INTRODUCTION

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Prolactin is the most functionally diversified pituitary hormone that exerts a wide range of biological actions involving lactation, reproduction, growth and metabolism, water-salt balance, immune regulation, and maternal behavior through specific prolactin receptors (PRLR)¹ present on various prolactin target tissues (1-3). The functional diversity of this hormone and the wide distribution of its receptors have suggested that a complex form of regulation determines the levels of PRLR expression in individual prolactin target tissues. This has been revealed by the recent demonstration of multiple and tissue-specific promoter control of the prolactin receptor gene expression in rat gonadal and non-gonadal tissues (4, 5). PRLRs are present as long and short forms and are encoded by multiple PRLR mRNA transcripts, which are derived from alternative transcription initiation from different PRLR promoters at the 5'-end, alternative splicing of the 5'-noncoding exon (exon 2), and the coding exons (3, 4). Three PRLR gene promoters have been identified in the rat. Promoter I, specifically utilized in gonadal cells, contains a functional SF-1 consensus element that binds steroidogenic factor-1 (SF-1), a specific zinc finger DNA-binding protein also known as Ad4BP (6), which is an essential transcriptional activator for this promoter (5). Promoter II is specifically utilized in the liver (4, 7) and its activity is regulated by the liver-specific hepatic nuclear factor 4 (HNF4) through binding to an HNF4 element in the promoter region (7). By contrast, the generic promoter III (PIII) is commonly utilized in the rat gonads and liver and as a sole promoter in the rat mammary gland (4, 5). Sequence analysis of PIII revealed C/EBP- and Sp1-binding elements. It is likely that this promoter is also utilized in other PRLR-expressing tissues that have not been examined. However, the mechanisms underlying regulation of the commonly utilized PRLR promoter III has not been investigated.

In the present study, we aimed to define the promoter domain of PIII and to identify the cis-elements and trans-factors that regulate its promoter activity in gonadal and non-gonadal cells. We demonstrated that C/EBP and Sp1 bind to the 5'-flanking region and regulate the PIII activity in homologous and heterologous cells. In addition, a proximal downstream sequence element (DSE) was essential for basal promoter activity. Finally, we have determined that the promoter III homolog in the mouse shares structural and functional similarity with the rat PIII, whereas in this species, the PI homolog exhibits minimal promoter activity due to mutation of the SF-1 element that renders this element non-functional. Taken together, these findings indicate that PIII is of central importance in the control of transcription of the prolactin receptor gene.

	MATERIALS AND METHODS

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Animals-- 24-Day-old immature female rats (Charles River, Wilmington, MA) were injected subcutaneously daily for 3 days with 1.5 mg/day 17-estradiol (Sigma). On the 4th day, the animals were sacrificed (CO₂ asphyxiation) and ovaries removed for preparation of ovarian granulosa cells. The studies were approved by the NICHD Animal Care and Use Committee (protocol 97039).

Culture of Cell Lines and Primary Rat Ovarian Granulosa Cells-- A stable steroidogenic cell line, mouse Leydig tumor cells (MLTC), which expresses prolactin and luteinizing hormone receptors (kindly provided by Dr. R. V. Rebois, National Institutes of Health, Bethesda, MD), was maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum and 1× antibiotic/antimycotic mixture (Life Technologies, Inc.). The human hepatoma cell line (HepG2, American Type Culture Collection, Rockville, MD), which expresses prolactin receptors, was maintained in minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, non-essential amino acids, sodium pyruvate, and antibiotic/antimycotic mixture. Drosophila SL2 cells (8) were maintained at room temperature in M3 medium (Quality Biological, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum and antibiotic/antimycotics. Primary rat ovarian granulosa cells were prepared from estrogen-primed immature female rat ovaries as described previously (5). Granulosa cells were suspended in Dulbecco's modified Eagle's medium/F12 (Life Technologies, Inc.) supplemented with 1% fetal bovine serum, 15 ng/ml ovine follicle-stimulating hormone (oFSH-20, National Pituitary Program, NIDDK), 10 ng/ml testosterone (Sigma) and plated at a density of 0.2 × 10⁶ live cells/cm² in 6-well plates and cultured at a CO₂ incubator for 3 days before transfection.

Construction of Reporter Plasmids with Deletions or Site-directed Mutagenesis-- DNA fragments with various lengths of 5'-flanking regions and partial first non-coding exon sequence were generated by PCR amplification. All synthesized oligodeoxynucleotides used were obtained from Genosys (The Woodlands, TX). DNA fragments with mutation of specific DNA element(s) were generated by introducing mutated nucleotides into the PCR primers or by two-step PCR when mutation was directed into the middle region of the DNA fragment. The DNA fragments generated by PCR were ligated to the vector pGL2 (Promega, Madison, WI). The pGL2 constructs were numbered relative to the translation initiation codon (PIII(n/n)LUC). All resulting plasmid constructs were restriction-mapped and sequenced for the inserts.

Rapid Amplification of cDNA 5'-Ends (5'-RACE)-- 5'-RACE analysis was performed as described previously (5) using total RNAs from MLTC cells that were transfected with luciferase reporter plasmids of PIII(437/179)/LUC or its mutation of the putative C/EBP element. First strand cDNA was synthesized using primer GS1 (5'-TTCACCTCGATATGTGCATCTG-3') (+128 to +149 of the luciferase coding region) and 3'-end-tailed with dCTP using terminal deoxynucleotidyltransferase, followed by PCR with reverse primer GS2 (5'-GCGTATCTCTTCATAGCCTTATGCAG-3') (+76 to +101 of the luciferase coding region) and forward dG-adaptor primer (5'-GCGAATTCTCGAGATCTGGGIIGGGIIGGGIIG-3'), where I represents inosine. The PCR products were resolved on 1.5% agarose gel and subjected to Southern blot analyses using a nested oligonucleotide probe (5'-CTGTCTCTGACAGGTAAAG-3', 197/179) within E1₃, labeled with [-³²P]ATP (3000 mCi/mmol, NEN Life Science Products) at the 5'-end with T4 kinase (Life Technologies, Inc.).

Preparation of Nuclear Proteins and Electrophoresis Mobility Shift Assay (EMSA)-- Nuclear proteins from MLTC, HepG2, and rat granulosa cells were extracted essentially as described previously (5). Synthesized oligomers or PCR-generated fragments were 5'-end-labeled with [-³²P]ATP (3000 mCi/mmol, NEN Life Science Products) and were used as probes in EMSA. 3-5 µg of nuclear proteins were incubated in 20 µl of reaction containing 12 mM HEPES, pH 7.6, 60 mM KCl, 4 mM Tris-HCl, 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 25 µg/ml polydeoxyinosinic-deoxycytidylic acid, with or without excess molar of unlabeled DNA competitors on ice for 15-30 min, followed by addition of 50,000 cpm of the probe. For supershift assays, specific antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) against C/EBP (pan-antibody) or its subtypes , , , and  or against Sp1 or Sp3 were added to the reaction mixture 30 min prior to the addition of the probe. DNA-protein complexes were resolved on 5% native polyacrylamide gel electrophoresis and were autoradiographed on Kodak X-Omat films.

Characterization of Mouse PRLR Genomic Clone from BAC (Bacterial Artificial Chromosome) Library-- A BAC DNA clone with ~120-kilobase insert containing the mouse PRLR genomic regions corresponding to both the rat promoters III and I was obtained from BAC library (Genome Systems, Inc. St. Louis, MO) by hybridization screening using rat PIII and PI region probes. The BAC DNA was prepared as described by the vendor's protocol (Genome Systems, Inc.). DNA was digested with EcoRI and BamHI and was further analyzed by Southern hybridization using rat promoters III or I region probes. Smaller fragments corresponding to the rat PIII or PI were subcloned into plasmid pGEM4Z (Promega) and were sequenced using Sequenase version 2.0 (U. S. Biochemical Corp.). PCR-generated DNA fragments from the mouse genomic clone with sizes corresponding to their rat counterparts were subcloned into luciferase reporter plasmid pGL2. The resulted reporter constructs of putative mouse PRLR promoters were transiently expressed in MLTC cells as described for the rat constructs (see above).

Transfection of Plasmids into Mammalian and Drosophila Cells-- A recently developed highly efficient transfectant Cytofectin GS3815 (Glen Research, Sterling, VA) (9) was utilized in this study for effective transfection of plasmids into both stable cell lines MLTC, HepG2 and Drosophila SL2 cells, and primary culture of rat granulosa cells. Cell lines were grown to ~70% confluence 36-48 h after plating into the six-well plates. Primary granulosa cells were maintained for 3 days before transfection. For transfection of each individual cell type, 3 µg of luciferase reporter plasmid DNA, 0.5 µg of pRSV--gal (galactosidase) plasmid, and 10 µg of cytofectin were used for each well (~9.5 cm²) of six-well plates. The luciferase activity values were normalized to internal control -galactosidase expression. Also, separate wells were transfected with SV40 promoter plasmid as a positive control. In cotransfection experiments, 0.5 µg of Sp1 cDNA (pPacSp1, kindly provided by Dr. Robert Tjian, Howard Hughes Medical Institute, Berkeley, CA) or 1 µg of C/EBP cDNA (pMEX-CRP2, kindly provided by Dr. Peter Jonhson, NCI, Frederick, MD) was mixed with the reporter plasmid DNA. DNA/liposome mixture was made by gently mixing 50 µl of DNA solution in serum-free Opti-MEM (Life Technologies, Inc.) and 50 µl of Cytofectin suspended in Opti-MEM for each well. The mixture was allowed to stand for 10-15 min at room temperature and was brought to 1 ml by adding 900 µl of 10% fetal bovine serum-containing media for respective cell lines. Cell culture media were replaced with this mixture (1 ml/well) and incubated 6 h for cell lines or 14 h for primary granulosa cells at 37 °C in a CO₂ incubator or at 22 °C, for SL2 cells only. For cell lines, the transfectant/DNA mixture was replaced with respective fresh media, and culture was continued for 36 h before termination. For granulosa cells, the mixture was replaced with Dulbecco's modified Eagle's medium/F12 medium supplemented with hCG (National Pituitary Program, NIDDK) or 8-bromo-cAMP (Sigma) at final concentrations of 0.5 nM or 1 mM, respectively, and culture was continued for 6 h before termination. Whole cell lysates were used for measurement of luciferase activity (10) and -galactosidase activity (11).

	RESULTS

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Promoter Domain of PIII Resides at 437 to 179-- 1.3 kilobases of genomic DNA (1427 to 179) containing 5'-flanking region and partial first exon sequence (E1₃) of the rat PRLR gene was initially examined for promoter activity in luciferase reporter gene construct in transiently transfected gonadal (MLTC) and non-gonadal (HepG2) cells. This fragment containing promoter III exerted strong promoter activity in both cell types, in contrast to PI or PII, which showed specific activation in MLTC or HepG2, respectively (3). Subsequently a series of deletions of this genomic fragment in luciferase reporter gene constructs were characterized in MLTC and HepG2 cells (Fig. 1). Deletions from 1427 to 437 had no significant effects on the luciferase activity in both MLTC and HepG2 cells (constructs 1-6). However, deletion from 437 to 344 reduced the promoter activity to 23 and 58% of full activities in MLTC and in HepG2 cells, respectively (construct 7), indicating that the 437 to 344 region is required for full promoter activation in both cell types. However, when the 5'-flanking fragment (506 to 345) containing this region was examined for promoter activity, it had only minimal activity in both cell types (~15% of full activity) (construct 8). These results indicated that both the 5'-flanking region and part of the exonic sequence downstream of the transcription start site (TSS) are also necessary for full promoter activation, and therefore the promoter domain of PIII resides at 437 to 179. This 258-bp promoter domain contains neither a canonical TATA box sequence nor consensus initiator sequences, although CT-rich sequences surrounding the translation start sites are present. A C/EBP-like element was identified at 398 in the 5'-flanking region of the promoter domain, 58 bp upstream of the major transcription start site at 340 (4). Three bp downstream of this C/EBP element resides a (CTC)₃ repeat, a variant Sp1-binding site, referred as Sp1_a (386). Two additional Sp1 consensus sequences downstream of the TSS are present at 273 and 261, referred as Sp1_b and Sp1_c, respectively. In addition, an AP2 consensus sequence 8 bp downstream of the major TSS is also present. These putative regulatory elements were further examined for their nuclear protein binding capability by EMSA and for their functionality in promoter activation by mutagenesis analysis (see below).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Basal luciferase activities of deletion constructs of the 5'-flanking region PIII-exon E13 in MLTC and HepG2 cells. Left, schematic representation of deletion constructs expressed in MLTC and HepG2 cells with the promoter domain and putative cis-regulatory elements shown above (see also Fig. 5). Luciferase reporter gene constructs with progressive 5'-deletions of the 5'-flanking region as well as the 3'-deletion of the adjacent first exon E13 are shown. Plasmid constructs are identified by nucleotide positions calculated from the first base of translation initiation codon (ATG, +1). In vivo transcription initiation sites are indicated by arrowheads. Right, activities of the constructs in MLTC and HepG2 cells are expressed as fold induction of luciferase activity over that of the promoter-less basic vector shown on the left y axis or as light units rLU/µg cellular protein shown on the right y axis. Results are expressed as mean ± S.E. of four to seven independent experiments in triplicate wells for each construct. The average luciferase activities of SV40 (pSV40Luc) in concomitant transfection experiments were 187,615 rLU/µg of protein (3.8 times of construct 437/179Luc, construct 6) in MLTC or 25,960 rLU/µg of protein (13 times of construct 6) in HepG2 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Determination of the transcriptional initiation site for the expressed PRLR-PIII/LUC gene constructs in MLTC cells. Above, Southern blot analysis of 5'-RACE products of reporter gene-derived mRNA transcripts using specific oligomer 5'-CTGTCTCTGACAGGTAAAG-3'(P#74). The left lane (WT) indicates mRNA derived from wild type (WT) PIII domain (437/179) and the right lane (C/EBPx) indicates mRNA from the same promoter region with mutation of the putative C/EBP element. The major extended product is 350 bp as indicated on the right side, and the DNA markers are shown on the left side in base pairs (bp). Note that the difference in signal strength of the bands does not represent quantitative changes of promoter activity from the two constructs, since the 5'-RACE analysis was utilized only for qualitative information of transcription initiation sites. Below, the scheme for 5'-RACE analysis. The sequences for GS1 and GS2 are 5'-TTCACCTCGATATGTGCATCTG-3' and 5'-GCGTAT CTCTTCATAGCCTTATGCAG-3', located at +149 and +101 of the luciferase coding region, respectively. The wavy line is a portion of the polylinker sequence from the vector (55 bp). CC indicates dC-tailing of the first strand cDNA synthesized with GS1. The sequence of dG adaptor primer is 5'-GCGAATTCTCGAGATCTGGGIIGGGIIGGGIIG-3'. P#74 is the oligomer located within the PIII domain and used for Southern blot hybridization as shown above. The calculated size of the transcript derived from the reporter constructs is 350 bp.

C/EBP Specifically Binds to the C/EBP Element in the 5'-Proximal Region of Promoter III in MLTC and HepG2 Cells-- To examine transcription factor-binding sites in the PIII domain, EMSA was employed using double-stranded DNA probes of various lengths. We first analyzed the putative C/EBP-like binding sequence GTTGCAACAC (398) (C/EBP consensus sequence RTTGCGYAAY where Py indicates) within the 258-bp promoter domain (Fig. 1). When a short probe (probe III 416/388) containing this site was used in EMSA, a prominent protein complex was revealed in the nuclear extracts from MLTC cells (Fig. 3A, lanes 1 and 9). This complex was completely inhibited by preincubation of the nuclear extract with a 250-fold molar excess of unlabeled DNA (lanes 2 and 10) but not with unlabeled mutated C/EBP sequence (C/EBPx m2, lanes 3 and 11). Similarly, this complex was inhibited by preincubation with unlabeled C/EBP consensus oligomer (CS, lane 4) but not by its mutant (CSx, lanes 5). These results indicated that this C/EBP-like sequence was specifically bound by a protein complex related to C/EBP family of proteins present in MLTC nuclear extract. To examine the nature of this protein complex, a panel of C/EBP antibodies including one that exhibits cross-reactivity to all subtypes (pan-antibody), and those specific for C/EBP subtypes , , , and  were used for gel supershift assay. This protein complex was clearly supershifted by C/EBP pan-antibody (lanes 6, 7, and 12) as well as by anti- subtype antibody (lane 14) but not by antibodies to , , and  subtypes (lanes 13, 15, and 16) or normal rabbit serum (lanes 8 and 17). These results demonstrated that the C/EBP-like element in the PIII domain binds specifically to C/EBP present in MLTC nuclear extracts.

View larger version (75K): [in this window] [in a new window]   Fig. 3.   Determination of C/EBP binding to the PIII region in nuclear extracts of MLTC and HepG2 cells by EMSA. A, EMSA of C/EBP binding in MLTC cells. Double-stranded DNA probes III and I containing C/EBP-like element (see C) were used for the EMSA. Probes were incubated with MLTC nuclear proteins in the absence (lanes 1, 9, 18, and 26) or presence of excess molar of unlabeled DNA competitors of wild type DNAs (lanes 2, 4, 10, 19, 27, and 29) or mutated DNAs (lanes 3, 5, 11, 20-22, 28, 30, and 31), or in the presence of normal rabbit serum (NRS, lanes 8, 17, 25, and 34) or specific antibodies against C/EBP (pan-antibody, lane 6, 7 and 12) or its subtypes , , , and  (lanes 13-16, 23, and 32), or against Sp1 protein (lanes 24 and 33). Specific C/EBP binding is indicated by arrows, and C1 and C2 are DNA-protein complexes that are not related to C/EBP but to Sp1 family proteins. B, EMSA of C/EBP binding in HepG2 cells. Probe III was incubated with HepG2 nuclear protein in the absence (lane 2) or presence of unlabeled DNAs with wild type (lanes 3 and 5) or mutated C/EBP element (lanes 4 and 6) or in the presence of normal rabbit serum (NRS, lane 8) or C/EBP antibody (lane 7). Lane 1 shows C/EBP binding in MLTC nuclear extract, which migrates differently as compared with that in HepG2 cells. C, double-stranded DNAs used as probes or competitors in EMSA, with wild type (in capital letters) or mutant (in lowercase letters) elements (C/EBP and Sp1a), derived from PIII domain (I, II and III) or C/EBP consensus oligomers (CS or CSx).

View larger version (98K): [in this window] [in a new window]   Fig. 4.   Determination of three Sp1-binding sites in the PIII domain in nuclear extracts of MLTC, HepG2, and rat granulosa cells by EMSA. A, EMSA of Sp1 family protein binding to Sp1a site in nuclear extracts of MLTC, HepG2, and rat granulosa (rGC) cells. Double-stranded DNA probe II (lanes 1-8 and 16-22) or probe II with C/EBP mutation (IIm1, lanes 9-15) and probe I (lanes 23-27) were used in EMSA. Probes were incubated with nuclear extracts in the absence (lanes 1, 9, 16, and 23) or presence of excess molar of DNA competitors of unlabeled probe sequence (lanes 2, 10, 17, and 24) or Sp1a-mutated sequence (lanes 3, 11, and 18), in the presence of NRS (lanes 8, 15, and 22) or specific antibodies against Sp1 and Sp3 (lanes 4-6, 12-14, 19-21, and 25-27), or against C/EBP (lane 7). C1 and C2 DNA-protein complexes are indicated by arrows, corresponding Sp1/Sp3 and Sp3, respectively. C/EBP detected in MLTC using probe II is also indicated. B, EMSA of Sp1 protein binding to Sp1b and Sp1c sites in nuclear extracts of MLTC, HepG2, and rat granulosa (rGC) cells. Double-stranded oligomer probe Sp1b, c (5'-CGGGTCTTCTGGGCTGGGCTTTCCCCGCCTTCCTGC-3') was used in EMSA. The mutated Sp1b, c sequence is 5'-CGGGTCTTCTGttaTGGGCTTTCCaatCCTTCCTGC-3'. Probes were incubated with nuclear extracts of MLTC, HepG2, and rat granulosa (rGC) in the absence (lanes 1, 8, 15, and 22) or the presence of DNA competitors of unlabeled probe sequence (lanes 2, 9, and 16) or Sp1 single or double mutated sequences (lanes 3-5, 10-12 and 17-19) or in the presence of normal rabbit serum (NRS)(lanes 7, 14, and 21), specific antibodies against Sp1 (lanes 6, 13, 20, and 23), or Sp3 (lane 24), or both (lane 25). Specific Sp1/Sp3 DNA complexes are indicated by arrows.

Sp1-binding Sites in the PIII Region-- As indicated above, a variant Sp1-binding sequence (Sp1_a, CTCCTCCTC, 386), 3' adjacent to the C/EBP element is present within the promoter domain (Fig. 3C). In EMSA of MLTC nuclear extracts using probe I, it was shown that the two complexes C1 and C2 were not associated with the C/EBP-binding site, since unlabeled DNA sequence I (Im1-Im3) or II (IIm1) with mutations of C/EBP showed competition for the C1 and C2 complexes (Fig. 3A, lanes 20-22 versus 19, and lane 31 versus 29). Moreover, unlabeled sequence III of wild type that only contains the C/EBP element or its mutant did not compete for the C1 and C2 complexes (lanes 27 and 28). However, C1 and C2 complexes were not competed by unlabeled sequence II with mutated (CTC)₃ repeat (IISp1ax) when using probe I (Fig. 3A, lane 30) or using probe II (416/373) (Fig. 4A, lane 3), indicating that C1 and C2 were specifically associated with this CTC repeat. A specific Sp1 antibody partially supershifted C1 (Fig. 3A, lanes 24 and 33, and Fig. 4A, lane 4 versus lane 3) but not C2. In contrast, C2 was completely supershifted by a specific Sp3 antibody (Fig. 4A, lane 5). When both Sp1 and Sp3 antibodies were used, C1 and C2 complexes were both completely supershifted (lane 6). These results indicated that C1 complexes contain both Sp1 and Sp3 proteins, whereas the C2 band corresponded to binding of Sp3 protein to the Sp1 element. Therefore, the CTC repeat (Sp1_a) in the PIII domain can bind to both Sp1 and Sp3 proteins present in MLTC nuclear extracts.

Transcriptional Activation of PIII by C/EBP and Sp1_a Elements in MLTC and HepG2 Cells-- To assess the functional roles of the nuclear protein-binding sites within PIII region, luciferase reporter genes directed by promoter domains of wild type or various mutations of the protein-binding sites or putative cis-elements were constructed and transiently expressed in MLTC or HepG2 cells (Fig. 5). In MLTC cells, single mutation of C/EBP element in the promoter domain decreased the promoter activity by nearly 50% (Fig. 5, construct 2). However, the same mutation only slightly reduced the luciferase activity (to ~82% of the wild type) in HepG2 cells. Single mutation of Sp1_a (construct 3) caused marked reduction in the promoter activity to 33% of the wild type in MLTC and smaller but significant reduction to 69% of the wild type in HepG2 cells (p < 0.01). Double mutation of C/EBP and Sp1_a elements caused further reduction in promoter activity to 22% of the wild type in MLTC cells (construct 4). This reduction was comparable to the decrease in activity observed (to 23%) in MLTC transfected with a deletion construct that excluded the region (437 to 344) containing both C/EBP and Sp1_a elements (Fig. 1, construct 7). These results indicated that in MLTC cells, both C/EBP and Sp1_a elements are important for PIII activity, the latter being dominant. In HepG2 cells, single mutation of either C/EBP or Sp1_a elements had much less impact on the activity of promoter III (82 and 69%, respectively), whereas double mutation of both elements caused a reduction in promoter activity to 38% of the wild type (construct 4). These results suggested that C/EBP and Sp1_a elements both contributed to the basal promoter activity, but there was some degree of redundancy in their function in HepG2 cells.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Functional analysis of PIII domain by mutation of putative regulatory cis-elements in MLTC and HepG2 cells. Left, above is shown schematic drawing of the PIII domain with putative regulatory elements (open symbols). Arrows indicate the transcription initiation sites (major at 340), and numbers indicate the nucleotide position relative to the translation initiation site. The promoter domain of PIII (437/179) (wild type, construct 1) and its mutants (filled symbols, constructs 2-8) were transiently expressed in MLTC and HepG2 cells. Promoter-less vector (Basic) was used as a control. Right, relative promoter activities are indicated as percentage of the wild type luciferase activity in MLTC or HepG2 cells. Data were collected from 4 to 10 independent transfections of triplicate wells and expressed as means ± S.E. The average luciferase activities in light units for the wild type promoter domain (100%) were 43,950 and 3,583 rLU/µg of protein in MLTC and HepG2 cells, respectively. The luciferase activities of positive control pSV40 were 164,812 and 24,850 rLU/µg of protein in the two cells, respectively.

Activation of PIII by Sp1 Protein through Sp1_a Element in SL2 Cells-- To examine whether PIII can be activated by Sp1 protein through Sp1_a site, Sp1 cDNA and PIII constructs were cotransfected into Sp1-deficient SL2 Drosophila cells. Cotransfection of SL2 cells with Sp1 cDNA yielded 2-3-fold induction of wild type promoter activity or C/EBP-mutated PIII domain (both containing intact Sp1_a site) and 5-fold that of the Sp1_a mutant (Fig. 6). This mutant displaying a basal activity lower than wild type in the absence of Sp1 cotransfection was not activated by cotransfection of Sp1 cDNA, reinforcing the notion that Sp1 protein binds specifically to the Sp1_a element and activates PIII. This finding also indicated that activation of this promoter by Sp1 in SL2 cells is independent of the C/EBP element.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Activation of PIII activity by Sp1 protein through Sp1a element in Drosophila SL2 cells. Luciferase constructs of PIII domain (437/179) of wild type, Sp1a-mutated (Sp1ax), or C/EBP-mutated (C/EBPx) were cotransfected into SL2 cells with 500 ng of Sp1cDNA (pPacSp1, black bar) or equal amount of empty vector (pPac, stippled bar). Promoter-less luciferase vector was also used as control (Basic). The promoter activities are indicated as percentage of activity (100%) of the wild type cotransfected with pPac, which had a luciferase activity of 3809 rLU/µg of protein. Data are expressed as mean ± S.E. of three independent transfections in triplicate wells.

C/EBP and Sp1_a Elements Are Transcriptional Activation Domains of PIII in Rat Granulosa Cells-- To examine the transcriptional activation and hormonal regulation of PIII in normal ovarian cells, primary cultures of rat granulosa cells were utilized for transient expression of PIII constructs of wild type or mutants (Fig. 7). Single mutation of C/EBP (C/EBPx) or Sp1_a (Sp1ax) elements decreased the basal promoter activity (in the absence of hormonal treatment and cotransfection) to 65.5 or 46.4% of the wild type, respectively. Double mutation of C/EBP and Sp1_a (CSx) further decreased the basal activity to 32.5% of the wild type. When the two upstream sites (C/EBP and Sp1_a) and the downstream DSE and Sp1_b, c were mutated altogether, the promoter activity decreased further to 12.8% of the wild type (Fig. 7, CSDSx). These results indicated that both C/EBP and Sp1_a are functional elements of promoter III in rat granulosa cells and that the downstream sequence is required for its promoter activity.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Functional analysis of PIII domain by mutation of C/EBP and Sp1 elements and by cotransfection of C/EBP cDNA in rat primary granulosa cells. Luciferase constructs of PIII domain (437/179) of wild type (WT), C/EBP mutated (C/EBPx), Sp1a mutated (Sp1ax), double mutation of C/EBP and Sp1a (CSx), or simultaneous mutation of C/EBP, Sp1a, DSE, Sp1b, and Sp1c (CSDSx) were cotransfected with the C/EBP cDNA (+) or with the empty vector () into the primary culture of rat granulosa cells. The transfected cells were treated with plain medium () or with 0.5 nM human chorionic hormone (+hCG), or 1.0 mM 8-bromo-cAMP (+cAMP), 6 h prior to cell harvest. Promoter activities are indicated as percentage of luciferase activity in wild type promoter-transfected cells without C/EBP cotransfection and without hormonal treatment (100%), which was 1916 rLU/µg of protein. The average luciferase activity of SV40 promoter was 2668 rLU/µg of protein. Data are expressed in means ± S.E. of three to five independent transfections in triplicate wells.

Structural and Functional Similarity between the Rat and Mouse PIII Region-- The mouse counterpart of rat E1₃ (non-coding exon 1₃ expression directed by PIII) of the PRLR mRNA was previously shown to be expressed in MLTC (5). A mouse genomic DNA fragment containing a sequence homologous to the rat PIII was isolated from a mouse BAC (bacterial artificial chromosome) library. There is a 93.6% similarity between the rat and mouse genomic region, which includes E1₃ and its 5'-flanking region (340 to 1019) as well as ~500 bp of the downstream intron sequence (Fig. 8A, above). The similarity between the promoter domain of PIII of the rat and mouse is 96.5%, in which the DNA elements for C/EBP, Sp1_a, Sp1_c, and DSE are identical between the two species (Fig. 8A, below). The activity of the mouse putative PIII was comparable to that of rat as indicated in MLTC cells by luciferase activities of the rat construct 437/179 and its mouse homologous construct (Fig. 8C). However, the mouse counterpart of the rat gonadal-specific promoter I has only 87.5% similarity with the sequence within the rat promoter domain (Fig. 8B). Although this putative mouse promoter is generally similar to the rat PI, it contains a mutated SF-1 element (CCAAGGTCA to CCACAGTCA) as determined by sequencing the genomic clone from the BAC library or independently by sequencing the PCR product of mouse liver genomic DNA (not shown). This difference in the SF-1-binding site abolished the function of this element (DNA binding and activation) as shown in mutational studies for the rat PI (5) and reduced the promoter activity of the mouse PI homolog to <10% of the rat PI (Fig. 8C). The conserved CCAAT box sequence in the mouse PI may contribute minimally to its activity, as previously shown for the rat PI (5). Since SF-1 is essential for PI activity in rat gonadal cells, lack of the functional SF-1 element in the mouse abrogates the function of this promoter (PI) and hence PRLR transcription from this promoter. It appears that PIII substitutes for PI-directed transcription of the PRLR in the mouse ovary. These findings indicate that PIII is both structurally and functionally conserved between the rat and mouse genes and are therefore of major importance in the control of PRLR gene transcription.

View larger version (47K): [in this window] [in a new window]   Fig. 8.   Sequence comparison of the promoter region of PIII and PI between the rat and mouse and their activities in MLTC cells. A, comparison of genomic regions of rat PIII with its mouse counterpart. The upper drawing represents the region including the exon E13 (open box) and its 5'-flanking region (thick lines and further upstream), as well as its 3'-intronic region of ~500 bp (in thin lines). The sequences diverge drastically between the rat and mouse upstream from 1019 (thin or broken lines). The regions defined by arrows share 93.6% homology between the two species. The nucleotide sequences (below) of the PIII domain (437 to 179) between the rat and mouse (GenBankTM accession number AF064034) are compared, with sequence similarity of 96.5% between the two. Underlined sequence elements are 100% conserved between the two species, whereas the boxed element Sp1b of the rat is less conserved. Circled nucleotides indicate the TSS from the rat PIII. B, sequence comparison of the promoter domain (700 to 549) of the rat PI with its mouse counterpart (GenBankTM accession number AF064033) is shown. The two sequences share 87.5% sequence similarity. Underlined is the CCAAT box that is 100% conserved, but the SF-1 element in the rat (boxed) is changed to a non-functional sequence in the mouse. The circled nucleotide is the TSS of the rat PI. C, transient expression of luciferase constructs containing the rat PIII (437/179) or PI (700/549) domains or their respective mouse counterparts in MLTC cells. Promoter activities are indicated as fold induction over the promoter-less vector, which had a luciferase activity of 1242 rLU/µg of protein in MLTC cells. Data are expressed in means ± S.E. of three independent transfections in triplicate wells. The average luciferase activity of SV40 (pSV40Luc) was 125,076 rLU/µg of protein in this experiment.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Our study has provided insights on the regulation of promoter III of the PRLR gene, a commonly utilized promoter of the prolactin receptor gene in a wide variety of tissues. The PIII activity is governed by activation of two functionally independent sites, a C/EBP element that binds to C/EBP and an Sp1 element (Sp1_a) that binds to Sp1 and Sp3, members of the same family of nuclear proteins. A DSE is required for basal functions of this promoter, and the two downstream adjacent Sp1 elements (Sp1_b, c) may substitute for DSE function.

Cell-type differences in activation of PIII by C/EBP and Sp1 were observed. In MLTC cells, single mutation of either binding site reduces the promoter activity by ~50% or more, with the role of Sp1 being more dominant than that of C/EBP, indicating that both factors are required to attain full promoter activity. However in HepG2 cells, mutation of either binding site caused only slight decrease in promoter activity (to 82 and 69% of the wild type, respectively), whereas double mutation reduced the activity to 38%, suggesting partial functional redundancy between the two factors. The differences in the C/EBP binding in EMSA analyses of nuclear extracts from MLTC (less retarded single band) and from HepG2 cells (more retarded double bands) may be related to the differential transactivation by the two factors in these two cell types. The close proximity of the two binding sites (C/EBP and Sp1_a) in the promoter domain may impose some spatial restraint to the extent that smaller complexes (in MLTC) may bind simultaneously to the two sites, whereas larger complexes (in HepG2) may bind in a mutually exclusive manner. Although synergistic effects were observed between C/EBP and Sp1 in other genes (13), synergism between the two factors was not observed on the promoter activity of promoter III of the PRLR gene. Therefore it appears that C/EBP and Sp1 contribute separately to the promoter activity, presumably through their association to specific coactivators or directly with members of the TATA-binding protein associated factor complexes. C/EBP was reported to bind to transcriptional coactivator p300/CBP (14), and Sp1 can directly associate with dTAFII110 or hTAFII130 (15) and TAFII55 (16).

Promoter III lacks a TATA box within the promoter domain and consensus initiator (Inr) sequences. The sequences encompassing the two main TSS (340 and 351) are TCAGAGA and TGAGCTT, respectively, and these represent mismatches by two nucleotides (underlined) of the consensus Inr sequence YYANt/aYY (17). Although it is not clear whether these sequences serve as variant Inr in PIII, the construct (506/345) lacking the downstream sequences including one of the two Inr-like sequences displayed minimal promoter activity. The downstream sequence element (DSE) (CGGCTGGCGA) is likely part of the core promoter elements required for efficient transcription initiation from this promoter, since its mutation reduced the promoter activity by ~50% in both MLTC and HepG2 cells. However, it appears that the DSE in PIII is distinct from DPE (downstream promoter element) by sequence as well as by its location. DPE was identified in a subset of TATA-less gene promoters with a distinct 7-nucleotide sequence (A/G)G(A/T)CGTG, located at +28 to +34 downstream of the transcription initiation sites (18). DPE can be bound by TFIID but not by TATA-binding protein and is defined as part of the core promoter components (18). Although the DSE in PIII does not conform to DPE, it is an integral part of the promoter domain and may participate in or facilitate the assembly of the basal transcription machinery.

Whereas the upstream Sp1_a is of major importance in PIII promoter activity, the two downstream Sp1_b and Sp1_c, which both bind to Sp1 protein, appeared to be non-operative or redundant in the presence of an intact DSE element, in that mutation of the two downstream Sp1 sites did not alter the promoter function in transient reporter gene assays. However, in the presence of altered DSE, mutation of both Sp1_b- and Sp1_c-binding sites caused further reduction in promoter activity, suggesting that SP1_b,c sites could partly compensate for the function of DSE when DSE was mutated. Both DSE and Sp1_b, c elements are GC-rich sequences, and when they were all mutated, the promoter activity was greatly compromised. Therefore, although the Sp1_b, c sites are normally redundant, they are potentially able to compensate or partially restore the promoter activity.

Unlike MLTC and HepG2 cells, we were not able to detect discernible C/EBP-binding complex(es) in the nuclear extract of rat ovarian granulosa cells using either the short probe containing only C/EBP site or the long probe containing both C/EBP and Sp1_a sites. However, functional analyses of the mutated C/EBP element and the transactivation of the promoter domain by cotransfection of the C/EBP cDNA into this cell (Fig. 7) indicated that C/EBP element in the PIII domain is a functional C/EBP-binding site in vivo, through an action that is independent of the Sp1 element.

In the C/EBP knock-out mice, ovarian follicles undergo normal growth but fail to luteinize after ovulation, suggesting that the ovary is deficient in expression of those genes that are participating in the process of luteinization and are regulated by C/EBP (19). It is well known that prolactin is required for the maintenance of luteinization as well as luteolysis in rodents (20). Expression of prolactin receptor is increased during follicular growth and during luteinization (21). C/EBP expression is increased after the luteinizing hormone surge when ovulation occurs (22). Therefore, up-regulation of both C/EBP and the prolactin receptor in granulosa cells is critical for luteinization. We now provide evidence that C/EBP is a transcriptional activator for this promoter, since overexpression of C/EBP in granulosa cells significantly increases the PIII activity. Because PIII activity is also controlled by Sp1/Sp3, the C/EBP knock-out mice would be expected to maintain a reduced level of PRLR, well below normal basal and hCG-stimulated levels. This marked reduction in transcription of the PRLR gene could in part explain the luteal deficiency observed in these mice.

Typical Sp1 DNA-binding sequences are GC box (GGGGCGGGC) and GT motif (GGGTGTGGC) present in most TATA-less and GC-rich promoters. A variant Sp1-binding site CTC repeat (CTCCTCCTC) was identified in TATA-less promoter regions of a number of other growth-related genes including Wilm's tumor-1 gene (WT-1) (23), platelet-derived growth factor-A chain (24), insulin-like growth factor-I, and epidermal growth factor receptors (25, 26), c-myc, c-myb, and vav (27-29). There are at least three close members of Sp family proteins, Sp1, Sp3, and Sp4, which share similar structural features (30, 31). The three proteins contain highly conserved DNA binding domains that all recognize the GC box and GT motif. Sp3 and Sp4 have also been suggested in transcriptional regulation. In Drosophila SL2 cells, Sp3 could suppress the Sp1-mediated transcriptional activation due to the competition with Sp1 for their common binding sites in some genes (32, 33). In other cases, Sp3 enhances the promoter activity (34-36) with equal potency (35) and sometimes is even a more effective activator than Sp1 (36). Therefore, there is differential regulation of different genes by Sp1 or Sp3. Although the activation of PIII in SL2 cells by Sp1 (2-3-fold) was not as dramatic as in some other genes, the fact that Sp3 is not present in these cells may also be reflective of the degree of activation observed in this study. In mammalian cells (CV-1, HeLa, and Ishikawa cells), it was also reported that the reporter promoters (uroglobin and SV40) containing GC box or GT motif were activated by Sp1 and Sp4 but not by Sp3 (37). In the current study, we demonstrated that like Sp1, Sp3 could also bind to CTC repeats in the PIII. Although it is clear that Sp1_a is a positive cis-element in this promoter, the individual contribution by Sp1 and Sp3 to this promoter activation is not certain. It is believed that Sp1 is an important activator to promoter III in gonadal and hepatic cells as in numerous other Sp1-activated genes (38, 39). Since Sp3 is also a ubiquitous factor and can bind to Sp1_a element, the regulatory roles of Sp3 in the PIII await further study.

The activity of the PIII domain (437/179) was induced significantly by hCG/cAMP in cultured rat primary granulosa cells. However, neither C/EBP- nor Sp1-binding sites were found to specifically mediate this response to hCG/cAMP, albeit they were important for basal promoter activity. Although both C/EBP and Sp1 have been shown to participate in cAMP induction of transcription in some genes (40-44) through phosphorylation by protein kinase A (40, 41, 43), this appears not to be the case for this promoter domain. However, there is no consensus cAMP response element or functional AP2 site present within the promoter domain that could mediate second messenger action, thus other elements may be involved in cAMP-mediated induction of promoter activity.

The mouse PIII is highly similar in structure and function to the rat promoter III as expected, suggesting that this promoter is well conserved. However, unexpectedly the homolog of the rat gonadal promoter I was critically altered in the mouse. The SF-1 element, which is essential for the rat PI activity, is a non-functional sequence in the mouse. This is reflected in the vestigial promoter activity observed in reporter assays and very low expression of the endogenous mouse counterpart of rat E1₁ (non-coding exon 1₁) (4) in both MLTC and mouse ovary as compared with the E1₃. Hence the essential promoter for the PRLR in the mouse ovary is promoter III and the expression of the mouse ovarian PRLR is primarily regulated by C/EBP and Sp1, whereas promoter I is inactive due to critical base pair substitution in the SF-1 element. It is therefore expected that PRLR transcription in the ovary is reduced in C/EBP knock-out mice and may contribute to the deficiency in luteinization in such animals (19).

Because C/EBP and Sp1 are the major transcriptional activators to promoter III of the rat PRLR gene, the wide expression of these two transcription factors may explain the common utilization of this promoter in multiple tissues and in different species. This is in contrast to PI and PII, which were specifically activated by SF-1 (5) and HNF-4 (7) in the gonads and the liver, respectively.

In summary, we have identified in promoter III multiple nuclear protein-binding sites including C/EBP and Sp1/Sp3 and a functional DSE. C/EBP and Sp1_a are the two major transcription factors regulating the PIII activity, and DSE is also required for the basal promoter activity. Promoter III is highly active and is well conserved in the rat and mouse; therefore, it is of central importance in the regulation of the PRLR gene expression in various tissues of different species.