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Endocrinology Vol. 140, No. 8 3478-3487
Copyright © 1999 by The Endocrine Society

ARTICLES

Identification of Essential Subelements in the hHSD17B1 Enhancer: Difference in Function of the Enhancer and That of the hHSD17BP1 Analog Is due to -480C and -486G1

Sinikka Leivonen, Yun-shang Piao, Hellevi Peltoketo, Pranee Numchaisrika, Reijo Vihko and Pirkko Vihko

Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu (S.L., Y.-s.P., H.P., P.N., R.V., P.V.), FIN-90401 Oulu, Finland; the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences (Y.-s.P.), Haidian, Beijing 100080, China; the Department of Obstetrics and Gynecology, Chulalongkorn Hospital (P.N.), Bangkok 10330, Thailand; and the Department of Biosciences, Division of Biochemistry, FIN-00014, University of Helsinki (P.V.), Finland

Address all correspondence and requests for reprints to: Prof. Pirkko Vihko, World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu, P.O. Box 5000, FIN-90401 Oulu, Finland. E-mail: pvihko{at}whoccr.oulu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The function of the gene encoding human 17ß-hydroxysteroid dehydrogenase (17HSD) type 1, the hHSD17B1 gene, is regulated by a cell-specific enhancer at position -662 to -392. The adjacent hHSD17BP1 gene, whose function is not known, contains an analogous region in its 5'-flanking region. The identity between the hHSD17B1 enhancer and the hHSD17BP1 equivalent is as high as 98%, i.e. they differ by only five nucleotides. Results from reporter gene analyses showed that the hHSD17BP1 analog, a pseudoenhancer, has only 10% the activity of the hHSD17B1 enhancer. Furthermore, the results indicate that the reduced function of the pseudoenhancer is a consequence of the presence of G and A at positions -480 and -486, whereas the hHSD17B1 enhancer contains -480C and -486G. In addition, three protected areas were localized to regions -495/-485 (FP1), -544/-528 (FP2), and -589/-571 (FP3) in deoxyribonuclease I footprinting analysis of the hHSD17B1 enhancer. Replacement of the footprinted regions with a nonsense sequence demonstrated that the FP2 region is the most critical for enhancer activity. Mutations of FP2 or a short palindromic region within it led to almost complete abolishment of enhancer activity. We have identified several subelements that are essential for appropriate function of the hHSD17B1 enhancer. The results also show that the hHSD17B1 and hHSD17BP1 genes operate differently despite the high homology between them.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERCONVERSION between low activity 17-ketosteroids and high activity 17ß-hydroxysteroids is catalyzed by 17ß-hydroxysteroid dehydrogenases (17HSDs), which thus modulate the biological activities of androgens and estrogens. Reductive 17HSDs are needed for the biosynthesis of testosterone and estradiol (E2), in particular, whereas the oxidative enzymes may protect tissues against excessive steroid action (1, 2, 3, 4). To date, eight different 17HSDs have been cloned (5, 6, 7, 8, 9, 10, 11, 12, 13), and each of them evidently plays a distinct role in steroid hormone action.

Human 17HSD type 1 mainly catalyzes the reduction of low activity estrone to high activity E2 (14, 15). The type 1 enzyme is the major 17HSD enzyme involved in E2 biosynthesis in developing ovarian follicles, and its amount correlates with E2 production from granulosa cells during the follicular phase of the menstrual cycle (16, 17). During pregnancy, the human type 1 enzyme is also abundantly expressed in placental syncytiotrophoblasts (18, 19, 20), in which placental E2 biosynthesis takes place. Variable concentrations of human 17HSD type 1 have also been found in benign and malignant breast tumors (21, 22), cell lines originating from breast epithelial cells (15), and endometrial tissue (20). Recent results suggest that expression of 17HSD type 1 is at least to some extent differentially regulated in ovarian granulosa, placental, and breast epithelial cells (16, 23, 24, 25, 26).

The gene encoding 17HSD type 1, hHSD17B1 (previously also called EDH17B2), contains two transcription start points, which results in two major 17HSD type 1 messenger RNAs (mRNAs), 1.3 and 2.3 kb in size (27). The concentration of the 1.3-kb transcript is correlated to the amount of 17HSD type 1 protein (15, 28), and this mRNA is also the target of several stimuli, such as growth factors (29, 30), retinoic acids (RAs) (26, 31), and cAMP analogs (32, 33). In previous studies our group has localized a cell-specific enhancer of hHSD17B1 between nucleotides -662 and -3932) and a silencer element in the region from -113 to -78 with respect to the transcription start point for the 1.3-kb mRNA (25). The elements are suggested to affect transcription of the 1.3-kb mRNA particularly. In addition, the fragment between nucleotides -78 and +9 is sufficient to promote transcription; thus, it is able to act as a basal promoter (25). Furthermore, our studies have shown that a RA response element (RARE) is situated in the hHSD17B1 enhancer, that the promoter region of hHSD17B1 contains activator protein-2 (AP-2)- and Sp-binding sites and that the silencer element includes a binding motif for GATA transcription factors (25, 34).

The hHSD17B1 gene is situated in chromosome 17 at region q21 in tandem with the gene hHSD17BP1 (6, 27, 35, 36) (Fig. 1Go). The gene hHSD17BP1 has also been found to be transcribed to some extent (37), but the possible role and function of the transcript, if any, are unclear. The differences between 17HSD type 1 and the putative protein product of hHSD17BP1 suggest that the latter does not have 17HSD activity (27, 38). Although hHSD17B1 and hHSD17BP1 share 89% overall identity, similarity between the hHSD17B1 enhancer and the analogous area in hHSD17BP1, hereafter called a pseudoenhancer, is as high as 98% (27, 38). In this study we show the significance of five dissimilar nucleotides in the hHSD17B1 enhancer and the pseudoenhancer with regard to enhancer activity. Furthermore, we have identified and analyzed three deoxyribonuclease I (DNase I) footprinted areas in the hHSD17B1 enhancer. A schematic presentation of the hHSD17B1 and hHSD17BP1 genes and hHSD17B1 regulatory elements is shown in Fig. 1Go.



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  		Figure 1. Schematic figure of hHSD17BP1 and hHSD17B1 genes. White and gray boxes represent the noncoding and coding areas in the hHSD17B1 gene and analogous areas in the hHSD17BP1 gene, and duplicate 1 and duplicate 2 show duplicated areas of the genes. The two transcription start sites in the 5'-region of the hHSD17B1 gene are indicated with arrowheads, and the enhancer region between them is marked with a black box. Ellipses depict the retinoic acid response element and binding sites for GATA, AP-2, and Sp factors, and the double headed arrow under AP-2 and Sp motifs symbolizes competition between the factors binding to the sites.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
The isotopes [{alpha}-32P]deoxy-CTP (3000 mCi/mmol) and [3H]acetyl coenzyme A [220 mCi/mmol, chloramphenicol acetyl transferase (CAT) assay grade] were purchased from Amersham Life Science (Aylesbury, UK). The transfection reagent N-[(1-(2,3-dioleoyloxy)propyl)]-N,N-trimethyl-ammoniummethylsulfate (DOTAP) and SuperFect Transcription Reagent were products of Boehringer Mannheim (Mannheim, Germany) and Qiagen, Inc. (Hilden, Germany), respectively. All media, buffers, supplements, and reagents for cell culture were obtained from Sigma Chemical Co. (St. Louis, MO) and Biochrom KG (Berlin, Germany), except for FCS, which was purchased from Bioclear UK Ltd. (Wiltshire, UK). The restriction endonucleases were from New England Biolabs, Inc. (Beverly, MA). Other reagents not mentioned in the text were obtained from Sigma Chemical Co., Boehringer Mannheim, and Merck A.G. (Darmstadt, Germany).

Preparation of the plasmid constructs for reporter gene analysis
The plasmid pCATBY and the nested deletion construct pCATBY-(-654/+9) were generated as described by Piao and co-workers (25). The expression plasmid pCATB3 was derived from pCAT-Basic vector (Promega Corp., Madison, WI) by replacing the original polylinker region HindIII-SphI-PstI-SalI-AccI-XbaI with HindIII-SphI-PstI-SalI-XmaI/SmaI-SacI-BglII-XbaI. An hHSD17B1 fragment, -662/-393, surrounded by HindIII and XbaI sites was inserted into pCATB3, and the construct was thereafter called pCATB3-(-662/-393). Fragment -660/-393 was linked by way of a SacII site to fragment -78/+9 as described previously (25) and further to pCATB3 backbone, resulting in the construct pCATB3(-660/-393)(-78/+9). pCAT-Control (pCATC) (Promega Corp.) containing simian virus 40 (SV40) promoter and enhancer and accomplishing high reporter gene expression, was used as a control vector. For the constructs, see Fig. 2aGo.



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  		Figure 2. Structure of the plasmid constructs used. A, At the top, the 5'-region of the hHSD17B1 gene is shown. The plasmid pCATBY-(-654/+9) contains hHSD17B1 fragment -654/+9 linked to the pCATBY vector, and in the construct pCATB3-(-660/-393)(-78/+9), the hHSD17B1 fragment -660/-393 has been connected to fragment -78/+9 and further to the pCATB3 backbone as the -662/-393 fragment of the hHSD17B1 gene in the construct pCATB3-(-662/-393). B, The construct pBLCAT4-(-662/-393) has been obtained by linking the -662/-393 fragment to the front of TK promoter in pBLCAT4. Arrows show the nucleotides of the hHSD17B1 enhancer that have been switched to the corresponding nucleotides of the hHSD17BP1 gene. Analogous constructs have also been prepared from the hHSD17BP1 gene in point and double mutated constructs. The locations of the protected areas in the DNase I footprinting assay of the hHSD17B1 enhancer replaced by a nonsense sequence in pBLCAT4-(-662/-393)FP1(-), -FP2(-), and -FP3(-) are shown by horizontal bars. C, Mutations introduced into position -539/-531 of the footprinted area FP2 are underlined.

 
The constructs pBLCAT4-(-662/-393) and -(-662/-393)p were obtained by linking the -662/-393 fragment of the hHSD17B1 and an analogous region from hHSD17BP1 to the front of the thymidine kinase (TK) promoter in pBLCAT4 vector, which was modified from pBLCAT2 (39) by deleting an Eco01091-NarI fragment according to the method of Kushner et al. (40). Mutations in the -662/-393 fragment were generated using the overlap-extension technique as described by Ho et al. (41). Five nucleotides in the hHSD17B1 enhancer were individually switched to those located in the hHSD17BP1 gene (-435 T->A, -452 A->G, -480 C->G, -486 G->A, or -615 T->C), and the mutated -662/-393 fragments were further linked to HindIII/XbaI sites of the pBLCAT4 vector. A double mutated construct pBLCAT4-(-662/-393)-(-480 C->G, -486 G->A) was generated similarly, as well as the construct pBLCAT4-(-662/-393)p-(-480 G->C, -486 A->G), in which nucleotides -480 and -486 of the (-662/-393)p fragment were replaced with those in the hHSD17B1 gene. The regions -495/-485 (FP1), -544/-528 (FP2), and -589/-571 (FP3), protected by nuclear extracts in the DNase I footprinting assay, were replaced with a nonsense sequence (GGGTTTCCCAAA)n, and as a result, pBLCAT4-(-662/-393)-FP1(-), pBLCAT4-(-662/-393)-FP2(-), and pBLCAT4-(-662/-393)-FP3(-) were obtained (Fig. 2bGo). The mutations (AATCAGATT -> GGAAACGAA and -> AGACACAAT) were introduced into the FP2 region at position -539/-531 of the -662/-393 fragment, and the resulting plasmids were named pBLCAT4-(-662/-393)-FP2m1 and pBLCAT4-(-662/-393)-FP2m2 (Fig. 2cGo). These constructs were prepared using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and reverse phase purified oligonucleotides (Kebo Lab AB, Stockholm, Sweden).

All constructs were verified by sequencing, using either a T7 Sequencing Kit (Pharmacia Biotech, Sollentuna, Sweden) or a PRISM AmpliTaq FS Dye Terminator Cycle Sequencing Kit (Perkin Elmer Europe B.V., Nieuwerkerk Ad Ijssel, The Netherlands) according to the instructions of the manufacturers. At least two independent purification preparations of each plasmid were mixed for transfection experiments.

Cell culture and transient transfection assays
Human choriocarcinoma cell lines (JEG-3 and JAR) and breast cancer cell lines (MCF-7 and T-47D) were obtained from American Type Culture Collection (Manassas, VA) and maintained according to the instructions of the supplier. For reporter gene analyses, JEG-3 cells were plated onto six-well plates, using 2.5 x 105 cells/well when transfected using DOTAP transfection reagent or 2.8 x 105 cells/well when SuperFect was used, 19–20 h before transfection. In the former case, 5.4 µg transfection reagent/ml medium and 2.7 µg plasmid construct were used for transfection. After 18 h the media were replaced, and the cells were cultured for a further 48 h before harvesting. In the transfections performed using SuperFect transfection reagent, 32 µg transfection reagent/ml medium and 1.5 µg plasmid construct were added to the cells. After 3 h the media were replaced, and the cells were cultured for a further 41–45 h before collection. All experiments were performed in duplicate and repeated at least three times.

CAT and protein assays
The harvested cells were subjected to four freeze-thaw cycles and to heat inactivation at 65 C for 20 min, after which the CAT activity of the samples was measured by fluor diffusion assay (42, 43). Protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Richmond, CA). CAT expression was described as picograms per mg total protein resulting from comparison of the CAT activity in samples with the CAT standard curve.

DNase I footprinting analysis and electrophoretic mobility shift assay (EMSA)
Double stranded fragments with 5'-protruding ends were labeled for DNA-protein interaction assays with [{alpha}-32P]deoxy-CTP, using Klenow fragments of DNA polymerase I. The preparation of nuclear extracts was previously described (34). DNase I footprinting analyses were based on the method described by Galas and Schmitz (44). Briefly, labeled -662/-393 fragments were incubated with 75 µg nuclear extract (JEG-3, JAR, T-47D, or MCF-7) or 50 µg BSA for 10 min at room temperature with 4 µg poly(dI-dC)·poly(dI-dC) in buffer containing 10 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 mM NaCl, 0.04% Nonidet P-40, and 2 mM phenylmethylsulfonylfluoride. Forty thousand counts per min of the 32P-labeled probe were then added to the reaction mixture, followed by further incubation for 20 min. Next, the binding mixtures were subjected to DNase I digestion for 1 min, in which 0.2 U DNase I for BSA samples and 2.0 and 3.0 U for other samples were used. Digestion was interrupted with DNase I stop solution (Pharmacia Biotech), after which the reaction mixtures were purified by phenol/chloroform extraction and ethanol precipitation. A sequencing ladder (A+G) was generated by the method described by Maxam and Gilbert (45). Purified samples were dissolved in FP loading dye (Pharmacia Biotech) and applied to 6% urea-polyacrylamide gel (Sequagel, National Diagnostics, Atlanta, GA). Finally, the gels were subjected to electrophoresis for 2 h at 1500 V, dried, and exposed to Kodak X-AR films (Eastman Kodak Co., Rochester, NY) for 24 h.

The EMSAs were performed as described by Bugge et al. (46) with modifications described by Piao et al. (25, 34). Ten micrograms of nuclear extract were first incubated at room temperature for 10 min in 20 µl binding buffer containing 10% glycerol, 20 mM HEPES (pH 7.9), 50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, 0.05% Nonidet P-40, and 2 µg poly(dI-dC)·poly(dI-dC). Next, 1.5–2.1 ng of a labeled probe (50,000 cpm) were added to the reaction mixture, and the incubation was continued for 30 min at room temperature. In competition experiments, a 100-fold excess of competitor was added together with the probe. Unrelated DNA was applied to some reactions to investigate the specificity of binding. The DNA-protein complexes were separated on a prerun (175 V, 20–25 min) 4% polyacrylamide gel (ratio of acrylamide to bisacrylamide, 19:1) containing 0.5 x TBE buffer (1 x TBE = 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA). The gels were subjected to electrophoresis for 3–4 h at 175 V, dried, and exposed to Kodak BioMax MR films (Eastman Kodak Co.) for 24–96 h. The sequence and position of each oligonucleotide used are illustrated in Table 1Go.


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  		Table 1. Sequences of the double stranded oligonucleotides used as a probe and competitors in EMSAs; the sequence of the sense strand has been shown

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Verifying the function of the -662/-393 fragment as an enhancer
The hHSD17B1 enhancer element, whose function is independent of its orientation, position, and the promoter type to which it is linked, has recently been localized between nucleotides -662 and -393 upstream of the hHSD17B1 gene (25). The region -113/-78, in turn, decreases the activity of the hHSD17B1 gene and contains binding sites for GATA-2 and -3 transcription factors (25, 34). To further verify the function of the hHSD17B1 enhancer, hHSD17B1 gene fragments containing the enhancer alone; the enhancer, silencer, and promoter together; or the enhancer linked to hHSD17B1 or TK promoter were linked in front of CAT genes, and the constructs obtained were transfected into JEG-3 choriocarcinoma cells. In JEG-3 cells, the enhancer efficiently increases promoter activity (25), the cells thus are suitable hosts for the experiments. As reported recently (25, 34), the absence of region -392/-79 resulted in enhanced reporter gene expression (Fig. 3Go). The hHSD17B1 enhancer and promoter linked together, construct pCATB3-(-660/-393)(-78/+9), drove reporter gene expression to the same degree as did a control vector, pCATC, which contains SV40 enhancer and promoter. Approximately 20 times greater expression was further achieved when the hHSD17B1 enhancer -662/-393 fragment was connected to TK promoter in the pBLCAT4 vector. However, when only the -662/-393 fragment was linked to the pCATB3 backbone, barely detectable reporter gene expression was obtained.



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  		Figure 3. Reporter gene analysis of the fragment -662/-393. Bars 1 and 7 show the background reporter gene expression derived from vectors pCATB3 and pBLCAT4, respectively. Bar 2 indicates the reporter gene expression induced solely by the enhancer fragment (-662/-393), whereas bar 3 shows reporter gene expression resulting from the hHSD17B1 fragment (-654/+9). In the construct pCATB3-(-660/-393)(-78/+9), the silencer element (-392/-79) has been eliminated by linking the enhancer adjacent to the proximal promoter, whereas in the construct pBLCAT4, the enhancer has been connected to the TK promoter (bar 6). pCATC (bar 5) was used as a positive control in reporter gene analysis. The CAT expression induced by pCATB3-(-654/+9) is defined as 100%, and those of all of the others are given as relative percentages. The results represent the mean ± SD of three independent experiments with duplicate samples in each.

 
Comparison of function of the hHSD17B1 enhancer and the pseudoenhancer and analysis of differences in the fragments
The -662/-393 fragment of the hHSD17B1 gene differs from the analogous region in hHSD17BP1, the pseudoenhancer, by only five nucleotides. However, the -662/-393 fragment operated, on the average, 10 times more effectively than the pseudoenhancer (-662/-393)p (Fig. 4aGo; see also Figs. 4bGo and 6Go). The five dissimilar nucleotides of the enhancer were then replaced one by one with those in the pseudoenhancer. Substitutions at -615 (T->C) and -435 (T->A) did not noticeably affect the activity of the enhancer, and replacement at -452 (A->G) augmented the function of the enhancer by 27%. However, changes at position -486 (G->A) and -480 (C->G) reduced enhancer activity to 31% and 55%, respectively, of the activity of the intact hHSD17B1 enhancer (Fig. 4aGo). After simultaneous changes of nucleotides -480 and -486 of the -662/-393 fragment, 8–17% of the original enhancer activity was achieved, which was as low as that of the pseudoenhancer, on the average (Fig. 4Go, a and b).



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  		Figure 4. Comparison of functions of the hHSD17B1 enhancer and the hHSD17BP1 pseudoenhancer. A, Identification of the nucleotides inflicting loss of activity of the pseudoenhancer. B, Verifying the importance of -480C and -486G in enhancer activity. In both panels, bar 1 indicates the background CAT activity induced by pBLCAT4 vector, and bars 2 and 3 show the CAT activities induced by hHSD17B1 enhancer (-662/-393) and the analogous region of hHSD17BP1 [(-662/-393)p]. Bars 4–9 of A show reporter gene expression derived from point or double mutated constructs, in which the dissimilar nucleotides of the hHSD17B1 enhancer have been switched to those located in the hHSD17BP1 pseudoenhancer. Bars 4 and 5 of B indicate the reporter gene expression originating from double mutated constructs, in which two dissimilar nucleotides in the hHSD17B1 enhancer have been replaced with the nucleotides of the pseudoenhancer and vice versa. In both panels, the CAT expression induced by pBLCAT4-(-662/-393) is defined as 100%, and those of all of the others are given as relative percentages. The results represent the mean ± SD of four or five independent experiments with duplicate samples in each.

 


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  		Figure 6. Reporter gene analysis of DNase I-protected areas. Bar 1 indicates the CAT activity originating from the pBLCAT4 vector, and bars 2 and 3 show the CAT activities induced by the hHSD17B1 enhancer (-662/-393) and the analogous region of hHSD17BP1 [(-662/-393)p]. Bars 4–6 demonstrate the reporter gene activities of mutated constructs in which the DNase I footprinted areas (FP1, FP2, and FP3) have been replaced with a nonsense sequence. The CAT expression induced by pBLCAT4-(-662/-393) is defined as 100%, and those of all of the others are given as relative percentages. The results represent the mean ± SD of three to five independent experiments with duplicate samples in each.

 
To further investigate the role of the nucleotides at positions -480 and -486 in the function of the hHSD17B1 and hHSD17BP1 enhancers, the nucleotides in hHSD17BP1 were replaced with those in the hHSD17B1 gene, resulting in a construct called pBLCAT4-(-662/-393)p-(-480 G->C, -486 A->G). As Fig. 4bGo shows, the reverse mutations in the pseudoenhancer resulted in reporter gene expression at the same level as that achieved by intact hHSD17B1 enhancer. The result confirms the significance of nucleotides C and G at positions -480 and -486, respectively, in the function of the hHSD17B1 enhancer.

DNase I footprinting analysis of the hHSD17B1 enhancer demonstrates three protected regions
To further locate subelements in the -662/-393 fragment that bind nuclear proteins and may thus be important for the function of the fragment, the sense strand end-labeled -662/-393 fragment was incubated with nuclear extract from JEG-3 and JAR choriocarcinoma cells and T-47D and MCF-7 breast cancer cells. The incubations were followed by DNase I digestion and phenol/chloroform extraction, after which the reaction mixtures were subjected to urea-polyacrylamide gel electrophoresis. As a result, three distinct regions of the -662/-393 fragment were observed to be clearly protected with the nuclear extracts used (Fig. 5Go). These areas are located within positions -485/-495 (FP1), -528/-544 (FP2), and -571/-589 (FP3). No apparent differences in protection between 17HSD type 1-expressing (JEG-3, JAR, T-47D) and nonexpressing (MCF-7) cell lines or between choriocarcinoma and breast cancer cell lines were detected.



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  		Figure 5. DNase I footprinting analysis of the hHSD17B1 enhancer. End-labeled sense strands of the hHSD17B1 enhancer region were incubated with nuclear extracts prepared from JEG-3 and JAR choriocarcinoma cells and T-47D and MCF-7 breast cancer cells. A BSA protein preparation was used to reveal nonspecific binding. The incubations were followed by DNase I digestion, after which the reaction mixtures were subjected to PAGE together with a sequencing ladder (G+A), which was generated using the method described by Maxam and Gilbert (45 ). With each nuclear extract, 2.0 and 3.0 U DNase I were used (see adjacent lanes). The positions of the protected nucleotides are shown with vertical bars (FP1, FP2, and FP3).

 
Significance of the DNase I footprinted areas with regard to the function of the hHSD17B1 enhancer
The importance of the protected areas was further studied with mutated hHSD17B1 enhancer constructs, in which the footprinted regions (FP1, FP2, and FP3) were replaced with a nonsense sequence. The nonsense sequence had been tested against the nuclear extracts used and was found to bind no proteins detectable by EMSA (data not shown). As shown in Fig. 6Go, substitution of the footprinted area FP2 with the nonsense sequence dramatically influenced enhancer activity, abolishing it almost completely, whereas mutation of the footprinted area FP3 had only a minor effect. Inefficiency of the nonsense sequence in the FP3 region further indicated that the nonsense sequence as such did not affect transcription rate. Replacement of the footprinted area FP1 decreased enhancer activity about 40%.

The nucleotide -486 is situated within the DNase I footprinted region FP1
The nucleotide -486G, whose replacement with A causes a reduction of enhancer activity by 69%, is localized within the DNase I footprinted region FP1 (-485/-495). To study the interactions between the FP1 region and the nucleotides -480C and -486G, a labeled (-511/-461) oligonucleotide (see Table 1Go) was incubated together with nuclear extracts prepared from JEG-3 and T-47D cells. The (-511/-461) oligonucleotide formed four complexes (complexes 1–4) with the nuclear extracts in EMSA (Fig. 7Go). The formation of the labeled complexes was prevented in the case of JEG-3 cells and was considerably decreased in the case of T-47D cells by an unlabeled oligonucleotide (-511/-461) and the analogous hHSD17BP1 oligonucleotide (-511/-461)p. The use of the labeled (-511/-461)p oligonucleotide also resulted in a complex pattern similar to that of the labeled (-511/-461) oligonucleotide (data not shown), additionally demonstrating that the replacement of -486G with A and -480C with G did not affect the binding seen in the EMSAs. The formation of complex 2 could be diminished by unrelated DNA, the (-552/-518) oligonucleotide, which indicated that the oligonucleotides (-552/-518) and (-511/-461) may bind the same factor. Instead, the (-511/-461)mut oligonucleotide containing a nonsense sequence as a replacement for the FP1, did not noticeably affect the formation of complexes, suggesting that the FP1 region of the (-511/-461) oligonucleotide is needed for binding the factors present in complexes 1–4. Similar results were obtained with the JAR and MCF-7 nuclear extracts (data not shown).



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  		Figure 7. Interactions between the FP1 region and the nucleotides -480C and -486G. Lane 1 represents the free probe, and lanes 2 and 7 demonstrate the binding between the (-511/-461) fragment and the JEG-3 and T-47D nuclear extracts. The unlabeled competitors have been identified above the lanes, and the influence of the oligonucleotides on the binding of nuclear proteins is illustrated in lanes 3–6 and 8–11. The complete sequences of the oligonucleotides used are listed in Table 1Go. The positions of the binding complexes, which were separated from each other in 4% polyacrylamide gel, are indicated by arrows on the left.

 
The DNase I footprinted area FP2 contains a palindromic region that is needed for the function of the hHSD17B1 enhancer
The FP2 region contains a palindromic area (5'-CCCACAATCAGATTAGA-3'), the role of which in the function of the enhancer was further studied by EMSA and reporter gene analysis. For these purposes, reporter gene constructs and EMSA oligonucleotides containing mutated palindromic areas (5'-AATCAGATT-3' -> 5'-GGAAACGAA-3', mut1, and -> 5'-AGACACAAT-3', mut2) were created. Reporter gene analysis with the mutated constructs pBLCAT4-(-662/-393)-FP2m1 and pBLCAT4-(-662/-393)-FP2m2 demonstrated the effects of the palindromic region on the function of the hHSD17B1 enhancer. Both of the constructs resulted in minor reporter gene expression, similar to that of pBLCAT4-(-662/-393)-FP2(-), in which the whole FP2 region had been replaced with a nonsense sequence (Fig. 8Go).



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  		Figure 8. Importance of the palindromic area within FP2 to hHSD17B1 enhancer activity. Bar 1 shows the CAT activity originating from the pBLCAT4 vector, and bars 2 and 3 indicate the reporter gene expression resulting from intact and FP2-mutated enhancer, respectively. The palindromic region located within FP2 has been mutated in the constructs pBLCAT4-(-662/-393)-FP2m1 and -FP2m2 (bars 4 and 5). The CAT expression induced by pBLCAT4-(-662/-393) is defined as 100%, and those of all of the others are given as relative percentages. The results represent the mean ± SD of three to five independent experiments with duplicate samples in each.

 
In EMSA, the labeled (-552/-518) oligonucleotide (see Table 1Go) formed two complexes, complex 5 and complex 6, with nuclear extracts prepared from JEG-3, JAR, MCF-7, and T-47D cells (Fig. 9Go). The formation of the complexes was hindered by the unlabeled oligonucleotide, but not by unrelated DNA in all cell lines. The presence of mutated oligonucleotides (-552/-518)m1 and (-552/-518)m2 (see Table 1Go) in the reaction mixture prevented the formation of complex 5, but not that of complex 6, from the JAR, MCF-7, and T-47D nuclear extracts. From the JEG-3 nuclear extracts, the mutated oligonucleotides were able to bind factors to some extent, as they decreased the formation of the labeled complexes.



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  		Figure 9. Effect of mutation of the palindromic area on the binding of nuclear proteins to the FP2 region. Lanes 1 and 12 represent free probe, whereas lanes 2, 7, 13, and 18 demonstrate the binding between the oligonucleotide (-552/-518) and JEG-3, JAR, MCF-7, and T-47D nuclear extracts. The influence of unlabeled competitors on the binding of nuclear proteins is shown in lanes 3–6, 8–11, 14–17, and 19–22, and each competitor has been marked above the corresponding lane. The complete sequences of the oligonucleotides used are listed in Table 1Go. The positions of the binding complexes, which were separated from each other in 4% polyacrylamide gel, are indicated by arrows on the left.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our recent studies have demonstrated that the regulation of hHSD17B1 gene expression is at least partially coordinated by the function of a cell-specific enhancer, silencer, and proximal promoter (25, 34). The enhancer is highly active in JEG-3 and JAR choriocarcinoma cells, and it also functions in T-47D breast cancer cells, but not in cell lines such as MCF-7, PC-3, and CV-1 (25), which do not express 1.3-kb 17HSD type 1 mRNA or 17HSD type 1 protein (15). In addition to the hHSD17B1 promoter, the enhancer is capable of increasing the action of other promoters, such as SV40 and TK promoters (Ref. 25 and the present study). The present results verify the enhancer-like characteristics of the -662/-393 fragment, as the fragment was not able to function properly without a promoter, i.e. it does not contain a promoter-like region itself.

The hHSD17B1 and hHSD17BP1 genes originate from the same ancestral gene as a result of duplication of a DNA fragment of about 6.2 kb in size (38). For an unknown reason the 5'-proximal regions of the genes, which include the hHSD17B1 enhancer, silencer, and promoter and the analogous areas of the hHSD17BP1 gene, have conserved an identity of 98%, whereas the overall identity between the genes is lower, 89%. However, a difference of one nucleotide in the TATA box-like sequence in the genes results in a reduction of promoter activity by half (47), and, as demonstrated here, the two dissimilar nucleotides in the enhancer and pseudoenhancer lead to a 10-fold difference in enhancer activity. Thus, variation of a few nucleotides is apparently enough to limit transcription of the hHSD17BP1 gene.

Using a sensitive RT-PCR, Touitou et al. (37) were able to show expression of both hHSD17B1 and hHSD17BP1 transcripts in carcinomatous cell lines and samples derived from breast, ovarian, endometrial, and cervical tissues. The method, however, does not distinguish between expression of 1.3- and 2.3-kb 17HSD type 1 mRNAs or several putative hHSD17BP1 transcripts; thus, the results do not necessarily demonstrate function of the hHSD17B1 enhancer and the hHSD17BP1 pseudoenhancer. In JEG-3 cells, transcripts of hHSD17BP1 have not been detected by ribonuclease protection analysis (33), whereas the cells evidently express 1.3-kb 17HSD type 1 mRNA and 17HSD type 1 enzyme (15). Hence, functioning of the hHSD17B1 enhancer and the hHSD17BP1 pseudoenhancer in JEG-3 cells is in line with expression of the hHSD17B1 and hHSD17BP1 genes, although the results do not exclude the role of other regulatory elements in the regulation of hHSD17B1 gene expression.

The two nucleotides, C and G, at positions -480 and -486, respectively, were demonstrated to determine the difference in the efficiency of function of the enhancer and the pseudoenhancer, and thus to be important for hHSD17B1 enhancer activity. The nucleotide -486G is actually situated within the FP1 region and adjacent to the RARE. Mutation of the whole FP1 region reduced enhancer activity by 40%, whereas replacement of G with A at -486 reduced this activity by almost 70% and the double mutation -480 (C->G)/-486 (G->A) reduced it by almost 90%. In line with that, the EMSA results showed that the FP1 region can form several complexes with proteins in the JEG-3, JAR, MCF-7, and T-47D nuclear extracts. The mutations in the nucleotides -480(C->G) and -486(G->A) did not affect the formation of the complexes seen in EMSA, whereas in reporter gene analyses the mutations almost abolished the enhancer activity, demonstrating their remarkable influence. The discrepancy between the binding demonstrated in EMSA and the effect of mutations on reporter gene analysis may be due to the slightly different binding conditions in the two assays or to the undetectable concentration of an essential factor interacting with the nucleotides -480C and -486G. The mutation of the RARE overlapping FP1, furthermore, affects only RA-dependent expression, not basal enhancer activity (25). All of the data together suggest that several factors can bind to the region, including RARE, FP1, and -480C, and at least part of them affect the hHSD17B1 enhancer activity. Comparisons of the FP1 region and the sequence around -486G against a database (Transcription Factor Database) yielded no promising matches for the known binding sites.

DNase I footprinting analysis of the hHSD17B1 enhancer revealed three protected areas among the bases -485/-495 (FP1), -528/-544 (FP2), and -571/-589 (FP3). No differences between the 17HSD type 1-expressing (JEG-3, JAR, and T-47D) and nonexpressing (MCF-7) cell lines were detected, indicating that the regions may be important for the function of the enhancer per se, but do not determine cell-specific expression. The FP2 region was found to be the most essential for enhancer action, which explains why shortening of the enhancer at its 3'-end from nucleotide -393 to -550 abolished reporter gene expression almost completely (25). Furthermore, the palindromic region within FP2 is evidently needed for proper functioning of the enhancer. The EMSA results additionally suggest that the binding between the (-552/-518) oligonucleotide and nuclear factor(s) from JAR, MCF-7, and T-47D cells can also take place outside the palindromic area, as the fragments containing the mutated palindromic area were able to prevent the formation of complex 5.

In conclusion, the present and previous results demonstrate that the hHSD17B1 enhancer consists of several essential subelements. Deletion of either the 5'-region -662/-551 or the 3'-region -393/-550 results in a dramatic drop in the efficiency of enhancer action (25), the latter part including the identified FP2 region and the nucleotides -486 and -480. The present results will facilitate the elucidation of the factors and mechanisms essential for the function of the enhancer and, furthermore, demonstrate differences in the operation of the hHSD17B1 and hHSD17BP1 genes.


   Acknowledgments
 
We thank Kristiina Kainulainen, M.Sc., for preparation of the nuclear extracts, and Ms. Helmi Konola for her skillful technical assistance.


   Footnotes
 
1 This work was supported by the Research Council for Health of the Academy of Finland (Projects 3314 and 40990) and Training Grant M8/181/4/P.201 from the WHO Special Program of Research, Development, and Research Training in Human Reproduction (to Y.-s.P.). The WHO Centre for Research on Reproductive Health is supported by the Ministries of Education, Social Affairs and Health, and Foreign Affairs of Finland. Back

2 Resequencing of the 5'-region of hHSD17B1 revealed a missed nucleotide at position -121; therefore, the numbering of the enhancer is different from that given by Piao et al. in 1995 (25 ). Back

Received October 15, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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