Mechanism of Gonadotropin Gene Expression

Regulation of the glycoprotein hormone α-subunit (GPHα) gene has been studied extensively in pituitary and placental cell lines, but little is known of the transcriptional regulators important for its ectopic expression. To investigate the molecular basis for ectopic expression, it was critical to define cis-regulatory elements and their cognatetrans-acting factors that modulate promoter activity in epithelial cell types that do not normally express GPH. DNA-mediated transient expression of promoter-reporter constructs was used to identify a novel negative regulatory element located at the GPHα gene transcription start site. Truncation or site-directed mutagenesis of this element produced up to a 10-fold increase in promoter activity. Electrophoretic mobility shift analysis detected a protein that binds specifically to a DNA motif encompassing the cap site. Based on competitive DNA binding studies with mutated oligonucleotides, it was determined that bases from −5 to −2 and +4 to +11 are critical for protein binding. The DNA sequence flanking the transcription start site from −9 to +11 is an imperfect palindrome; consequently, this motif is referred to as the cap site diad element (CSDE) and the cognate factor as the cap site-binding protein (CSBP). CSBP activity was present at different levels in nuclear extracts prepared from a variety of cell types. Significantly, the ratio of activities exhibited by the GPHα promoter with a mutated CSDE compared with the promoter with a wild-type CSDE was dependent on the transfected cell line and its content of CSBP. These results indicate that a negative regulatory element centered at the GPHα gene cap site and its cognate DNA-binding protein make a significant contribution to the production of α-subunit in a variety of tumor tissues. A detailed understanding of this cis/trans pair may further suggest a mechanism to explain, at least in part, how this gene becomes activated in nonendocrine tumors. Regulation of the glycoprotein hormone α-subunit (GPHα) gene has been studied extensively in pituitary and placental cell lines, but little is known of the transcriptional regulators important for its ectopic expression. To investigate the molecular basis for ectopic expression, it was critical to define cis-regulatory elements and their cognatetrans-acting factors that modulate promoter activity in epithelial cell types that do not normally express GPH. DNA-mediated transient expression of promoter-reporter constructs was used to identify a novel negative regulatory element located at the GPHα gene transcription start site. Truncation or site-directed mutagenesis of this element produced up to a 10-fold increase in promoter activity. Electrophoretic mobility shift analysis detected a protein that binds specifically to a DNA motif encompassing the cap site. Based on competitive DNA binding studies with mutated oligonucleotides, it was determined that bases from −5 to −2 and +4 to +11 are critical for protein binding. The DNA sequence flanking the transcription start site from −9 to +11 is an imperfect palindrome; consequently, this motif is referred to as the cap site diad element (CSDE) and the cognate factor as the cap site-binding protein (CSBP). CSBP activity was present at different levels in nuclear extracts prepared from a variety of cell types. Significantly, the ratio of activities exhibited by the GPHα promoter with a mutated CSDE compared with the promoter with a wild-type CSDE was dependent on the transfected cell line and its content of CSBP. These results indicate that a negative regulatory element centered at the GPHα gene cap site and its cognate DNA-binding protein make a significant contribution to the production of α-subunit in a variety of tumor tissues. A detailed understanding of this cis/trans pair may further suggest a mechanism to explain, at least in part, how this gene becomes activated in nonendocrine tumors. The glycoprotein hormone (GPH) 1The abbreviations used are: GPH, glycoprotein hormone; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; CSBP, cap site-binding protein; CSDE, cap site diad element; CRE, cAMP-responsive element; DTT, dithiothreitol; EB, E-box; EMSA, electrophoretic mobility shift analysis; FSH, follicle-stimulating hormone; GPHα, glycoprotein hormone α-subunit; GSE, gonadotrope-specific element; LH, luteinizing hormone; PGBE, pituitary glycoprotein hormone basal element; PMSF, phenylmethylsulfonyl fluoride; TSE, trophoblast-specific element; TSH, thyroid-stimulating hormone. family consists of four members, chorionic gonadotropin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). These hormones are heterodimers, sharing a common α-subunit but having unique β-subunits that are thought to confer their biological specificity. LH, FSH, and TSH are produced in the anterior pituitary, whereas chorionic gonadotropin is synthesized in the developing placenta. Thus, it is significant that the isolated α- or β-subunits are also synthesized by a variety of tumors (1Weintraub B.D. Rosen S.W. J. Clin. Invest. 1973; 52: 335-342Google Scholar, 2Blackman M.R. Rosen S.W. Weintraub B.D. Adv. Int. Med. 1978; 23: 85-113Google Scholar, 3Braunstein G.D. Vaitukaitis J.L. Carbone P.P. Ross G.T. Ann. Intern. Med. 1973; 78: 39-45Google Scholar, 4Rees J. Scholar, J. and cell The are by cell from and tumors Scholar, J. Clin. 1978; the are to ectopic of a cell that from the tumor was for α-subunit at of the is by α-subunit production and tumor in J. Scholar, J. and the of tumor cell in Scholar, J. The molecular α-subunit gene expression in the been studied has been that elements in the of DNA from the transcription start site are to the gene in a J.L. These are in promoter elements and at and elements that from to Scholar, J.L. J. Scholar, The sequence is an that is also in cAMP-responsive J. M.R. Scholar, Scholar, M.R. Scholar, J. Scholar, J. Scholar, J. to the in the GPHα gene a located from to that basal levels of GPHα gene expression in placental Scholar, J.L. J. Scholar, J.L. J. Scholar, J.L. G.D. The trophoblast-specific is a and DNA-binding for at least The the to its on transcription Scholar, J.L. J. Scholar, J.L. G.D. The and are referred to as and regulatory J.L. Scholar, J.L. J. Scholar, J.L. J. Scholar, J.L. G.D. and the and a binding as is to to J. been that specifically with regulatory elements Scholar, J.L. J. J.L. J. Scholar, J.L. J. Scholar, J. Scholar, J. of expression of the α-subunit gene in the pituitary from that in the J.L. Scholar, J.L. J. Scholar, J.L. G.D. Scholar, J. Scholar, Scholar, J. Scholar, Regulation of GPHα gene expression in the cell line and in the pituitary cell line is dependent on the Scholar, Scholar, the trophoblast-specific not to and binding activity detected in or cell of the been for expression in pituitary Scholar, Scholar, The the gonadotrope-specific element is located in the gene from to and is in α-subunit A protein that binds this element was detected in but not in and was as The to to and to that confer expression to the α-subunit gene do not to the Scholar, The sequence and is referred to as the pituitary glycoprotein basal element and an imperfect centered and has been to a transcription factor that a protein expression of of the of pituitary with the to in the protein binding are located in the GPHα promoter to and to of the These are referred to as and and their basal activity of the promoter in pituitary the and of basal and elements that confer of the GPHα gene in and the molecular basis for its expression in nonendocrine cell types the α-subunit is and at levels with in the studies that the α-subunit gene promoter is not as in as it is in and that do not the binding protein to with the Scholar, To investigate the ectopic production of the gonadotropin in nonendocrine the of GPHα gene expression was studied in a detailed analysis of the GPHα gene promoter an imperfect centered at the transcription start site was is in this that the cap site sequence a negative element that to promoter activity in a variety of cell types on the of a nuclear factor that binding to the cap site The element and its cognatetrans-acting factor are referred to as the cap site diad element (CSDE) and cap site-binding protein and pituitary in with and and in with The cell line was in with and also with and as and in at in a of and A from to was from a GPHα J. by with and The was with and to the with DNA the chloramphenicol gene of the wild-type α-subunit gene To the was and the was isolated and the site of To the cap site a DNA from to of the GPHα J. was and DNA was isolated to as a for mutagenesis Scholar, bases at −5 to −2 from to and bases at +4 and from and to and The from to the from and A from to with was in and site of to and To and and and the and by or the cap site the imperfect at a and the and of in constructs by of the wild-type and cap site used as or in DNA binding the wild-type and mutated the is in and the diad and are in a that of the are mutated in the are at the transcription start site by the at in a the wild-type and mutated the is in and the diad and are in a that of the are mutated in the are at the transcription start site by the at in J. A of the expression and of the the gene of the and cell for activity as by G.T. Scholar, and for activity as by J. A activity was to activity for The for at for to for activity of cell extracts was determined to a by the of activities for a by by with of or of the and from a a in present and in nuclear extracts from and prepared as in a and in a and The cell was in of nuclear and phenylmethylsulfonyl for on the cell with the the and the to and at for at in of and for on of a DTT, and the protein extracts of a DTT, and for the was with an of and was in nuclear extracts was determined as by used as binding and are in was as by with in a of of nuclear and was to protein binding. the with of up to a of the to the a at was and was for on in and and by of the and with and with the in a with and in as The by of DNA in of for at with of in of and The was by of a and of The was by The was to EMSA, and to and and The with of with and of by with and in of The DNA was at for and with The was with in of a and and for of from and to on a that transfected with or from by and and in the by The in and by of by was prepared from the by of and as The for of was is located at to to the gene start site. and DNA and to for to the of was to of and of DTT, and The and by the at for by at for for by of of of and of to the was at for The with of and at for A of and of DNA a The was at in for and to The of promoter activity for by the elements in the promoter of and by the of their DNA-binding a of regulatory factors also been that or the transcription start site and make significant to promoter activity Scholar, J. Scholar, Scholar, Scholar, in the the GPHα gene promoter is elements in the DNA that with nuclear isolated from J.L. Scholar, Scholar, J.L. J. Scholar, J.L. J. Scholar, J.L. J. Scholar, J. and pituitary Scholar, Scholar, Scholar, the activity of the GPHα gene promoter from to was to in to the activity in To an understanding of the to expression of the GPHα promoter in and cell types as it was important to identify regulatory elements that for this promoter activity. of the the of the cap this was to for regulatory of a site centered at the of the GPHα a was that at on the the of the of the α-subunit gene promoter activity by gene expression in transfected with and in activity from was to that from These results suggest the of a negative regulatory element located in the of the GPHα The element is located or the transcription start as the promoter activity by from to the GPHα gene in this an imperfect was is located with the diad at the transcription start site and referred to as the To a for the CSDE in expression of the GPHα the cap site to the diad on from −5 to −2 and to the diad and has at and to and to and diad The DNA from to mutated cap site sequence from DNA and The and with activity was to activity to for in in activity from and as was and to that from the as at the the wild-type results indicate that CSDE as a negative regulatory of the diad to the as in the and at the The was that the of expression exhibited by from a in the transcription start site as a of the analysis was used to the of from the wild-type and A of the of is in the GPHα gene cap site is the of the are for the wild-type and for the These determined by the of or for wild-type and and or for wild-type and The results in A that from and the in that at the to of the GPHα is also that the of was that of further that the activity increase produced by the is at the of The of site-directed and in the CSDE on transcriptional activity of the α-subunit gene promoter the that this site with nuclear to GPHα gene To this was A that from to to the transcription start site of the α-subunit gene was and with cell nuclear of the binding by a the CSDE was by the of (CSDE) but it was not by the of GPHα DNA from to wild-type or mutated cap site used as of protein binding to the CSDE the different in the levels of DNA are in as the was with of wild-type whereas the of a at −5 to −2 to not with the of at the Thus, of the was dependent on an CSDE The of a with a the GPHα cap site diad that the CSBP a binding activity for the GPHα promoter and may as a to promoter activity. To the binding site for this a of that the of diad synthesized and used in a the wild-type and and used as for protein binding to a CSDE A was was by with of CSDE whereas and as by at the A and Thus, the from −5 to −2 and to in the CSDE are important for with CSBP. promoter activity and it was that bases may also important for binding To this of on as as the diad sequence was and binding The wild-type and and used as for protein binding to was was by of that the in and by and the of to by and in and on as to at with the results in indicate that the from −5 to −2 and +4 to +11 are the critical for binding of CSBP, and that bases to and to may also to but at a To identify that to CSBP nuclear extracts with this of in with the but not at the binding The results in that at and on the in the as at and +4 on the These are with in of at on the and at and on the with CSBP and this is by in was by of at on the These results of the mutagenesis to the diad sequence as a protein binding of the CSDE produced a GPHα promoter that was the wild-type it was of to the diad element to the and it it was in an or an To investigate this of the CSDE was of the promoter in and to and These constructs with and transfected The activity to activity is in levels for and and of for Thus, the CSDE in activity of the α-subunit gene but was by CSDE in the the promoter activity of constructs that of that activity of the negative element may the cap site diad or that the may CSBP and factors in the transcription The constructs the diad of the CSDE of the cap site. To in its the sequence by the and with and the to and for the of the of the cap site from and The at the transcription start of the GPHα promoter and at the diad in the CSDE of the and an CSDE in the wild-type and with of and at and and a and transfected the activity from and was that from but that from These results suggest that sequence from to of the GPHα gene may also to negative of the and that the CSDE to a not increase its activity. tumors in a variety of the α-subunit J. Scholar, Scholar, Scholar, M.R. Scholar, Rosen S.W. Weintraub B.D. J. 1973; Scholar, Scholar, Scholar, S.W. Weintraub B.D. J. Med. it was of to the activity of wild-type and in of cell types. the and with different cell lines, and and levels to of in the The in that the activities from and from the wild-type in of the cell types. and a for the mutated whereas the cell line little or to the wild-type and expression and in their expression of wild-type and promoter activity and the of CSBP in a variety of tumor cell activity binding transfected as with the expression and for and The activity to activity in of the are determined for in The the binding activity of CSBP in nuclear extracts prepared from the cell The of was determined by of The to extracts at are the of different nuclear in a transfected as with the expression and for and The activity to activity in of the are determined for in The the binding activity of CSBP in nuclear extracts prepared from the cell The of was determined by of The to extracts at are the of different nuclear the CSDE a negative on promoter activity in transient expression and the to a in shift it was that the of CSDE in the cell from in the levels of CSBP that are of cell To this in the of cell by the CSDE was used as a a was from nuclear extracts prepared from of the cell but their with and that the in cell not The of was by and the for cell to that of at are in results with nuclear and with not that an of CSBP binding is present in nuclear extracts from cell with binding as the of by of and extracts of protein was to the of the levels produced by not that the levels of CSBP not a of as DNA-binding not significant in binding or the binding activity in a from that of CSBP levels in in and The of the and least was in cell line by the increase in activity for the compared with the wild-type this is as the the ratio of levels produced from the in the of cell to the GPHα promoter for the contribution from the CSDE or from sequence of of activity to that of for in their the ratio of promoter activities in cell line is their of CSDE binding activity. is a with the levels of CSBP activity and a increase in transcription from to that from exhibited the levels of CSBP produced levels of activity from expression by the wild-type and These results suggest that the increase in activity of the promoter to the wild-type promoter is at least in part, on the levels of CSBP activity in cell of the glycoprotein hormone α-subunit gene is by a promoter that regulatory elements that nuclear DNA-binding J.L. Scholar, J.L. J. Scholar, J.L. J. Scholar, J.L. J. Scholar, J. elements transcription are located of the transcription start site. regulatory elements that with factors in basal transcription of the GPHα gene in are located and to the transcription start site it has that a of and elements for transcriptional that are located or the cap site Scholar, J. Scholar, Scholar, Scholar, of regulatory elements located in B.D. J. Scholar, in DNA J. and in and J. J.L. as or expression of gene constructs the GPHα gene promoter was to in in that for expression in is not present in in levels of activity produced from a GPHα DNA from to produced from the GPHα promoter DNA to that a the cap site a cis-regulatory element at least in part, for transcription of the GPHα promoter in transfected for the from the analysis of site-directed and with the that sequence and of the transcription start site to the activity of the diad element centered at The CSDE is as a negative regulatory element for the GPHα promoter of the activity of the to that of the The that the increase in production is by in the of the a in J. or by the of a transcription start site been on the in of the diad sequence also exhibited the activity expression from of an on or as this is of the transcription start site. of that the not the transcription start site and that activity for by a increase in levels Thus, activity of the CSDE is at the transcriptional transcriptional regulatory elements studied to as DNA elements for Electrophoretic mobility shift to the GPHα CSDE from to or a with nuclear from and The of CSDE of of the DNA sequence for protein binding and of bases from −5 to −2 and from +4 to +11 in this element binding to its cognate whereas mutated bases to in or to in on binding to of the diad element is important for promoter activity. a the activity The in transcriptional activity of the may an of the binding or the diad may to the is with the that an with an to an with a and analysis also that the with CSBP in the to of the CSDE the do not this it that the diad element in a the by is a and a was the wild-type CSDE at promoter activity. suggest that protein binding to CSDE is for its negative of the GPHα A of the transient expression and shift results that CSDE activity in different cell is to the levels of the factor (CSBP). The electrophoretic mobility shift that the CSBP is present in a variety of cell lines, and but at different levels These cell also a significant in their to a gene it was of a GPHα promoter at as compared with at Significantly, the of the CSDE compared with the wild-type diad element in a cell line was to the of CSBP activity in nuclear extracts prepared from that cell line with levels of CSDE binding activity a significant increase in their to the promoter to the wild-type and with CSDE binding activity a in their to the wild-type and mutated cap in transient expression These results are to the of CSBP as a the activity of at least in part, for the activity of the GPHα promoter in cell types. is that activity of the CSDE a the protein that binds to this element is present at different levels or with different binding by analysis of CSDE binding activity that of may as and been for CSBP in nuclear from different cell and the CSDE is located at the transcription start site of the GPHα it a for gene expression. a detailed understanding of its mechanism of further the are by the results The that in the CSDE the negative that of CSBP with DNA is to transcription of the GPHα The results in that production from the promoter was the CSDE was in a site of the cap that the GPHα CSDE in an as of a of CSDE at or at a of GPHα has not been the motif has of a The levels of CSDE binding activity in different cell and suggest that the levels of CSBP may at least mechanism by it transcription the of CSBP to transcription may further by its or the CSDE is an imperfect CSBP may a and the activity of the CSDE may on the to it is by or of are CSBP at transcription may with basal transcription factors for binding to a common DNA the cap site diad and the of a CSBP and or basal transcription factors and or may to of the transcription at the of or promoter CSBP may as to the GPHα promoter and the A was of sequence bases for the of the CSDE motif in of of the elements with or compared with the CSDE of to located at or the transcription start site. it of to for an on expression of the of the GPHα cap site sequence a to an located and of the promoter J. element the promoter by protein and protein is the that this element is to a sequence but with the GPHα cap site diad also a with to the of and in transient expression by and the with and the of pair also the CSDE or CSDE of or or was for expression not that the GPHα CSDE and the element are The GPHα gene is in the pituitary of and in the of and was of to the CSDE to the cap site sequence of α-subunit The sequence from to for and are in with a sequence of the bases that at the bases diad centered at are with and the and The sequence also as an imperfect in the and by or of a are with the The of bases that diad in sequence and the of compared with the sequence are at the The cap site are in the to the of bases diad this is to the of not to an the cap site sequence and placental expression. the of the is as it the of on the of in different with the that is to cap site sequence of GPHα are at the transcription start site and a sequence was cap site the of bases diad and the of compared to the sequence in the are with a on the and a on the bases that from the sequence are with The of in the to was determined by the of bases in the The and the sequence an imperfect in a at the transcription start site and a sequence was cap site the of bases diad and the of compared to the sequence in the are with a on the and a on the bases that from the sequence are with The of in the to was determined by the of bases in the The and the sequence an imperfect was the of this that expression with the GPHα promoter at the and is in the transient expression activity of wild-type and mutated CSDE are compared in cell a variety of CSBP the activities of and a the wild-type promoter as the levels of CSBP whereas the exhibited activity at least that of the promoter in the and of The basis for this is but at least an of CSDE may also to the negative of on GPHα an element in but not in and at to protein that with DNA from to been may to of the are for A is by the results of and that a important in the and of their transcription start that a sequence and and was important for this activity. of the GPHα wild-type and that at to was to the A to by the Thus, to a sequence for binding may also a on transcription the negative CSDE is by The results in that CSBP not to an to the sequence of the and in not it has been determined that at is for CSBP binding. further suggest the that an A to at in tumor may to GPHα gene expression, or the negative contribution that CSBP at the CSDE and sequence with for the of its mechanism of the of the CSDE, at least in part, as a transcription levels in cell types in the GPHα gene is normally and and its transcription in nonendocrine cell types in α-subunit production is to the of CSBP, a of negative regulatory on GPHα gene transcription been These by the at the an binding site the to J.L. and by of a for the J. may by a factor that binds a motif in and DNA The an for expression of the GPHα gene in cell it is and expression in cell the gene is the of negative regulatory in to the variety of and and elements and suggest the by to regulatory of GPHα gene expression. for with and for the of this