to a clearer understanding of how they modulate one another in the gut. Not surprisingly, there are GATA motifs upstream of other genes whose expression is restricted to enterocytes, including those which encode the intestinal fatty acid binding protein (25) and SI (32). It will be of interest to determine whether an intestinal GATA binding protein(s) plays a functional role in regulating the transcription of these and/or other promoters that are active in intestinal cells.