An Upstream Regulator of the Glycoprotein Hormone α-Subunit Gene Mediates Pituitary Cell Type Activation and Repression by Different Mechanisms
1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês
10.1074/jbc.274.22.15526
ISSN1083-351X
AutoresWilliam M. Wood, Janet M. Dowding, David F. Gordon, E. Chester Ridgway,
Tópico(s)Adrenal Hormones and Disorders
ResumoTargeting of α-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived α-TSH and α-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in α-TSH and α-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal α-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in α-TSH and α-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence. Targeting of α-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived α-TSH and α-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in α-TSH and α-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal α-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in α-TSH and α-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence. Expression of the gene for the common α-subunit of the pituitary glycoprotein hormones is restricted to two cell types within the anterior pituitary gland. In combination with the specific β-subunits produced by thyrotropes and gonadotropes, it forms the biologically active hormones TSH, 1The abbreviations used are: TSH, thyroid-stimulating hormone; bp, base pair(s); PGBE, pituitary glycoprotein hormone basal element; GSE, gonadotrope-specific element; PCR, polymerase chain reactionlutenizing hormone, and follicle-stimulating hormone. Many reports have described areas within the proximal promoter region of the α-subunit gene that are important for expression in both pituitary cell types (1Sarapura V.D. Wood W.M. Gordon D.F. Ocran K.W. Kao M.Y. Ridgway E.C. Endocrinology. 1990; 127: 1352-1361Crossref PubMed Scopus (39) Google Scholar, 2Delegeane A.M. Ferland L.H. Mellon P.L. Mol. Cell. Biol. 1987; 7: 3994-4002Crossref PubMed Scopus (283) Google Scholar, 3Bokar J.A. Keri R.A. 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Keri R.A. Farmerie T.A. Fenstermaker R.A. Andersen B. Hamernik D.L. Yun J. Wagner T. Nilson J.H. Mol. Cell. Biol. 1989; 9: 5113-5122Crossref PubMed Scopus (118) Google Scholar, 8Heckert L.L. Schultz K. Nilson J.H. J. Biol. Chem. 1995; 270: 26497-26504Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Silver B.J. Bokar J.A. Virgin J.B. Vallen E.A. Milsted A. Nilson J.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2198-2202Crossref PubMed Scopus (174) Google Scholar, 11Steger D.J. Altschmied J. Buscher M. Mellon P. Mol. Endocrinol. 1991; 5: 243-255Crossref PubMed Scopus (84) Google Scholar), where it is also a component of chorionic gonadotropin. We have recently discovered a more distal area of the 5′-flanking region of the mouse gene, located approximately 4 kilobase pairs upstream from the start of transcription, that directs high levels of expression of a β-galactosidase reporter that was restricted to the thyrotropes and gonadotropes of transgenic mice (12Kendall S.K. Gordon D.F. Birkmeier T.S. Petrey D. Sarapura V.D. O'Shea K.S. Wood W.M. Lloyd R.V. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1994; 8: 1420-1433PubMed Google Scholar). Subsequent studies demonstrated that when an 859-bpKpnI-BglII fragment derived from the region between −4600 and −3700 was fused directly to a proximal α-subunit promoter from −341 to +43, the high level of cell-specific transgene expression was maintained (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar). Furthermore, we also demonstrated that the 859-bp region was also capable of stimulating proximal promoter activity in transient transfections of cells derived from thyrotropes (α-TSH) and gonadotropes (α-T3), both of which express the α-subunit gene endogenously (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar). More recently, our laboratory (14Wood W.M. Dowding J.M. Sarapura V.D. McDermott M.T. Gordon D.F. Ridgway E.C. Mol. Cell. Endocrinol. 1998; 142: 141-152Crossref PubMed Scopus (12) Google Scholar) has shown that the functional interaction between the upstream enhancer and the proximal promoter in transfections of both cell lines is dependent on an intact pituitary glycoprotein hormone binding element (PGBE) located from −337 to −330, an area within the proximal area that was shown to bind a LIM homeodomain factor (15Roberson M.S. Schoderbek W.E. Tremml G. Maurer R.A. Mol. Cell. Biol. 1994; 14: 2985-2993Crossref PubMed Google Scholar, 16Bach I. Rhodes S.J. Pearse I.I. Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (293) Google Scholar). In addition, interaction at the more proximal gonadotrope-specific element (GSE) from −213 to −200, which binds the steroidogenic factor SF-1 (17Barnhart K.M. Mellon P.M. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar), is also required for the enhancer to function in gonadotrope cells but not in thyrotrope cells (14Wood W.M. Dowding J.M. Sarapura V.D. McDermott M.T. Gordon D.F. Ridgway E.C. Mol. Cell. Endocrinol. 1998; 142: 141-152Crossref PubMed Scopus (12) Google Scholar). To identify the sites of functional interaction within the upstream region, we now report that the most proximal 125 bp of the previous 859-bp enhancing area are sufficient to account for all of the proximal promoter stimulatory activity in both pituitary-derived cell types that express the α-subunit. We demonstrate that the native orientation of the region with respect to the proximal promoter is crucial to maintain activation. We also show that several areas within this region bind proteins present in nuclear extracts from pituitary-derived cells including somatotrope-derived GH3 cells and that the areas of interaction contain potential binding sites for known transcription factors that also bind at important functional areas within the proximal promoter (17Barnhart K.M. Mellon P.M. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar, 18Steger D.J. Hecht J.H. Mellon P.L. Mol. Cell. Biol. 1994; 14: 5592-5602Crossref PubMed Google Scholar, 19Jackson S.M. Gutierrez-Hartmann A. Hoeffler J.P. Mol. Endocrinol. 1995; 9: 278-291PubMed Google Scholar). Interestingly, we further show that the same upstream region inhibits proximal α-promoter activity in GH3 cells. The mechanism whereby the upstream region exerts its repressive effect differs from activation in that it is not dependent on interaction at two proximal promoter binding sites and on the orientation of the upstream region with respect to the proximal region. The construction of luciferase vectors containing the region from −341 to +43 of the α-subunit promoter without and with mutated PGBE and GSE sites and the same plasmids with the 859-bp upstream enhancer fused upstream were described previously (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar, 14Wood W.M. Dowding J.M. Sarapura V.D. McDermott M.T. Gordon D.F. Ridgway E.C. Mol. Cell. Endocrinol. 1998; 142: 141-152Crossref PubMed Scopus (12) Google Scholar). As a first approach, the 859-bp region was subdivided by digestion at PstI restriction sites. After separation on agarose gels and purification using Qiaex II resin (Qiagen), each fragment was inserted into the PstI site of pGEM5zf+ (Promega). Subsequent excision with EcoRV andHindII generated a blunt-ended fragment that was ligated into the SmaI site of a plasmid that contained the region from −341 to +43 of the α-subunit promoter inserted between theBamHI and HindIII sites of pSELECT (Promega). After checking by sequencing for the correct forward orientation of each PstI fragment, the fused enhancer/promoter region was excised by digestion with KpnI and HindIII and inserted between the same sites of the promoterless pA3LUC mammalian expression vector (20Wood W.M. Kao M.Y. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar, 21Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar). This resulted in luciferase expression plasmids with the areas from 70 to 246, from 241 to 654, and from 694 to 833 (renumbered as 1–184 in Fig. 6, also see the area between the underlined PstI sites in Fig. 2) fused upstream of the proximal α-subunit promoter region from −341 to +43. Progressively shorter 5′ prime truncations of the enhancer region were generated using a PCR strategy that employed sense oligonucleotides with the following sequences: (a) 5′-GCCGGTACCCTGCAGGTCTGCACATAAATTC-3′, (b) 5′-GCGGGTACCCACTCAGTCAATATCTTATCTCT-3′, (c) 5′-GCCGGTACCCAGAAGCAATTAAGCAGTCA-3′, (d) 5′-GCCGGTACCCTGCAGAATAAAAGCTCTTTG-3′, (e) 5′-GCGGGTACCACAGGTGTTAGGAACTC-3′, (f) 5′-GCGGGTACCCAGCCCGTGACCTCAT-3′, and (g) 5′-GCGGGTACCCTGCAGTCTAGGAGATTTG-3′.Figure 2Nucleotide sequence of the functional 210-bp region. The nucleotide sequence of the most proximal 210 bp of the 859-bp upstream enhancing area is shown. Positions 1–210 are identical to those from 649 to 859 of the GenBankTM sequence AFF044976. Two PstI sites used to generate a 3′ deleted fragment for both functional and DNase footprint assays areunderlined. Sequences homologous to binding sites for various transcription factors are boxed and identified. Thegray box denotes a 60-bp sequence that is repeated with 65–70% homology in other areas of the mouse genome.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These oligonucleotides comprise sequences originating at positions 241, 421, 552, 649, 733, 798, and 827, respectively, of the upstream 859-bp enhancer sequence, respectively (GenBankTM accession number AFF044976). The last three represent positions 85, 150, and 179 of the renumbered 210-bp region shown in Fig. 2. They were designed with a 5′KpnI site (italicized) to facilitate subsequent subcloning. Each oligonucleotide was used in conjunction with a common antisense 22-nucleotide amplimer that is complementary to the sequence coding for amino acids 4–10 of luciferase and used to amplify the appropriate truncated enhancer/promoter fragment from the full-length 859-bp enhancer/promoter fusion in pA3LUC. The resulting fragments were then digested with KpnI and HindIII and reinserted between the same sites of pA3LUC. A 5′ deletion plasmid originating at position 85 and fused to a truncated Rous sarcoma virus promoter was similarly constructed starting with the previously described 859-bp enhancer Rous sarcoma virus fusion vector (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar). To generate an enhancer/promoter plasmid containing sequences that extended downstream of position 859, we first subcloned, into pGEM7zf (Promega), a fragment that extended from −4600 to −3400 that was derived by KpnI and SphI digestion from a plasmid containing α-subunit sequences from −5000 to +43 (12Kendall S.K. Gordon D.F. Birkmeier T.S. Petrey D. Sarapura V.D. O'Shea K.S. Wood W.M. Lloyd R.V. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1994; 8: 1420-1433PubMed Google Scholar). After sequencing this downstream 300-bp extension, we utilized PCR with this plasmid and a 5′ oligonucleotide that spanned the KpnI site at −4600 along with an antisense oligonucleotide that originated 237 bp downstream of the BglII site at −3700 with 5′-GCGGGATCCATAATTCACCTTTAGGGAGG-3′. Incorporation of aBamHI site (italicized) allowed the amplified product to be excised with KpnI and BamHI and inserted in the forward orientation between the same sites of the pSELECT plasmid containing the −341 to +43 α-subunit promoter sequence. The 3′ extended enhancer/promoter fusion region was then excised withKpnI and HindIII and cloned into pA3LUC as before. An α-subunit proximal promoter luciferase plasmid containing the 210-bp region inserted in the opposite orientation was constructed using the following PCR strategy. An oligonucleotide was synthesized that had the same sequence as the one described above that originated at position 649 of the 859-bp enhancer sequence, except that aBamHI site was incorporated at the 5′ end instead of aKpnI site. When this was used in PCR with an antisense oligonucleotide that spanned the downstream BglII site (at 859), it allowed the amplified product to be digested withBamHI and BglII and inserted at theBamHI site upstream of position −341 in the pSELECT plasmid containing this proximal promoter region. Because BamHI andBglII overhangs can anneal, it allowed the generation of enhancer/promoter fusions with the 210-bp region in both orientations. The reverse orientation was identified by sequencing, and the enhancer/promoter fragment was recloned into pA3LUC as described above. Transient transfections using electroporation for α-TSH (3 × 106), α-T3 (4 × 106), and GH3 (4 × 106) cells were performed as described previously (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar,22Haugen B.R. Gordon D.F. Nelson A.R. Wood W.M. Ridgway E.C. Mol. Endocrinol. 1994; 8: 1574-1582PubMed Google Scholar). 20 μg of α-subunit promoter and enhancer/promoter luciferase constructs were used for the α-TSH and α-T3 cell transfections, and 2 μg of a cytomegalovirus β-galactosidase plasmid were included as an internal control of transfection efficiency. Transfections were carried out in duplicate with a Rous sarcoma virus promoter luciferase vector and a promoterless pA3LUC vector as positive and negative controls (20Wood W.M. Kao M.Y. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar). Experiments were performed a minimum of three times with at least two preparations of each plasmid. After 16–24 h, luciferase and β-galactosidase activities were measured from duplicate aliquots of freeze-thawed cytoplasmic lysates. Luciferase activities of the various enhancer/promoter constructs were normalized to the corresponding β-galactosidase value and expressed as the fold stimulation ± S.E. of the normalized activity of the −341 to +43 promoter in the various cell types. Nuclear extracts were prepared from dispersed TtT-97 thyrotropic tumors or α-TSH, α-T3, and GH3 cells as described previously (23Sarapura V.D. Wood W.M. Gordon D.F. Ridgway E.C. Thyroid. 1992; 2: 31-38Crossref PubMed Scopus (13) Google Scholar, 24Wood W.M. Dowding J.M. Bright T.M. McDermott M.T. Haugen B.R. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1996; 271: 24213-24220Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Cultured cells were placed in medium containing 10% charcoal-stripped fetal calf serum for 48 h before harvesting for extract preparation. Protein concentrations were determined by Bio-Rad DC Protein Assay (Bio-Rad) using bovine serum albumin (Roche Molecular Biochemicals) as a standard. The previously described pGEM5zf plasmid containing the 184-bp PstI fragment from position 649 to position 833 of the 859-bp enhancer region was digested withNotI and NdeI to generate a fragment for footprinting analysis that could be labeled uniquely at the upstream end using [α-32P]dGTP and dCTP to fill in theNotI overhang (GGCC). To generate a footprinting fragment that was labeled at the downstream position, we constructed a plasmid that contained a fragment extending from 649 to a position 100 bp 3′ of the BglII site. This was amplified by PCR from the previously described KpnI to SphI fragment containing vector using the KpnI 5′ tagged sense primer originating at 649 and an antisense amplimer with a 5′HindIII site (italicized) with the following sequence: 5′-GCGAAGCTTGGGGGAAATATCACTGCATG-3′. As before, excision with EcoRI and MluI enabled unique filling of theMluI overhang (CGCG) with [α-32P]dGTP and dCTP. This 3′ extension allowed the area immediately 5′ of theBglII site to be displayed in an optimal area of the footprint gel. DNase I protection assays were carried out as described previously (24Wood W.M. Dowding J.M. Bright T.M. McDermott M.T. Haugen B.R. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1996; 271: 24213-24220Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Briefly, radiolabeled probes were allowed to interact with 10 μg of bovine serum albumin (no extract) or 60–70 μg of nuclear extract protein derived from TtT-97, α-TSH, α-T3, or GH3 cells, subjected to DNase I digestion under defined conditions, and analyzed on a denaturing 5% polyacrylamide-8 m urea gel. Previous transgenic and transient transfection data indicated that sequences required for the enhancement of the α-subunit proximal promoter specifically in thyrotropes and gonadotropes were present in a KpnI–BglII fragment located between −4.6 and −3.7 kilobase pairs upstream of the transcriptional start site (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar). To further localize the sequences responsible for enhancement within this 859-bp region, subfragments of the KpnI–BglII fragment were fused to the −341 to +43 proximal promoter region and analyzed independently for their ability to enhance in both thyrotrope- and gonadotrope-derived cells. The results of this analysis are shown in Fig.1. A strategy that involved progressively deleting sequences from the 5′ end demonstrated that the enhancing effect of the 859-bp area previously reported in both thyrotrope-derived α-TSH cells and gonadotrope-derived α-T3 cells (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar) could be entirely accounted for by the most proximal 210 bp from 649 to 859. The lack of other areas eliciting any enhancing capability was further confirmed by showing that neither of two PstI fragments, which comprised all but the terminal 69 bp of the sequence upstream of position 654, exhibited any capacity to stimulate the proximal promoter (Fig. 1). Because the sequences responsible for the stimulatory effect appeared to map to the region immediately 5′ of theBglII site at −3700 (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar), we thought it important to determine if sequences immediately downstream of the BglII site could further augment the previously observed enhancement. Therefore, a larger fragment from −4600 to −3400 was generated from a previously described α-subunit genomic clone (12Kendall S.K. Gordon D.F. Birkmeier T.S. Petrey D. Sarapura V.D. O'Shea K.S. Wood W.M. Lloyd R.V. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1994; 8: 1420-1433PubMed Google Scholar), and PCR was used to amplify a fragment that extended more proximally to a position 237 bp downstream of the BglII site. This was inserted upstream of −341 in the proximal α-subunit promoter, and the stimulatory effect of this 3′ augmented enhancer region was compared with the previous 859-bp area. The results of this analysis revealed no further enhancement in either cell type when the enhancer region fused to the proximal promoter-included sequences 3′ of the BglII site (data not shown). Therefore, the sequences mediating enhancement appear to be entirely contained within the previously determined 210-bp area. The nucleotide sequence of the 210-bp region that accounts for all of the enhancer activity of the upstream 5′ region of the mouse α-subunit gene is shown in Fig. 2. Nucleotides 649–859 of the previously described 859-bp enhancer region redesignated 1–210 are shown. The sequence shaded in graydenotes the location of a 60-bp sequence that is 60–70% homologous to other sequences that occur within several reported rodent genes as revealed by a GenBankTM search. Boxed andlabeled sequences correspond to consensus binding sites for several transcription factors that have been reported previously to play a role in the activity of the proximal α-subunit promoter (17Barnhart K.M. Mellon P.M. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar, 18Steger D.J. Hecht J.H. Mellon P.L. Mol. Cell. Biol. 1994; 14: 5592-5602Crossref PubMed Google Scholar, 19Jackson S.M. Gutierrez-Hartmann A. Hoeffler J.P. Mol. Endocrinol. 1995; 9: 278-291PubMed Google Scholar) as well as in other pituitary genes (25Ingraham H. Chen R. Mangalam H.J. Elsholtz H.P. Flynn S.E. Lin C.R. Simmons D.M. Swanson L. Rosenfeld M.G. Cell. 1988; 55: 519-529Abstract Full Text PDF PubMed Scopus (792) Google Scholar, 26Tansey W.P. Catanzaro D.F. J. Biol. Chem. 1991; 266: 9805-9813Abstract Full Text PDF PubMed Google Scholar, 27Howard P. Maurer R.A. J. Biol. Chem. 1995; 270: 20930-20936Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Bradford A.P. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar, 29Schaufele F. West B.L. Reudelhuber T.L. J. Biol. Chem. 1990; 265: 17189-17196Abstract Full Text PDF PubMed Google Scholar). These include several binding sites for GATA and ETS factors. Also present are two E-box motifs (CANNTG) and single sites that could potentially interact with Pit-1, Sp1, and nuclear hormone receptor family members such as the steroidogenic factor SF-1. A proximal promoter area from −213 to −200 that binds SF-1 (the GSE) has been reported by our group (14Wood W.M. Dowding J.M. Sarapura V.D. McDermott M.T. Gordon D.F. Ridgway E.C. Mol. Cell. Endocrinol. 1998; 142: 141-152Crossref PubMed Scopus (12) Google Scholar) and by others (5Horn F. Windle J.J. Barnhart K.M. Mellon P.L. Mol. Cell. Biol. 1992; 12: 2143-2153Crossref PubMed Google Scholar, 17Barnhart K.M. Mellon P.M. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar) to be critical for both basal and enhancer-stimulated α-subunit promoter activity in gonadotrope cells. To begin to investigate which proteins are interacting with the 210-bp region and could perhaps be playing a role in the enhancement of the proximal promoter, we performed a comparative DNase I footprinting analysis using nuclear extracts derived from pituitary cells that express the endogenous α-subunit gene (TtT-97 thyrotropic tumor, α-TSH and α-T3 cells) as well as those that do not (somatotrope-derived GH3 cells). Fig. 3shows the results of this analysis using fragments encompassing the 210-bp region that have been labeled at either the upstream (A) or downstream (B) position. In Fig.3 A, which utilized a 184-bp PstI fragment that terminates 26 bp from the proximal end of the fully enhancing region (see Fig. 2), protection from DNase I digestion can be seen in three general areas. Using DNA size standards loaded in a parallel lane (Stds) the approximate location of the footprinted areas are from 77 to 112, from 132 to 150, and from 152 to 178 on the antisense strand. No protection by any extract was evident upstream of position 77. The area from 77 to 112 appears to be similarly protected by the two thyrotrope cell extracts (TtT-97 and α-TSH), but the footprint generated by α-T3 nuclear extracts, although overlapping the thyrotrope footprint, appears to be less well protected for the most proximal 10 bp, suggesting that a different factor(s) present in each cell type may be interacting at this region. As shown in Fig. 2, this area contains sequences that correspond to binding sites for both E-box and Ets factors. When the sense strand labeled at the downstream position was analyzed, the same overall area was protected (Fig.3 B), but the area from 74 to 129 was subdivided into three clearly defined footprints from 74 to 86, from 92 to 105, and from 109 to 129. The first two were seen with all of the extracts, including those from GH3 cells. These encompass the upstream E-box and Ets sites. The protected area from 109 to 129, which contained a second Ets site, was observed with both thyrotrope-derived cell nuclear extracts (TtT-97 and α-TSH) but appeared not to be protected by either α-T3 or GH3 nuclear extracts. Two other more downstream footprints from 133 to 147 and from 152 to 178 that were also seen with the other strand were also protected by all of the extracts, including those from GH3 cells. These footprints contain a second E-box motif and potential binding sites for GATA factors, Sp1 and SF1. It is of interest that parts of the area of the enhancer that interact with the pituitary cell nuclear extracts colocalize with the 60-bp region that is repeated elsewhere in the mouse genome, shown in Fig. 2 as a gray box. A surprising observation resulting from these protein-DNA binding experiments suggests that non-α-subunit-expressing pituitary GH3 cells may contain factors that interact with the enhancer region, and thus such factors are not confined to cell types that express the endogenous gene, i.e. thyrotropes and gonadotropes. The finding that nuclear proteins from somatotrope-derived GH3 cells, which do not express the α-subunit, bind to sequences within a region that is involved in the activation of proximal α-subunit promoter activity in homologous α-TSH and α-T3 cells prompted us to investigate whether the upstream 210-bp area was also capable of enhancement in GH3 cells. We had previously shown that in non-pituitary CV-1 monkey kidney cells, the larger 859-bp region only modestly stimulated the proximal α-subunit promoter, although it was capable of enhancing the activity of a viral promoter in a cell type-independent fashion (13Brinkmeier M.L. Gordon D.F. Dowding J.M. Saunders T.L. Kendall S.K. Sarapura V.D. Wood W.M. Ridgway E.C. Camper S.A. Mol. Endocrinol. 1998; 12: 622-633PubMed Google Scholar). Fig. 4shows the effect of the 210-bp area on the proximal α-subunit promoter in GH3 cells compared with that previously seen in α-TSH and α-T3 cells. As shown before, the 210-bp area confers a 25- and 7-fold stimulation to the proximal promoter in α-TSH and α-T3 cells, respectively. However, in the non-α-subunit-expressing pituitary-derived GH3 cells, the upstream 210-bp region dramatically inhibits the activity of the proximal promoter by 75% (note that the scale of the x axis in Fig. 4 is not linear). Therefore, the upstream regulatory region serves a role not only to activate α-subunit expression in α-subunit-expressing cell types but also to repress it in another pituitary cell type that does not express t
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