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Molecular Endocrinology 12 (5): 622-633
Copyright © 1998 by The Endocrine Society

Cell-Specific Expression of the Mouse Glycoprotein Hormone {alpha}-Subunit Gene Requires Multiple Interacting DNA Elements in Transgenic Mice and Cultured Cells

Michelle L. Brinkmeier, David F. Gordon, Janet M. Dowding, Thomas L. Saunders, Susan K. Kendall, Virginia D. Sarapura, William M. Wood, E. Chester Ridgway and Sally A. Camper

Department of Human Genetics (M.L.B., T.L.S., S.K.K., S.A.C.) University of Michigan Ann Arbor, Michigan 48109-0638 Department of Medicine (D.F.G., J.M.D., V.D.S., W.M.W., E.C.R.) University of Colorado Health Sciences Center Denver, Colorado 80262


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glycoprotein hormone {alpha}-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between -4.6 and -3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse {alpha}-subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact -4.6 kb/+43 construct. Deletion of sequences between -341 and -297 resulted in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a -341/+43 {alpha}-promoter in both {alpha}TSH and {alpha}T3 cells, but it did not enhance {alpha}-promoter activity significantly in CV-1 cells. Removal of sequences between -341 and -297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the {alpha}-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from -341 to -297 are critical for restricting expression to the anterior pituitary.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The anterior pituitary gland is composed of five cell types that are distinguished by the hormones they produce: thyrotropes produce TSH, gonadotropes produce LH and FSH, somatotropes produce GH, lactotropes produce PRL, and corticotropes produce ACTH. Three of these hormones, TSH, LH, and FSH, belong to a pituitary glycoprotein hormone family that consists of heterodimeric proteins with a common {alpha}-subunit and unique ß-subunits. The requirement for regulated expression of the {alpha}-subunit in gonadotropes and thyrotropes in response to different stimuli suggests that different cis-acting DNA sequence elements may be involved. Studies with transgenic mice have supported this idea. Two large human {alpha}-subunit transgenes containing more than 5 kb of 5'-sequences were sufficient for pituitary-specific expression and targeted oncogenesis of both gonadotrope and thyrotrope lineages (1, 2). In contrast, 1.5–1.8 kb of the human {alpha}-subunit 5'-flanking sequence directed transgene expression solely to gonadotropes, suggesting the possibility of a more upstream element necessary for expression in thyrotropes (3, 4). Consistent with this idea, gonadotrope specificity was also observed in transgenic mice with -315 bp of the bovine {alpha}-subunit promoter driving reporter gene expression (5, 6). However, the potential for divergence in the location of cell-specific elements was suggested by the observation that both gonadotrope and thyrotrope expression can be obtained with only -480 bp of mouse {alpha}-subunit 5'-flank (7).

Transfection studies using cell lines representative of thyrotropes ({alpha}TSH) and gonadotropes ({alpha}T3), as well as a murine TtT-97 mouse thyrotropic tumor cell model (8), have implicated individual regions of the {alpha}-subunit gene in cell specificity and hormone regulation. Most of these studies have focused on defining important cis-acting elements within the proximal 500 bp upstream of the transcription initiation site in the mouse and human genes (Fig. 1Go) (9). Deoxynuclease I (DNase I) protection studies using TtT-97 thyrotropic nuclear extracts have identified cell-specific protein-DNA interactions between -474 and -419 bp (10). A lin11, Isl1, mec3 (LIM) homeodomain factor-binding site, functionally important in gonadotropes and thyrotropes, has been localized to the area between -342 and -329 bp (11, 12) of the mouse gene and a similar element, pituitary glycoprotein hormone basal element (PGBE), was identified in the human gene (13). Recently, two basal elements located just downstream of the PGBE site within the human {alpha}-promoter, {alpha}BE1 and {alpha}BE2, were demonstrated to be functionally important for expression in {alpha}T3 cells (13). Additional gonadotrope-specific elements have been localized between positions -445 to -438 (11), and a GnRE at -406 to -399 (14) of the mouse gene. These elements were shown to be important for basal activity or GnRH responsiveness, respectively. Two other elements within the human gene important for expression in gonadotropes bind the transcriptional activators SF-1, -225 to -205 bp (15), and GATA2/3, -161 to -146 (16).



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  		Figure 1. Deletion Constructs Designed to Test Importance of Sites of Protein-DNA Interaction

A schematic representation of -480 bp of m{alpha}GSU 5'-flanking sequence summarizes data identifying thyrotrope-specific (T) and gonadotrope-specific (G) protein-DNA interaction sites (9). Some factors that bind these regions have been identified: GnRE, GnRH responsive element; Ptx-1/P-OTX, pituitary transcription factor; Lhx2, LIM homeodomain protein; {alpha}BE1 and 2, {alpha}-basal element 1 and 2; SF-1, steroidogenic factor 1; GATA2/3. Transfection studies identifying Ptx1, {alpha}BE1, and {alpha}BE2 were performed with either the rat {alpha}GSU promoter (PTX1) or the human {alpha} GSU promoter ({alpha}BE1, {alpha}BE2) although consensus binding sites for these factors can be found in mouse {alpha} GSU promoter sequence. The location of potential interaction sites with these factors within the -480 bp is noted. The -381-, -341-, and -297-bp constructs were designed for transfection and transgenic mouse assays based on the deletion of binding sites important for gonadotrope and thyrotrope specificity.

 
Transgenic mice provide a rigorous in vivo test of the relevance of individual sites for cell-specific expression in the context of chromatin while transient transfection of cell lines allows the testing of many different constructs and a measurement of their differential effects on promoter activity in homologous and heterologous cells. We generated and analyzed transgenic mice bearing deletion constructs that were designed to test the importance of specific regions thought to play a role in gonadotrope and thyrotrope specificity. Previous studies have suggested that an element between -4.6 and -2.7 kb is important for high-level expression in transgenic mice (7). We have used transgenic mice and cell transfection to refine and characterize this enhancer region. The results illustrate that the region between -4.6 and -3.7 kb functions as an enhancer element and contributes to the restriction of {alpha}-subunit gene expression when juxtaposed to a proximal {alpha}-promoter construct. Our data also indicate that cell-specific expression in thyrotropes and gonadotropes is due to interactions between multiple elements. Although neither the LIM- nor {alpha}BE1-binding sites are essential for transgene expression in gonadotropes and thyrotropes, one or both are critical for restriction of expression to these two pituitary cell types.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Deletion Analysis Demonstrates the Importance of Sequences between -4.6 and -3.7 kb for High-Level Expression
Levels of transgene expression in mice carrying constructs of the mouse {alpha}-subunit promoter (m{alpha}) with 5'-termini at -3.7 and -3.1 kb were compared with previously published data from -4.6 and -0.48 m{alpha}-ßgal transgenics to narrow the 5'-flanking region necessary for high-level expression in the anterior pituitary (7). Fragments (-3.7 and -3.1 kb) of m{alpha} were joined within the 5'-untranslated region of exon 1 to a lac Z reporter gene containing a nuclear localization signal and used to generate transgenic mice. Twelve and 11 transgenic mouse lines were generated with the -3.7 and -3.1 kb m{alpha}-ßgal constructs, respectively. The level of transgene expression in each line was quantitated in pituitary gland homogenates using a fluorometric assay for ß-galactosidase activity (Materials and Methods). ß-Galactosidase levels in each transgenic line were expressed relative to the level in the highest expressing -4.6 kb m{alpha}-ßgal transgenic line, 8365 (7). None of the transgenic mice with the -3.7 or -3.1 kb m{alpha}-ßgal transgenes exhibited high-level expression (Fig. 2Go). In contrast, half of the lines with the -4.6 m{alpha}-ßgal construct expressed at a high level (7). While the position of integration influences the level of expression in each founder, the difference between the -4.6 and the smaller -3.7 and -3.1 m{alpha} constructs is statistically significant (P = 0.0017 and P = 0.0036, respectively) according to the Fisher’s Protected Least Significant Difference ANOVA post hoc test. These data suggest that the sequences between -4.6 and -3.7 kb are important for high levels of expression in the anterior pituitary.



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  		Figure 2. Deletion Analysis in Transgenic Mice Reveals the Ability of the -4.6- to -3.7-kb Region to Function as an Enhancer

Transgenic mice generated with 3.1 and 3.7 kb of m{alpha}-subunit 5'-flank joined to the ßgal reporter gene were used to narrow the enhancer region to the 859 bp between -4.6 and -3.7 kb. This enhancer region (E) was tested in transgenic mice in conjunction with various fragments of m{alpha}-subunit promoter 5'-flank fused to the ßgal reporter gene (E/-480, E/-381, E/-341, E/-297). Transgenic mice generated with enhancerless constructs served as a control for enhancer activity (-381 and -341). ßgal activity was measured in homogenates of individual pituitary glands. Each circle represents an individual transgenic founder. The open circles represent founders that tested positive for the transgene by PCR but have no ßgal activity detectable in homogenates or in Xgal-stained pituitary sections. The ßgal activity of each founder is expressed as a percentage of the ßgal activity detected in the highest expressing founder with the -4.6-kb construct (100%) (7). The dashed line indicates the arbitrary lower limit of high expressing transgenic mouse lines. Stars next to the constructs indicate those that are statistically different from the highest expressing -4.6-kb transgenic line (*, P < 0.05; **, P < 0.01; ***, P < 0.001; exact P values are stated in the text). Results of -4.6 kb and -480 bp are reproduced here for ease of comparison (7).

 
The 5'-Flanking Region between -4.6 to -3.7 kb Can Function as an Enhancer in Transgenic Mice
To test whether an 859-bp fragment between -4.6 and -3.7 kb could function in vivo as a position-independent enhancer, we fused it 5' of several more proximal m{alpha} promoter fragments containing or lacking sequences thought to be important for gonadotrope and thyrotrope expression (9). Based on 5'-deletion and mutagenesis studies in transfected cell lines, we used proximal promoter fragments of -480, -381, -341, and -297/+43 bp fused to a ßgal reporter gene containing a nuclear localization signal (Fig. 1AGo). These constructs were termed E/-480, E/-381, E/-341, and E/-297. Additionally, -480, -381, and -341 m{alpha}-ßgal constructs lacking the 859-bp sequence were tested in transgenic mice. Transgenic founders were identified by PCR of genomic DNA obtained from tail biopsies. The initial screening was carried out with primers designed to amplify a portion of the lac Z gene. The integrity of the transgene was confirmed by PCR amplification with primers specific for the m{alpha} enhancer-promoter fusion region. The identity of these PCR products was verified by restriction enzyme digestion and DNA sequence analysis. The pituitary glands from individual transgenic mice were analyzed for ßgal activity using a chemiluminescent assay on tissue homogenates and Xgal staining on tissue sections.

Interestingly, the enhancer functioned most effectively when fused upstream of the -341/+43 bp fragment. The levels of transgene expression in lines analyzed from the E/-341 m{alpha}-ßgal construct were generally high and exhibited a similar pattern to those observed with the -4.6 kb m{alpha}-ßgal construct (Fig. 2Go) (7). In contrast, the range of expression levels detected in transgenic lines bearing the -341 m{alpha}-ßgal construct was consistently low, similar to that observed in the -480 bp m{alpha}-ßgal transgenics (Fig. 2Go) (7). This suggests that sequences contained within the 859-bp fragment are required to obtain high levels of expression in the pituitary.

Little or no enhancement was evident when the 859-bp segment was fused 5' of the -480 and -381 bp m{alpha}-promoters (Fig. 2Go). Although one high expressing line was generated from each of these constructs, the majority of the founders did not express the transgene in the anterior pituitary by two independent assay methods. This suggests that the enhancer functions optimally when juxtaposed to the -341/+43 proximal promoter fragment and is inhibited by an additional 40 or 139 bp of m{alpha}-5'-flanking sequence. The lack of enhancement when fused to some promoter fragments may result from incorrect spacing between elements within the enhancer and proximal promoter or from differences in chromatin structure. Transgenics generated from the E/-297 construct exhibited very poor expression in all 19 animals tested (Fig. 2Go). The range of expression levels was similar to the -480 bp m{alpha}-ßgal transgenics (7). Low transgene activity may result from the loss of an important LIM-binding site or the adjacent {alpha}BE1 site (12, 13). The differences in ßgal activity between both -4.6 vs. E/-297 and -4.6 vs. -341 m{alpha}-ßgal transgenics were verified using Fisher’s Protected Least Significant Difference ANOVA Post hoc test (P = 0.0008 and 0.0027, respectively). Levels of ßgal activity in transgenics from m{alpha} -4.6 and E/-341 were not significantly different (P = 0.4). The results indicate that the 859-bp upstream enhancer can function in vivo in a position-independent manner and that its effectiveness is influenced by its context with respect to other proximal promoter sequences.

E/-297 Transgenic Mice Show a Loss of Cell-Type Restriction in the Pituitary
Previous studies had demonstrated that both -4.6 kb and -0.48 kb were sufficient for cell-specific expression even though -0.48 kb were expressed at a much lower level (7). Six of six 4.6-kb transgenics exhibited appropriate cell specificity and four of four 0.48-kb transgenics as well. Each of the enhancer-promoter deletion constructs was assessed for the ability to confer pituitary cell specificity. Half of the pituitary gland from each transgenic founder was stained with Xgal, embedded in paraffin, and sectioned. The penetrance of transgene expression was determined by quantitating the average number of nuclei stained with Xgal in a set field of approximately 650 cells (n = 12). Founders exhibiting a penetrance of expression of 1% or greater were examined for cell specificity by immunohistochemistry with antibodies to the pituitary hormones: TSH, LH, ACTH, GH, and PRL. Cell specificity was calculated by dividing the number of cells that exhibited colocalization of the transgene and the endogenous pituitary hormone gene by the total number of immunostained cells and reporting it as a percentage. These percentages were compared with those obtained from -4.6 kb and -480 bp m{alpha}-ßgal transgenic lines. The E/-480, E/-381, -381, and E/-341 constructs all retained appropriate transgene expression in gonadotropes and thyrotropes (Table 1Go). Transgene expression was consistently seen in a few somatotropes and may reflect GH/TSH double positive cells that can be found in wild-type pituitary (17). No transgene expression was detected in corticotropes or lactotropes. In contrast, three of three transgenics analyzed from the -341 construct exhibited a loss of pituitary cell specificity. Three lines from the -341 construct and two from the E/-341 construct were examined by immunohistochemistry. Each of the E/-341 lines expressed the transgene only in gonadotropes and thyrotropes (Fig. 3Go, A and B). In contrast, transgene expression was detected in corticotropes and somatotropes in addition to gonadotropes and thyrotropes in all three lines from the -341 construct (Fig. 3Go, C–F). One of these lines also expressed the transgene in lactotropes (Table 1Go). Similarly, the E/-297 transgene was expressed in pituitary cells that stained positively for TSH and LH as well as in those cells secreting ACTH, GH, and PRL (Fig. 4Go and Table 1Go). These data suggest that in the absence of the enhancer, sequences between -381 and -341 are important for the restriction of m{alpha}-subunit expression to pituitary gonadotropes and thyrotropes. The enhancer conferred the ability to restrict expression to the appropriate cell types when fused to the -341 construct. However, even in the presence of the enhancer region, cell specificity was lost when the region from -341 to -297 was removed. Thus, both the enhancer and proximal promoter sequences contribute to restrict expression to the appropriate pituitary cells.


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  		Table 1. Cell Specificity of Mouse {alpha}-Subunit Transgene Constructs

 


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  		Figure 3. Colocalization of the -341 m{alpha}-ßgal Transgene to {alpha}GSU Expressing and Nonexpressing Pituitary Cells

Paraffin sections of Xgal-stained pituitaries from transgenics generated with either the E/-341 or -341 constructs were immunostained with hormone-specific antibodies. Cells with blue nuclei represent those expressing the transgene. Cells costained with Xgal and the indicated pituitary hormone (brown cytoplasm) are marked with arrows. A, E/-341 and TSH; B, E/-341 and LH; C, -341 and TSH; D, -341 and LH; E, -341 and ACTH; and F, -341 and GH.

 


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  		Figure 4. Nonspecific Expression of the E/-297 m{alpha}-ßgal Transgene in Anterior Pituitary Cells

Paraffin sections of Xgal-stained pituitaries from transgenic mice generated with the E/-297 construct were immunostained with antibodies raised against TSH, LH, ACTH, GH, or PRL. Cells with blue nuclei represent those expressing the transgene. Cells with a brown cytoplasm represent those producing the respective pituitary hormone. Representative cells costained with Xgal and a particular pituitary hormone are marked with arrows.

 
E/-297 Transgenics Exhibit Inappropriate Transgene Expression in Peripheral Tissues
For each transgene construct, we examined several lines for ectopic transgene expression in nonpituitary tissues. Peripheral tissues from transgenic founders were sectioned and stained with Xgal to test for inappropriate expression of the transgene. Nontransgenic mice were used to determine background levels of Xgal staining in the selected tissues (Fig. 5AGo). Extrapituitary transgene expression was detected in six of the nine founders with the E/-297 construct but not with other constructs (Table 2Go). The particular tissues, cell types, and levels of ectopic expression varied between transgenics. For example, Xgal staining revealed abundant transgene expression in the kidney (Fig. 5BGo) and regional expression in the brain (Fig. 5Go, C and D). Rare scattered cells staining with Xgal were observed in peripheral tissues of nontransgenic mice and a few transgenic mice with the other constructs (E/-480, E/-341, -381, and E/-381) (Table 2Go). These data support the importance of the promoter region between -341 and -297 in directing expression of the {alpha}-subunit exclusively to the anterior pituitary.



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  		Figure 5. Ectopic Expression of the E/-297 m{alpha}-ßgal Transgene in Peripheral Mouse Tissues

Frozen sections from nontransgenic kidney (A), E/-297 transgenic line 20087 kidney (B), tg line 19983 brain (C), and tg line 20087 brain (D) were stained with Xgal to determine transgene expression in peripheral tissues. A low level of background Xgal stain is evident in panel A (see Table 2Go).

 

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  		Table 2. Peripheral Tissue Expression

 
The 859-bp Region Can Function as an Enhancer in Transfected Gonadotrope and Thyrotrope Cell Lines
Constructs containing the m{alpha} promoter sequences of -480, -381, -341, and -297/+43, with or without the 859-bp enhancer, were fused to the firefly luciferase reporter gene and transiently transfected into both {alpha}T3 and {alpha}TSH cell lines. The mean promoter activities were expressed as light units normalized to the activity of a cotransfected cytomegalovirus (CMV)-ßgal plasmid to control for variations in transfection efficiency. In constructs lacking the enhancer, deletion of sequences from -480 to -381 led to a 20-fold decrease in promoter activity in {alpha}TSH cells and a 5-fold decrease in {alpha}T3 cells (Fig. 6Go, A and C). The lowered activity may result from the loss of binding sites for thyrotropic nuclear proteins, -474 to -419, and elements important for expression in {alpha}T3 cells, -445 to -438 and -406 to -399 (10, 11, 14, 18). Further deletion to position -341 resulted in a slight increase in promoter activity while deletion to position -297 lowered activity 5-fold in {alpha}TSH cells and 13-fold in {alpha}T3 cells (Fig. 6Go, A and C). The latter deletion eliminates the binding site (-342 to -329) for a LIM homeodomain protein, Lhx2, shown to be important in both cultured gonadotropes and thyrotropes as well as the adjacent {alpha}BE1 element important for full activity in {alpha}T3 cells (12, 13).



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  		Figure 6. The 859-bp Region Shows Enhancer Activity in Transiently Transfected {alpha}TSH and {alpha}T3 Cells

Recombinant m{alpha}-luciferase constructs in the presence or absence of the 859-bp enhancer were transfected by electroporation, as described in Materials and Methods, into cell lines representing mouse pituitary thyrotropes ({alpha}TSH cells) (A and B) and gonadotropes ({alpha}T3 cells) (C and D). The panels to the left show the activity levels in light units of constructs transfected with and without the enhancer in both cell lines ± SEM (A and C). The black bars in the panels to the right represent the fold stimulation of promoter activity in the enhancer-promoter construct relative to the enhancerless construct (B and D). At least three independent experiments were done in triplicate for each construct.

 
The fold stimulation achieved using the 859-bp enhancer in both {alpha}TSH and {alpha}T3 cells is shown in Fig. 6Go, B and D. The highest stimulation was found with the E/-341 construct where the enhancer increased promoter activity 35- fold in {alpha}TSH cells and 8-fold in {alpha}T3 cells. Differences in the magnitude of the response may be due to higher promoter activity of the enhancerless constructs in {alpha}T3 cells. This is consistent with the data from transgenic mice that show high expression levels of the E/-341 construct in pituitary gonadotropes and thyrotropes. The E/-381 m{alpha}-luc construct resulted in a less robust stimulation of 12-fold and 4-fold in {alpha}TSH and {alpha}T3 cells, respectively. The E/-480 and E/-297 constructs showed minimal stimulation. This may result from differences in spacing of the enhancer with more proximal elements or from the loss of key basal response elements. These data demonstrate that the 859-bp enhancer can strongly stimulate reporter gene activity in cultured gonadotrope and thyrotrope cells when placed upstream of proximal {alpha}-promoter sequences.

Comparison of Enhancer Activity in Homologous and Heterologous Cell Types
Transgenic mice containing the E/-297 construct demonstrated inappropriate expression of the ßgal reporter gene in nonpituitary tissues such as the kidney and brain (Fig. 5Go and Table 2Go) while the E/-341 construct targeted expression solely to the pituitary gonadotropes and thyrotropes. To test whether such leaky expression would also occur in cultured nonpituitary cells, we transiently transfected these constructs and their enhancerless counterparts into heterologous CV-1 kidney cells (Fig. 7AGo). The overall expression of these four constructs was very low in CV-1 cells relative to that observed in the pituitary cell lines. This is consistent with the presence of pituitary-specific elements in both the enhancer and promoter regions. The E/-341 m{alpha}-luc construct resulted in a lower level of stimulation in CV-1 kidney cells (Fig. 7BGo) than that observed in both {alpha}T3 and {alpha}TSH cells (2-fold vs. 8- to 35-fold). The reduction of enhancer and promoter function in heterologous cells suggests that optimal function of the E/-341 {alpha}-promoter requires the participation of pituitary-specific factors. The E/-297 construct, which had low activity (2-fold) in the pituitary cell lines, stimulated promoter activity by 8-fold in CV-1 cells (Fig. 7BGo). These results further support the importance of the 44 bp between -341 and -297 for maintenance of pituitary cell specificity. Promoter elements are necessary to restrict expression to pituitary gonadotropes and thyrotropes in both cell culture and in vivo assays.



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  		Figure 7. E/-297 Exhibits an Increased Promoter Activity in CV-1 Cells Compared with E/-341

The -341, E/-341, -297, and E/-297 luc constructs were transfected into a heterologous CV-1 cell line to test for promoter activity in a nonpituitary cell line. Panel A shows activity in light units for each construct ± SEM, and panel B shows fold stimulation of the enhancer-promoter constructs compared with the enhancerless construct. The E/-297 luc construct, which was activated 2-fold over the -297 luc construct in {alpha}TSH and {alpha}T3 cells, illustrated a 7-fold stimulation in CV-1 cells. The 859-bp region demonstrates enhancer activity on a heterologous RSV180-promoter in both pituitary and nonpituitary cells: recombinant constructs with the RSV180 promoter driving expression of a luciferase reporter gene, in the presence or absence of the 859-bp distal enhancer, were transfected into {alpha}TSH, {alpha}T3, CV-1 cell lines. The activity of each construct was quantitated with luciferase assays and normalized to a cotransfected CMV-ßgal plasmid as an internal control (C), and the fold enhancement was determined (D). The black bars in panel D represent the fold stimulation of promoter activity of the E/RSV-luciferase construct relative to the RSV-luciferase construct. Panel C presents the activity in light units of each construct ± SEM. At least three independent experiments were executed in triplicate for each construct.

 
To test whether the 859-bp fragment would stimulate a heterologous promoter, it was fused to an Rous sarcoma virus (RSV)180 promoter (Fig. 7CGo). Figure 7DGo shows that the enhancer was able to stimulate the RSV promoter by about 7- to 10-fold in {alpha}TSH, {alpha}T3, and CV-1 cells. Thus, the 859-bp fragment displayed independent enhancer activity when fused to a heterologous promoter in pituitary and nonpituitary cells. These data show that the enhancer interacts with ubiquitous and pituitary-specific factors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous studies in transgenic mice have suggested that the sequences between -4.6 and -2.7 kb of the m{alpha}-subunit are necessary for high levels of expression to the anterior pituitary (7). The current investigations have localized the critical element to 859 bp between -4.6 and -3.7 kb. This sequence is able to function as an enhancer in context with the m{alpha}-subunit promoter both in the pituitary gonadotropes and thyrotropes of transgenic mice and in transfected {alpha}TSH and {alpha}T3 cell lines. It also directed high levels of expression of an RSV promoter in the appropriate pituitary-specific cell lines and in a heterologous CV-1 cell line.

Both cell transfection and transgenic mice were used to analyze a series of m{alpha}-promoter deletion constructs in conjunction with the 859-bp enhancer for expression levels and cell specificity. Previous transfection studies in {alpha}TSH cells showed no difference in expression of -5-kb m{alpha}-ßgal and -0.48-kb m{alpha}-ßgal constructs. However, the 859-bp enhancer region functioned as an enhancer when juxtaposed to the 480-bp m{alpha} promoter sequences in both {alpha}TSH and {alpha}T3 cells. While enhancer elements are defined as position independent, reduction in enhancer strength has been observed as the distance from the promoter increases (19). This may explain the difference in the -5-kb m{alpha}-ßgal phenomena and E/-480 constructs. The enhancement observed in cell transfection was not as great as that observed in transgenic mice (2- to 3-fold vs. 10-fold). This could be attributable to many factors including alterations in the composition of transcription factors in cell lines relative to normal pituitary cells and the influence of higher order chromatin structure and developmental history on expression. More dramatic enhancement in transgenic mice than cell transfection has been observed previously. In spite of these potential problems, the results obtained in cell culture and transgenic mice are generally consistent.

The ability of the upstream 859-bp sequence to function in a cell-specific fashion is critically dependent on proximal elements within the m{alpha}-subunit promoter. Both transgenic mouse analysis and cell transfection studies demonstrated that the enhancer is most effective in conjunction with a -341 bp fragment. In contrast, when fused to the -297 m{alpha}-promoter, the enhancer exhibited low expression levels in both cell transfection and transgenic studies. The fact that the enhancer was not as effective on the -480- and -381-bp fragments was an unexpected finding. The low level expression of the E/-480 and E/-381 constructs is not likely to be due to a repressor element located between -480 and -341 because the -480, -381, and -341 transgenics all have a similar low level of transgene activity (Fig. 2Go). Although it is unlikely, an insulator sequence may have been created at the junction of both the E/-381 and E/-480 constructs that is not present in the -4.6 or E/-341 transgene. Insulator sequences may function by blocking communication between an enhancer and more proximal promoter sequences (20). We cannot rule out the possibility that a suppressive element is not functional in the context of the -4.6-kb fragment or that another positive stimulatory element overrides its influence. The region between -480 and -341 contains interaction sites for thyrotrope protein(s), several basal gonadotrope and GnRH-responsive elements, a TRH- responsive element, and established binding sites for the transcription factors P-OTX/Ptx-1 and Msx1 (11, 14, 18, 21, 22, 23, 24, 25). We favor the possibility that the spacing necessary for interaction between these elements and the enhancer is impaired. The lack of optimal function in the E/-480 and E/-381 constructs could be attributable to unfavorable stereospecific protein-protein contacts.

In addition to testing regions important for the level of expression in the anterior pituitary, deletion constructs provided important information on the role of cis-acting elements on cell specificity. Immunohistochemical data showed that the E/-341, E/-381, and -381 transgenics retain restriction of expression to pituitary gonadotropes and thyrotropes. This is in contrast with the loss of thyrotrope expression in transgenic mice generated to study portions of the human and bovine {alpha}-subunit genes (5, 6). Species divergence also exists in the expression of the {alpha}-subunit in the placenta. Human and equine {alpha}-subunit genes contain a region, approximately 180 bp from the initiation start site, with two distinct cis-acting regulatory elements, a cAMP-responsive element and a trophoblast-specific element, that confer expression in the placenta (26, 27). Although transgene expression was detected in the gonadotropes and thyrotropes in -341 transgenics, a loss of restriction of specificity resulted in additional transgene detection in corticotropes, somatotropes, and lactotropes. This suggests that the sequences between -381 and -341 may contain elements that limit expression in other pituitary cell types. These data also show that the enhancer is able to rescue the restriction of cell specificity of the -341 m{alpha}-promoter. The anterior pituitary of E/-297 transgenics displayed a loss of restriction of specificity, and ectopic transgene expression was observed in a variety of peripheral tissues. Expression of the E/-297 construct in nonpituitary cell types was confirmed by cell transfection data demonstrating E/-341 m{alpha}-promoter activity only in {alpha}TSH and {alpha}T3 cells while E/-297 m{alpha}-promoter activity occurred in CV-1 cells in addition to the pituitary cell lines. Thus, the enhancer cannot overcome the loss of tissue specificity acquired by deletion of the region between -341 to -297.

Transfection data in cultured cells have suggested that the LIM homeodomain factor-binding site is important for basal levels of expression in transfected gonadotropes and thyrotropes and, when deleted, results in a loss of cell-specific enhancer function (Fig. 6Go). We have shown that it may also play a role in vivo in the restriction of expression to these two cell types (Tables 1Go and 2Go). The LIM site at -342 to -329 interacts with closely related homeodomain factors such as Lhx2 and P-lim (Lhx3) (12, 28, 29). In addition, an adjacent element has been located in the human {alpha}-subunit gene, {alpha}BE1, important for high level gonadotrope expression (13). Mutations in this region at positions -310 to -303 of the mouse gene show a 30% drop in promoter activity in {alpha}TSH cells and had no effect in {alpha}T3 cells (11). Considering the importance of these factors suggested by transfection data, one or more may assist in restricting expression of the {alpha}-subunit to the gonadotropes and thyrotropes.

The 859-bp enhancer region facilitates restriction of expression to gonadotropes and thyrotropes as well as serving as an enhancer. These multiple functions may be exerted by distinct elements within the 859-bp enhancer region. Sequences similar to those present between -480 and -341 of the m{alpha}-promoter can also be found within the 859-bp enhancer (GenBank accession number AF044976). These include consensus sites for interaction with thyrotrope and gonadotrope proteins, SF1, GATA2/3, GnRE, and Ptx1. If these putative elements are functional, the redundancy could explain the appropriate gonadotrope and thyrotrope expression in the E/-341 transgenics and the loss of restriction in the -341 transgenics. Sequences corresponding to the consensus binding sites for LIM homeodomain proteins and {alpha}BE1, located between -341 and -297 in the m{alpha}-promoter, are not evident within the enhancer. Removal of binding sites for these factors in the promoter could explain the loss of restriction of expression to the appropriate anterior pituitary cell types in the E/-297 transgenics. Therefore, with the exception of the LIM homeodomain proteins and {alpha}BE1, it is possible that similar factors interacting with both the enhancer and proximal promoter sequences contribute to cell specificity.

Our studies suggest that thyrotrope as well as gonadotrope specificity of the m{alpha}-subunit is a result of interactions between multiple elements within the promoter and enhancer. A trend toward reduced basal levels of promoter activity in {alpha}TSH cells and {alpha}T3 cells (Fig. 6Go, A and C) was seen with the m{alpha}-promoter deletion constructs. In both cell types, the enhancer worked optimally with the -341 promoter (Fig. 6Go, B and D). It did not function when fused to the -297 m{alpha}-promoter fragment despite the intact SF-1 site. The importance of SF-1 for gonadotrope cell specification and gonadotrope transcription is controversial (30, 31, 32). The significance of SF-1 in gonadotrope expression was not addressed in these studies; however, we have evidence that the Lhx2 and {alpha}BE1 binding sites in the promoter are not required for expression in the anterior pituitary. In the context of the enhancer, one or both of these sites are necessary for cell-specific restriction of expression.

The loss of pituitary cell-type restriction evident in the m{alpha}-subunit gene is typical of that observed with other pituitary-specific genes such as PRL, GH, and POMC (33, 34, 35). In each case, constructs tested in transgenic mice were selected based on regions determined in transfected cells to be important for specific expression. The PRL, GH, and POMC transgenics all exhibited position effects. The requirement for synergistic interactions between multiple elements to achieve appropriate cell-specific expression was demonstrated in each case.

Our experiments demonstrate the involvement of multiple DNA sequence elements in high-level expression and cell specificity. No regions important exclusively for thyrotrope or gonadotrope expression were identified. Our results also implicate sequences in the {alpha}-subunit promoter (-341 to -297) for organ-specific and cell-specific expression. These sequences bind one or more LIM homeodomain factors (12). This is intriguing because two LIM homeodomain genes, Lhx3 and Lhx4, have been demonstrated to play important roles in pituitary organogenesis (36). It will be intriguing to dissect the roles of individual LIM homeodomain genes on activation and regulation of {alpha}GSU transcription.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of the Recombinant Plasmids
The -3.7 kb m{alpha}-ßgal transgene was generated by digesting the 5.0 m{alpha}-ßgal plasmid (7) with BglII. The -3.1 m{alpha}-ßgal transgene was generated from the same plasmid by digesting with SpeI and HindIII. Four different fragments from the mouse {alpha}-subunit promoter (-480/+43, -381/+43, -341/+43, and -297/+43 bp) were isolated by digesting the appropriate fragments within the pA3luc plasmid (37) with BamHI and HindIII. These fragments were subcloned into pSELECT1 (pALTER-1, Promega Corporation, Madison, WI) utilizing the BamHI and HindIII sites. An 859-bp fragment, -4.6/-3.7 kb, of the mouse {alpha}-subunit promoter was isolated by digesting a -5 kb/+43 m{alpha} promoter fragment in pGEM7Zf+ (7) with KpnI and BglII. This fragment was fused at the 5'-end of the four different mouse {alpha}-subunit promoter fragments by subcloning between the KpnI-BamHI sites of pSELECT1. The fragment containing the 859-bp region fused to the pieces of the mouse {alpha}-subunit promoter was isolated by cleaving with EcoRI and HindIII for the transgenic constructs. The ends were blunted using HMV RT (Promega Corporation), and the fragments were cloned into the SmaI site of the pnlacF reporter plasmid (kindly provided by Jacques Peschon and Richard Palmiter). The transgenes were isolated from plasmid sequences by digestion with the appropriate restriction enzymes, separated by agarose gel electrophoresis, and purified for microinjection with the NucleoSpin Extract Kit (The Nest Group, Inc., South Borough, MA). In a similar manner, m{alpha}-promoter constructs with or without the 859-bp enhancer sequence were isolated from the appropriate pSELECT1 vector by digestion with SmaI and HindIII and directionally reinserted into the pA3luc vector. Plasmid preparations were purified from bacterial lysates by double CsCl gradient centrifugation. At least two independent plasmids from each construct were tested.

Generation of Transgenic Mice
The purified inserts were microinjected into F2 hybrid zygotes from (C57BL/6J x SJL/J) F1 parents at a total concentration of approximately 2–3 ng/ml. After overnight incubation, embryos at the two-cell stage were transferred to day 0.5 postcoitum pseudopregnant CD-1 females. (C57BL/6J x SJL/J) F1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and CD-1 mice were obtained from Charles River (Wilmingon, MA). All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals. All experiments were conducted in accord with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals.

Identification of Transgenic Mice
Genomic DNA was prepared from tail biopsies (38) and pituitary sections and screened for the presence of the transgene by PCR. A 364-bp fragment corresponding to the nucleotides 15–379 of the ß-galactosidase gene was amplified using a 30-bp sense oligo (5'-TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA-3') and a 30-bp antisense oligo (5'-ATG TGA GCT AGT AAC AAC CCG TCG GAT TCT-3'). The PCR reactions were performed under standard conditions using 100–200 ng genomic DNA, 0.5 pmol/ml primers, 2.5 mM MgCl2, and 1.7 U Taq DNA polymerase per reaction. Reactions were carried out for 30 cycles of denaturation at 94 C for 1 min, annealing at 65 C for 30 sec, extension at 72 C for 30 sec, and a final extension at 72 C for 10 min. The identity of the mouse {alpha}-subunit 5'-deletion constructs was confirmed for each transgenic animal using PCR. Two 20-bp oligos [forward (5'-CTC ATT TTT TAA GGC ACT GC-3'); and reverse (5'-GAT CAT ATC ACA TTG CAA CC-3') were used to amplify 430-, 331-, 291-, and 247-bp fragments corresponding to E/-480, E/-381, E/-341, and E/-297, respectively. The reactions were carried out for 30 cycles of denaturation at 92 C for 1 min, annealing at 60 C for 1 min, extension at 70 C for 1 min, and a final extension at 72 C for 10 min. The identity of the PCR product was further confirmed by digesting with RsaI to yield fragments of 242 and 188 bp in the E/-480 PCR product and fragments of 242 and 89 bp in the E/-381 PCR product. In addition, the identity of the PCR products from all of the constructs was verified by sequence analysis.

Quantitation of ß-Galactosidase Activity with Fluorimeter and Luminometer Assays
One lobe of the anterior pituitary was excised using a scalpel and placed in 250 µl of a lysis buffer containing 100 mM potassium phosphate (pH 7.8) and 0.2% Triton X-100. The samples were freeze-thawed by incubation on dry ice and 37 C alternately for three cycles and then sonicated 2 x 20 sec on ice (550 Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA). The cell lysates were centrifuged at 12,000 x g for 3 min, and the supernatant was removed to new tubes. Transgene expression was quantitated in tissue extracts from -3.7 and -3.1 m{alpha}-ßgal transgenics using a fluorometric assay for ß-galactosidase (7). A chemiluminescent reporter assay for ß-galactosidase was used to quantitate transgene expression in tissue extracts from -381, -341, E/-480, E/-381, E/-341, and E/-297 transgenics. ß-Galactosidase activity was detected using the Galacto-Light (Tropix, Bedford, MA) chemiluminescent reporter assay system. Two microliters of the lysate were incubated for 1 h in a reaction buffer containing Galacton, a chemiluminescent substrate cleaved by ßgal. The samples were placed in a luminometer chamber (Auto Lumat LB 953, EG&G Wallac, Gaithersburg, MD), and 100 µl of a light emission accelerator were added to the samples. After a 5-sec delay, light production was measured over a 10-sec interval. The level of ßgal activity in each sample was compared with a standard curve of 0.15, 0.38, 0.96, 2.4, 3.8, 4.8, 6, and 8 U x 106 ßgal (Sigma, St. Louis, MO).

Reactions were performed in triplicate, luminometer readings were averaged, and nontransgenic background levels were subtracted. ß-Galactosidase activity was normalized for the amount of protein in each homogenate. The Bradford protein assay (39) was performed with the Coomassie brilliant blue dye reagent on 20 µl of homogenate in microtiter plates as described (Bio-Rad Laboratories, Hercules, CA). Absorbance of the samples and the BSA protein standard was measured at 630 nm using an EL311 sx Auto Reader (Bio-Tek Instruments, Wihooski, VT).

Histology and Immunohistochemistry on Adult Pituitary and Peripheral Tissues
Peripheral tissues were removed and frozen immediately in OCT embedding media (Miles Scientific, Elkhart, IN) on dry ice. Sections were cut (12 µM) on a cryostat (Bright Instruments Company, Huntingdon, U.K.) and mounted on poly-L-lysine-coated slides. Sections were fixed for 5 min in 0.2% glutaraldehyde, containing 0.1 M NaH2PO4 (pH 7.3), 5 mM EGTA, and 3 mM MgCl2, washed three times in wash buffer [0.1 M NaH2PO4 (pH 7.3), 2 mM MgCl2, 0.02% NP-40], and incubated at 37 C overnight in an Xgal solution containing 1 mg/ml Xgal (Boehringer Mannheim, Indianapolis, IN), 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6·3 H2O dissolved in wash buffer (above). The counterstain was 0.5% neutral red (Sigma).

The cell specificity of transgene expression was evaluated by an Xgal enzymatic assay followed by immunohistocytochemistry for individual pituitary hormones. Pituitaries were fixed for 1 h in 4% paraformaldehyde in pH 7.2 sodium phosphate buffer and incubated at room temperature overnight in Xgal solution as above, except that Xgal was reduced to 0.1 mg/ml for pituitaries from the -4.6 m{alpha}-ßgal transgenic lines and the highest expressing E/-341 m{alpha}-ßgal transgenics. After 3–6 h postfixation in buffered formaldehyde, the samples were embedded in paraffin and 3–4 µM sections were prepared. Immunostaining was performed as previously described (7).

Cell Culture, Transient Transfection, and Reporter Gene Assay
Transient transfections using calcium phosphate into CV-1 monkey kidney cells were performed as described previously using 10 µg reporter plasmid (40). Approximately 750,000 cells were added per 100 mm x 20 mm tissue culture dish 20 h before transfection. {alpha}TSH (3 x 106 cells) and {alpha}T3 cells (4 x 106 cells) were transfected by electroporation as described (40). The pituitary cells were transfected with 20 µg of the indicated luciferase construct and also contained 2 µg of pCMV-ßgal DNA as an internal transfection control. Each set of transfections was done in triplicate and also contained a Rous sarcoma virus promoter-luciferase plasmid transfected in parallel as a positive control and a promoterless pA3luc vector as a background control. After 24–48 h, luciferase activity was measured in a Monolight 2010 luminometer from duplicate aliquots of freeze-thaw cytoplasmic lysates (41) from the cells while ß-galactosidase activity was measured using a colorimetric assay (42) and were compared with a standard curve of enzymatic activity. Light units were normalized to the ß-galactosidase activity.


   ACKNOWLEDGMENTS
 
We would like to thank Mark Berard, Dwayne Petry, and Maggie Van Keuren from the University of Michigan Transgenic Animal Core for their work in the generation of transgenic mice. We would like to thank the University of Michigan Morphology Core, especially Kaye Brabec, for their assistance in paraffin embedding and sectioning, Amy Young for her help in typing mice, and Juanita Merchant, Audrey Seasholtz, and Jeff Chamberlain for the use of their equipment. We acknowledge the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Child Health and Human Development, and the US Department of Agriculture for providing the PRL, GH, LHß, and TSHß antibodies.

Work done in the laboratory of Dr. Sally Camper is supported by NIH Grant RO1-HD34283-01. The Transgenic Core Facility is supported by the University of Michigan Centers for Arthritis, Cancer, and Organogenesis, and National Institutes of Health/National Science Foundation grants. Support for the Morphology Core is provided by Grant P30-HD-18258. Additional support was provided by NIH Grant RO1-DK47407 and a generous gift from the Markey Foundation to E. C. Ridgway. Cultured cells were grown by the Tissue Culture Core Laboratory at the University of Colorado Health Sciences Center supported by NIH Grant CA-46934.


   FOOTNOTES
 
Address requests for reprints to: Dr. Sally A. Camper, Division of Molecular Medicine and Genetics, University of Michigan Medical School, 4301 MSRB III, Ann Arbor, Michigan 48109-0638.

Received for publication October 31, 1997. Revision received December 22, 1997. Accepted for publication January 21, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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