Identification of cis-acting promoter elements important for expression of the mouse glycoprotein hormone alpha-subunit gene in thyrotropes.

Identification of C/s-Acting Promoter Elements Important for Expression of the Mouse Glycoprotein Hormone a-Subunit Gene in Thyrotropes

Kenneth W. Ocran*, Virginia D. Sarapura, William M. Wood, David F. Gordon, Arthur Gutierrez-Hartmannf, and E. Chester Ridgway Division of Endocrinology Department of Medicine University of Colorado Health Sciences Center Denver, Colorado 80262 Division of Clinical Endocrinology Medizinische Hochschule Hannover (K.W.O.) D-3000 Hannover, West Germany

the human a-subunit gene in the placenta. (Molecular Endocrinology 4: 766-772, 1990)

The glycoprotein hormone a-subunit gene is expressed in a cell-specific manner in the anterior pituitary and placenta. Previous studies have shown that the region between -178 to - 1 1 1 is indispensable for placental-specific expression of the human a-subunit gene. Using gene transfer techniques with chimeric luciferase plasmids, this report identifies regions of the mouse a-subunit promoter that are important for transcriptional activation in primary thyrotropic cells. Transient expression of a series of 5' flanking DNA deletions resulted in stepwise reductions of basal promoter activity between -480 to -417 (4-fold), -254 to -177 (5-fold), and -177 to -120 (3.5-fold). DNase-l protection analysis with nuclear extracts from thyrotropic tumor cells revealed specific protein-DNA interactions within each of these functionally defined regions. These were mapped to positions -474 to -452, -447 to -419, -213 to -170, and -158 to - 1 0 1 within the 5' flanking region. In contrast, in mouse fibroblast Lcells no significant difference in a-subunit promoter activity was found by deleting the region from -480 to -177. However, a 3-fold decrease, similar to that found in primary thyrotropes, was found by deleting the region from -177 to -120. Further, a smaller region between -138 and -122 was the only area detected by the DNase-l protection assay using Lcell nuclear extracts. Thus, several c/s-acting promoter elements located up-stream of position -177 are important for expression in thyrotropes. These elements also bind nuclear factors present in thyrotropes but not in nonpituitary fibroblasts and, therefore, differ from those mediating expression of

INTRODUCTION

TSH is a member of the glycoprotein hormone family, which also includes FSH, LH, and hCG. These hormones consist of two nonidentical noncovalently linked subunits termed a and j8. The /3-subunit is unique and confers specific biological activity to each dimeric hormone (1). The a-subunit is common to all members of the family, but has a characteristic pattern of secretion and distinct regulatory responses. The underlying mechanism for differential expression and regulation of the a-subunit in thyrotropes, gonadotropes, and placental cells is not well understood. Expression and regulation of the human and bovine a-subunit genes have been extensively studied in human placental choriocarcinoma cell lines (2-5). Two major regulatory elements have been described. One is a tandem copy of an 18-basepair (bp) cAMP response element (CRE) located at positions -146 to -111 (6, 7) of the human a-subunit gene which resembles CREs found in other genes (8-10). In the human a-subunit gene this element mediates cAMP responsiveness in a nontissue-specific fashion, but has no effect on basal expression (7). In addition, an up-stream regulatory element (URE), located immediately 5' of the CRE, has no independent activity, but participates in the tissuespecific expression of the human a-subunit gene in choriocarcinoma cell lines by interacting with the CRE. Simultaneous binding by two or more proteins to these two promoter elements is necessary for a-subunit gene activation (5, 7). In contrast, little is known about c/s-acting DNA

0888-8809/90/0766-0772$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

766

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 November 2015. at 07:14 For personal use only. No other uses without permission. . All rights reserved.

Promoter Elements of Mouse «-Subunit Gene

elements and frans-acting factors, which are important for a-subunit gene activation in pituitary thyrotropes and gonadotropes. Recent experiments in transgenic mice suggest that factors that interact with c/s-acting sequences of the a-subunit gene are different in placental cells from those in the pituitary (11,12). Previously, this laboratory isolated and characterized the mouse a-subunit gene from a thyrotropic tumor (13). The TtT-97 thyrotropic tumor (14, 15) expresses both TSH subunits and responds to physiological regulators, such as thyroid hormone (T3), and TRH (16, 17). This tumor has proven to be an excellent source of thyrotropic cells to study TSH subunit gene regulation (18,19) within a homologous cellular environment. Recently, Sarapura et al. (20) have reported a dramatic reduction in transient expression of 5' deletions of the mouse a-subunit gene between -480 and -120 bp up-stream of the transcriptional start site. In the current studies nuclear proteins extracted from primary murine thyrotropic cells were used to identify areas of protein-DNA interaction within this region of the mouse a-subunit gene promoter. Additional gene transfer experiments with plasmid constructs containing or omitting these protected regions suggest that these sequences are important for expression of the mouse asubunit gene in thyrotropes.

767

Constructs -120A43 1 I) n-9 -177A43

1

-254/-43

1

^

-417/*43 1

^

n-9

•1

1

1

n-24

n-24

-4807*43 1 1000 2000 3000 4000 5000 17000 Relative Luciferase Activity (Light Units)

Fig. 1.5' Deletional Analysis of a-Subunit Luciferase Expression Constructs in TtT-97 Thyrotropes Primary TtT-97 thyrotropes were transfected with 20 ^g chimeric plasmid by electroporation. Plasmids contained the firefly luciferase reporter gene fused to fragments of the mouse a-subunit gene, the 5' and 3' extents of which are indicated on the left. Luciferase activities for 10 ng protein are plotted as solid bars and are expressed as light units relative to the activity of 1 million light units pA3RSVIuc. Values are the mean of n determinations, as indicated, ± SEM.

basal promoter activity. These data suggest that at least three functionally important promoter elements are necessary for transcriptional activation of the mouse a-subunit gene in thyrotropes.

RESULTS Localization of Sequences Involved in Transcriptional Activation

Nuclear Protein Binding to the Proximal Region of the a-Subunit Gene Promoter and Correlation with Expression in Primary Thyrotropes

To map sequences involved in basal promoter activity and expression of the murine a-subunit gene in thyrotropes, various fragments of the 5' flanking region fused to the firefly luciferase reporter gene were transiently transfected into TtT-97 cells and mouse L-cell fibroblasts by electroporation. In parallel, a promoterless construct (pA3LUC) was used to determine background activity, and a plasmid containing the Rous sarcoma virus (RSV) long terminal repeat (pA3RSVLUC) was used as a positive control. Sarapura et al. (20) have demonstrated that sequences between -480 to -120 bp of the mouse a-promoter have an important function in activating the expression of the a-subunit gene in thyrotropes. The current studies used a series of 5' deletions of the mouse a-subunit promoter from -480 to -120 bp and extending to position +43 within the first exon. These deletions resulted in stepwise reductions of luciferase expression: between -480 and -417 (4-fold), -254 and -177 (5-fold), and -177 and -120 (3.5-fold; Fig. 1). No drop was observed between -417 and -254. Overall, luciferase activity was 64-fold higher in the -480 construct pA3ma(-480/+43)LUC than that in a plasmid containing 120 bp of the mouse a-promoter pA3ma(-120/+43)LUC. The -120-bp construct still was 10-fold higher than the promoterless pA3LUC construct, probably reflecting the presence of both a TATA box and a CAAT sequence necessary for nonspecific

To more precisely identify c/s-acting sequences between -480 and -120 that are recognized by transacting factors, DNase-l protection analysis was performed. Nuclear extracts from both TtT-97 and L-cells were prepared and incubated with a Hae\\\/Pvu\\ fragment extending from -238 to - 6 3 of the mouse asubunit gene. This region accounts for a 10- to 15-fold change in transient expression in thyrotropes (Fig. 1). Two regions protected from DNase-l digestion, spanning nucleotides -158 to -101 (FP1) and -213 to -170 (FP2) on the noncoding strand, were detected using TtT-97 nuclear extracts (Fig. 2). With L-cell nuclear extracts, nucleotides between -138 and -122 showed partial protection; however, no protection was observed with L-cell extracts between -213 to -170 (Fig. 2). To further assess the role of FP1 and FP2 in asubunit expression in thyrotropes, promoter activities of constructs containing or lacking these regions were compared between TtT-97 and L-cells. The first construct, pA3ma(-177/+43)LUC, which included FP1, but not FP2, sequences, increased basal expression 3-fold in both TtT-97 thyrotropes and L-cells (Fig. 3) compared to another construct, pA3ma(-120/+43)LUC, which exhibited the same minimal basal promoter activity in both cell types. A second construct, pA3ma(-254/+43)LUC, which included both FP1 and FP2, increased basal


Vol 4 No. 5

MOL ENDO-1990 768

1 2

3

4

0

Constructs

-120A43

-254A43 1000

2000 3000 4000 S000 Relative Luciferase Activity (Light Units) I L-cells

! TtT 97

Fig. 3. Luciferase expression of proximal a-promoter sequences in L-cells and TtT-97 thyrotropes. L-Cells (solid bars) and TtT-97 cells (hatched bars) were assayed for luciferase expression after transfection with the indicated 5' truncated (-254, -177, and -120) fusion plasmids, all extending to position +43. Values are the mean of n determinations, as indicated, ± SEM and are normalized to 1 million light units pAaRSVLUC.

occurring within both FP1 and FP2 are important for activation of the murine a-subunit promoter in thyrotropes.

FP2

Protein-DNA Interactions with the Distal a-Subunit Promoter and Correlation with Expression in Primary Thyrotropes

-213

Fig. 2. DNase-l Protection Analysis of the Proximal Mouse aSubunit Gene 5' Flanking Region A 32P-labeled (5' end) -238 to - 6 3 Haelll-Pvull DNA fragment of the mouse a-subunit gene was treated with limiting amounts of DNase-l in either the absence (lane 0) or presence of varying amounts of crude nuclear extracts from TtT-97 cells (lane 1, 9 ng protein; lane 2,18 ng; lane 3, 24 ^g) and mouse fibroblast L-cells (lane 4, 33 »g protein). The location of the DNase-l-protected regions was determined by comparison with a Maxam and Gilbert sequencing ladder (not shown). The position relative to the transcriptional start site of regions protected from DNase-l cleavage are indicated by hatched boxes on the left.

expression 17-fold over that of the minimal promoter construct after transfection into TtT-97 cells (Fig. 3). In contrast, mouse L-cell fibroblasts exhibited similar levels of expression with DNA fragments containing FP1 alone or FP1 and FP2 together (Fig. 3). These gene transfer data suggest that protein-DNA interactions

To further assess the contribution of up-stream sequences for expression in thyrotropes, a fragment extending from -646 to -342 bp (Haelll/ftsal) was assayed for protein interactions by DNase-l protection (Fig. 4). This fragment was chosen to evaluate potential protein-DNA interactions in the region between -480 and -417, which accounts for a 4-fold change in transient expression. TtT-97 nuclear extracts protected the regions from -447 to -419 (FP3) and from -474 to -452 (FP4; Fig. 4). No DNase-l footprints were detected using L-cell nuclear extracts. A chimeric construct, pA3ma(-480/+43)LUC, was used to measure the activities of these two footprints in transiently transfected TtT-97 cells. This construct was compared with another fusion plasmid, pA3ma(-417/+43)LUC, which excluded both FP3 and FP4. As depicted in Fig. 5, the -480/+43 fusion plasmid showed a 4-fold increase in expression compared to that of the -417/+43 construct only in TtT-97 primary thyrotropes. In contrast, there was no significant difference between the expression of the two constructs when transfected into Lcells. This suggests that in addition to the previously identified FP1 and FP2 regions, FP3 and FP4 are important for activation of the a-subunit gene in thyrotropes, but the latter three regions do not influence its activity in nonpituitary fibroblasts.

DISCUSSION The a-subunit gene of the glycoprotein hormones is expressed in thyrotropes and gonadotropes of the pi-


769

Promoter Elements of Mouse a-Subunit Gene

3

4

S000 10000 15000 20000 Relative Luciferase Activity (Light Units)

Fig. 5. Luciferase Expression of Distal a-Promoter Sequences in L-Cells and TtT-97 Cells L-Cells (solid bars) and TtT-97 cells (hatched bars) were assayed for luciferase activity after transfection with 5' truncated (-417 and -480) plasmids extending to +43. Values are the mean of n determinations, as indicated, ± SEM and are normalized to 1 million light units PA3RSVLUC.

FP3

-419

§••m

Fig. 4. DNase-l Protection Analysis of the Mouse a-Subunit Distal Promoter Region A 32P-labeled (3' end) fragment (-646 to -342) of the 5' flanking region was incubated with either TtT-97 cell nuclear protein (lanes 1 -3) or crude nuclear extracts from L-cells (lane 4). The following amounts of protein were used: lane 1, 9 ^9; lane 2, 18 ^g; lane 3, 24 ^g; and lane 4, 33 fig. Chemical cleavage (Maxam and Gilbert; not shown) at purine residues (G+A) was used to determine the borders of DNase-l protection. The positions relative to the transcriptional start site of regions protected from DNase-l cleavage are indicated by hatched boxes on the left.

tuitary and in placental cells. A unique set of regulatory signals is probably involved in expression and regulation of the gene in each of these cell types. Activation of eucaryotic gene expression is initiated through binding of transcription factors to c/s-acting DNA regulatory elements or by inhibiting the action of transcriptional repressors (21, 22). It can, therefore, be hypothesized

that a family of unique frans-activating factors is responsible for the characteristic regulated expression of the a-subunit gene in each cell type. In the current study activation of the mouse a-subunit gene was studied using murine thyrotropic cells harvested from thyrotropic tumors. This laboratory has previously validated this model for studying TSH /?subunit gene transcription and T3 regulation (18, 19). Transient expression of a series of 5' flanking deletions resulted in stepwise reductions of basal promoter activity between -480 to -417 (4-fold), -254 to -177 (5fold), and -177 to -120 (3.5-fold). Using the DNase-l protection assay and nuclear extracts from TtT-97 thyrotropes, this study revealed four areas of protein-DNA interaction within these functionally important regions of the mouse a-subunit gene promoter; three of these were not observed with nuclear extracts from murine L-cell fibroblasts. These four areas have been denoted FP1 through FP4. FP1 (-158 to -101) contains a CCAAT sequence (-109 to -105) and also spans sequences that are similar to a single copy of the duplicated human a-subunit CRE and the URE which were both shown to be sufficient for a-subunit expression in placental JEG3 cells. However, the C/T transition seen in the fourth position of the core palindrome, which is highly conserved in a number of cAMP-responsive genes (23), has been found in the bovine a-subunit gene to substantially decrease the binding activity of CREB, a CRE-binding protein, and functionally does not respond to cAMP in JEG3 cells (12). Since the mouse sequence has the same C/T transition, it is likely to have a much lower binding affinity for placental CREB than the human CRE. Further experiments will be required to define the cAMP responsiveness of the mouse CRE homolog in thyrotropic cells. However, it is clear from the current studies that this 17-bp sequence is protected by thyrotropic nuclear proteins, even at low concentration, but whether this is due to CREB or a related protein remains to be elucidated. Furthermore, deletion of the FP1 region decreases the activity of the a-subunit promoter 3-fold in both TtT-97 and L-cells.


Vol 4 No. 5

MOL ENDO-1990 770

Thus, factor interactions at FP1 may contribute to basal expression, but may not confer cell-specific activation of the a-subunit gene in thyrotropic cells. In contrast, inclusion of the FP2 region in chimeric constructs enhances expression of the a-subunit promoter in thyrotropes 5-fold, a result not found in mouse L-cell fibroblasts. Similarly, constructs containing the FP3 and FP4 regions demonstrate further increases in promoter activity of 4-fold in thyrotropes, but not in L-cells. Recently, Horn et al. (24) reported several DNase-lprotected regions on the human glycoprotein hormone a-subunit gene with nuclear extracts from «T3-1 cells, a murine pituitary tumor line with gonadotropic properties that expresses the endogenous a-subunit gene. One of these protected regions maps to position - 2 1 8 to -205 of the human a-subunit gene. This corresponds to nucleotides - 2 1 4 to - 2 0 1 of the mouse gene and is contained within FP2. On the human gene this sequence was found to correlate with specific expression of chimeric plasmids in «T3-1 cells. A recent report (12) found that both human and bovine a-subunit promoters were transcriptionally active in the pituitary of transgenic mice, whereas only the human transgene was expressed in the placenta. This indicated that a different set of frans-acting factors is necessary for pituitaryand placenta-specific a-subunit gene activation. Whether FP2 represents a thyrotrope-specific enhancer element still remains to be elucidated. Certainly, this c/s-acting element (FP2) could play an important role in activation of the a-subunit gene in thyrotropes. The relationship between FP2 and FP3 or FP4 is also unclear. There are no striking sequence homologies between these DNA areas; thus, identical or dissimilar proteins may be interacting in a complementary fashion with different c/s-acting elements for full thyrotrope-specific activation. Additional studies using point mutations of FP2, FP3, and FP4 will be necessary to ascertain their relative importance. In summary, we have identified three c/s-acting transcriptional regulatory elements (FP2-FP4) on the mouse glycoprotein hormone a-subunit gene in addition to the sequences (FP1) corresponding to the previously identified human a-subunit gene enhancer elements (CRE and URE), that are important for transcriptional activation in thyrotropic cells. Comparative experiments with placental, gonadotropic, and thyrotropic cells will be necessary to further elucidate the roles of these c/sacting elements in cell-specific a-subunit gene regulation.

MATERIALS AND METHODS Construction of Specific Mouse a-Subunit Probes The fragments used in the DNase-l protection assays, 5'm-a Haelll-flsal (-646/-342) and 5'm-a tfaelll-Pvull (-238/-63), were subcloned into pGem7Zf(+) (Promega, Madison, Wl) at the Smal site, and junctions were verified by dideoxynucleotide sequencing (25). Fragments were excised by digestion with EcoRI and Mlu\ (Boehringer Mannheim, Indianapolis, IN) sites

bordering the insert. The 357- and 228-bp fragments, respectively, were size-separated on a 4.5% polyacrylamide gel, eluted, and purified by diethylaminoethyl-cellulose (DE52) chromatography (Whatman Bio Systems, Maidstone, Kent, England). The resultant Mlu\ and EcoRI 5' protruding ends allowed the probes to be preferentially end labeled at a unique terminus with reverse transcriptase (Promega) and either [«32P]dCTP plus [«32P]dGTP or [a32P]dATP plus [a32P]TTP (ICN Radiochemicals, Irvine, CA), respectively. Preparation of Nuclear Extracts Nuclear extracts were prepared using previously described methods (26, 27) with the following modifications. TtT-97 thyrotropic tumors (Biomeasure, Inc., Hopkinton, MA) were dissected from LAF1 mice (Jackson Laboratory, Bar Harbor, ME), weighed, and immediately placed on ice. All subsequent operations were carried out at 4 C. Tissue was minced and washed several times with buffer A [0.25 M sucrose, 15 rriM Tris-CI (pH 7.9), 60 mM KCI, 2 mM EDTA, 15 HIM NaCI, 0.5 mM spermine, 0.15 mM spermidine, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethlysulfonylfluoride (PMSF)], followed by passage through a 1-mm stainless steel mesh to disperse cells and remove connective tissue. Cells were then broken with a motor-driven Teflon pestle in a glass homogenizer, and the homogenate was filtered through two layers of cheesecloth. The filtrate was adjusted to 0.3% Triton X-100, and homogenization was repeated. The homogenate was carefully mixed with an equal volume of buffer B (buffer A plus 2.6 M sucrose) and layered over 5-ml cushions each of buffer Cl (buffer A plus 2.1 M sucrose) and buffer Cll (buffer A plus 1.7 M sucrose). After centrifugation at 25,000 rpm for 50 min (at 0 C) in a Beckman (Palo Alto, CA) SW28 rotor, the supernatants were aspirated and discarded. The pelleted nuclei were transferred to a Dounce homogenizer (Kontes Co., Vineland, NJ) and resuspended in buffer D [100 mM KCI, 10 mM Tris-CI (pH 7.9), 2 mM MgCI2> 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF]. This nuclear suspension was then transferred to an ultracentrifuge tube. To lyse the nuclei and dissociate nuclear proteins, 4 M ammonium sulfate was added dropwise to a final concentration of 0.4 M, followed by gentle shaking for 30 min (4 C). DNA was then removed from the mixture by centrifugation at 30,000 rpm for 60 min at 4 C in a Beckman Ti70 rotor. The supernatants were pooled, and proteins precipitated with 0.33 g ammonium sulfate/ml supernatant, followed by gentle stirring for 30 min. The precipitate was collected by centrifugation at 30,000 rpm for 20 min, resuspended in TGMEDK buffer [25 mM Tris-CI (pH 7.9), 10% glycerol, 5 mM MgCI2,100 mM KCI, 0.1 mM EDTA, 1 mM DTT, and 1 mM PMSF] and subsequently dialyzed twice against 500 ml TGMEDK buffer for 3 h each. Extracts were cleared by centrifugation at 10,000 rpm for 5 min, and aliquots were frozen in dry ice and stored at - 7 0 C. Protein concentrations were determined by the method of Bradford (28) (Bio-Rad, Richmond, CA), using BSA (Boehringer Mannhein) as a standard. DNase-l Protection Assay of Protein-DNA Interactions The DNase-l protection assay was performed as previously described by Jones et al. (29). Reactions were carried out in a total volume of 50 n\. The final KCI concentration was kept between 60-80 mM. Nuclear extract was preincubated in binding buffer [25 mM Tris-CI (pH 7.9), 5 mM MgCI2, 0.1 mM EDTA, 10% glycerol, and 1 mM DTT] in the presence of 500 ng salmon sperm DNA on ice for 15 min. The binding reaction was initiated by the addition of 0.3-0.7 ng uniquely end-labeled DNA fragment, and incubation was continued at room temperature for 20 min. Freshly diluted DNase-l (Cooper Biomedical, Malvern, PA), an amount empirically determined, was added in a volume of 50 fi\. After 60 sec of digestion at room temperature, the reaction was terminated with 100 M' 0.5% sodium dodecyl sulfate, 0.2 mM NaOAc (pH 5.0), 30 mM EDTA, and 100 Mg/ml mussel glycogen (Sigma, St. Louis, MO). The


771

Promoter Elements of Mouse a-Subunit Gene

labeled DNA was extracted with phenol-chloroform and ethanol precipitated, and pellets were dried in vacuo. Fragments were dissolved in denaturing dye mixture containing 90% formamide and 0.1% xylene cyanol, heated at 90 C for 3 min, and size-separated by electrophoresis through a 7% polyacrylamide sequencing gel containing 8 M urea. Gels were dried and exposed to Kodak X-AR-5 film (Eastman Kodak Co., Rochester, NY) for 1 - 3 days using intensifying screens. The precise location of protected regions was determined by the chemical sequencing method of Maxam and Gilbert (30).

Received January 15,1990. Revision received February 19, 1990. Accepted February 19,1990. Address requests for reprints to: Dr. E. Chester Ridgway, UCHSC Box B-151, 4200 East Ninth Avenue, Denver, Colorado 80262. This work was supported in part by NIH Grants DK-36843 and CA-47411 (to E.C.R.) and NIH Grant DK-37667 (to A.G.H.). * Supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Oc 17/1-1). t Pew Scholar in the Biomedical Sciences.

Construction of Luciferase Expression Vectors Standard protocols were used for all plasmid constructions (31). The control promoterless construct PA3LUC and the strong promoter-containing plasmid pA3RSVLUC were constructed as previously described (18). Chimeric plasmids containing 1700, 480, 254, and 120 bp of the 5' flanking region of the mouse a-subunit gene promoter (13) and extending to position +43 within the first exon were inserted into the promoterless expression vector pA3LUC. The detailed construction of these plasmids is described by Sarapura, V.D., W.M. Wood, D.F. Gordon, K.W. Ocran, M.Y. Kao, and E.C. Ridgway (manuscript in preparation). To generate expression vectors containing 417 and 177 bp of the 5' flanking region and extending to position +43, plasmid pA 3 ma(-1700/ +43)LUC was linearized by digestion with AflW (-580) or Ba/I (-238), followed by digestion with exonuclease-lll (Boehringer Mannhein) and mung bean nuclease (Promega). After Sma\ digestion, plasmids were recircularized by ligation under dilute conditions. This generated a series of deletions from the indicated sites; two of these were chosen, pA3m«(—417/ +43)LUC and pA3ma(-177/+43)LUC, and their junctions were verified by DNA sequencing using the chain termination method (25). Plasmids were prepared by alkaline lysis (32), followed by two CsCI density centrifugations (33). Preparation of Primary Thyrotropes and L-Cells for Transfection TtT-97 thyrotropic tumors were propagated in hypothyroid male LAF1 mice and cultivated into primary cell dispersion, as previously described (18). Briefly, LAF1 mice were killed, and TtT-97 tumor was excised and finely minced. After washing the tissue filtrate with Hanks' Balanced Salt Solution, cells were collected by centrifugation. The cells were then suspended in Hanks' Balanced Salt Solution and digested with trypsin (Sigma) and DNase-l (Sigma) for 30 min at 37 C, followed by exhaustive washing to remove these enzymes. The cells were finally suspended to a density of 3 million viable cells/100 n\ Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) supplemented with 10% charcoal-stripped fetal calf serum (Gibco). Transfection and luciferase assays were carried out as previously described (18). Monolayer cultures of mouse L-cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum. Experimental Animals Studies using LAF1 mice for the propagation of TtT-97 thyrotropic tumors were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Acknowledgments We thank Angela Nelson for excellent technical assistance. We are also indebted to the Tissue Culture Core Laboratory of the University of Colorado Cancer Center (NIH Grant CA46934) for growing the L-cell fibroblasts.

REFERENCES 1. Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465-495 2. Bokar JA, Roesler WJ, Vandenbark GR, Kaetzel DM, Hanson RW, Nilson JH 1988 Characterization of the cyclic AMP responsive elements from the genes for the asubunit of the glycoprotein hormones and phosphoenolpyruvate carboxykinase (GTP). J Biol Chem 263:1974019747 3. Jameson JL, Deutsch PJ, Gallagher GD, Jaffe RC, Habener JF 1987 Trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin a gene. Mol Cell Biol 7:3032-3040 4. Jameson JL, Jaffe RC, Deutsch PJ, Albanese C, Habener JF 1988 The gonadotropin a-gene contains multiple protein binding domains that interact to modulate basal and cAMP-responsive transcription. J Biol Chem 263:98799886 5. Jameson JL, Albanese C, Habener JF 1989 Distinct adjacent protein-binding domains in the glycoprotein hormone a gene interact independently with a cAMP-responsive enhancer. J Biol Chem 264:16190-16196 6. Silver BJ, Bokar JA, Virgin JB, Vallen EA, Milsted A, Nilson JH 1987 Cyclic AMP regulation of the human glycoprotein hormone a-subunit gene is mediated by an 18-base-pair element. Proc Natl Acad Sci USA 84:2198-2202 7. Delegeane AM, Ferland LH, Mellon PL 1987 Tissue-specific enhancer of the human glycoprotein hormone asubunit gene: dependence on cyclic AMP-inducible elements. Mol Cell Biol 7:3994-4002 8. Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682-6686 9. Short JM, Wynshaw-Boris A, Short HP, Hanson RW 1986 Characterization of the phosphoenolpyrvate carboxykinase (GTP) promoter-regulatory region. II. Identification of cAMP and glucocorticoid regulatory domains. J Biol Chem 261:9721-9726 10. Comb M, Birnberg NC, Seasholtz A, Herbert E, Goodman H 1986 A cyclic AMP- and phorbol ester-inducible DNA element. Nature (Lond) 323:353-356 11. Fox N, Solter D 1989 Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone a-subunit gene is restricted to the pituitary in transgenic mice. Mol Cell Biol 8:5470-5476 12. Bokar JA, Keri RA, Farmerie TA, Fenstermaker RA, Andersen B, Hamernik DL, Yun J, Wagner T, Nilson JH 1989 Expression of the glycoprotein hormone a-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different c/s-acting element mediates pituitary-specific expression. Mol Cell Biol 9:5113-5122 13. Gordon DF, Wood WM, Ridgway EC 1988 Organization and nucleotide sequence of the mouse a-subunit gene of the pituitary glycoprotein hormones. DNA 7:679-690 14. Furth J 1955 Experimental pituitary tumors. Recent Prog Horm Res 11:221-257


MOL ENDO-1990 772

Vol 4 No. 5

15. Condliffe PG, Mochizuki M, Fontaine YA, Bates RW 1969 Purification and properties of thyrotropin from functional pituitary tumor in mice. Endocrinology 85:453-464 16. Chin WW, Shupnik MA, Ross DS, Habener JF, Ridgway EC 1985 Regulation of the a and thyrotropin /3-subunit messenger ribonucleic acids by thyroid hormones. Endocrinology 116:873-878 17. Cacicedo L, Pohl SL, Reichlin S 1981 Pituitary tumor cells in vitro. Endocrinology 108:1012-1091 18. Wood WM, Kao MY, Gordon DF, Ridgway EC 1989 Thyroid hormone regulates the mouse thyrotropin /3-subunit gene promoter in transfected primary thyrotropes. J Biol Chem 264:14840-14847 19. Alexander LM, Gordon DF, Wood WM, Kao MY, Ridgway EC, Gutierrez-Hartmann A 1989 Identification of thyrotroph-specific factors and c/s-acting sequences of the murine thyrotropin 0-subunit gene. Mol Endocrinol 3:1037-1045 20. Sarapura VD, Kao MY, Gordon DF, Wood WM, The mouse glycoprotein hormone a-subunit gene promoter exhibits both negative and positive regulation by thyroid hormone when transfected into murine thyrotropic tumor cells. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 267 (Abstract) 21. Maniatis T, Goodboum S, Fischer JA 1987 Regulation of inducible and tissue-specific gene expression. Science 236:1237-1245 22. Akerblom IE, Slater EP, Beato M, Baxter JD, Mellon PL 1988 Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. Science 241:350-353 23. Roesler WJ, Vandenbark GR, Hanson RW 1988 Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 263:9063-9066

24. Horn F, Windle JJ, Mellon PL, Tissue-specific and hormone-regulated expression of the human glycoprotein hormone a-subunit gene in gonadotrope pituitary cells. Cold Spring Harbor Laboratory Meeting, Cold Spring Harbor NY, 1989, p 92 (Abstract) 25. Sanger E, Nicklen S, Coulson A 1977 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 26. Graves BJ, Johnson PF, McKnight SL 1986 Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell 44:565-576 27. Parker CS, Topol J 1984 A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity. Cell 36:357-369 28. Bradford MM 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248254 29. Jones KA, Yamamoto KR, Tjian R 1985 Two distinct transcription factores bind to the HSV thymidine kinase promoter in vitro. Cell 42:559-572 30. Maxam AM, Gilbert W 1980 Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499-560 31. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 32. Birnboim HC, Doly J 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513-1523 33. Radloff R, Bauer W, Vinograd J 1967 A dye-buoyantdensity method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci USA 57:1514-1521

The Endocrine Society's 72nd Annual Meeting ATLANTA June 20-23, 1990 Georgia World Congress Center For program and registration information please contact: The Endocrine Society 9650 Rockville Pike Bethesda, MD 20814

(301)571-1802 FAX (301) 571-1869 Meet Us On Peachtree, June 20-23,1990


Thyrotrope expression and thyroid hormone inhibition map to different regions of the mouse glycoprotein hormone alpha-subunit gene promoter.

Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter.

Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus.

The function of conserved elements in the promoter of the mouse angiotensinogen gene.

Upstream promoter elements of the herpes simplex virus type 1 glycoprotein H gene.

Identification of functional glucocorticoid response elements in the mouse FoxO1 promoter.

Two promoter elements are necessary and sufficient for expression of the sea urchin U1 snRNA gene.

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA.

Protein factors in thyrotropic tumor nuclear extracts bind to a region of the mouse thyrotropin beta-subunit promoter essential for expression in thyrotropes.

Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene.

Promoter elements and regulation of expression of the cysD gene of Escherichia coli K-12.

A combination of upstream and proximal elements is required for efficient expression of the mouse renin promoter in cultured cells.

Identification and Analysis of Regulatory Elements in Porcine Bone Morphogenetic Protein 15 Gene Promoter.

Different combinations of regulatory elements may account for expression of the glycoprotein hormone alpha-subunit gene in primate and horse placenta.

Identification and characterization of the promoter for the cytotactin gene.

Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns.

Identification of an up-stream promoter of the human parathyroid hormone-related peptide gene.

Identification of the promoter region and gene expression for human acid alpha glucosidase.

A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

Analysis of sequence elements important for expression and regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium.

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.

Comparison of the mouse L32 ribosomal protein promoter elements in mouse myoblasts, fibers, and L cells.

Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.