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Molecular Endocrinology, doi:10.1210/me.2006-0245
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Molecular Endocrinology 21 (1): 172-185
Copyright © 2007 by The Endocrine Society

Differential Utilization of Transcription Activation Subdomains by Distinct Coactivators Regulates Pit-1 Basal and Ras Responsiveness

Dawn L. Duval, Matthew D. Jonsen, Scott E. Diamond, Patience Murapa, Annie Jean and Arthur Gutierrez-Hartmann

Departments of Medicine (M.D.J., A.G.-H.), Biochemistry and Molecular Genetics (A.G.-H.), and Clinical Pharmacy (D.L.D., A.J.), University of Colorado Health Sciences Center, Aurora, Colorado 80045; and Department of Physiology (S.E.D., P.M.), University of Kentucky College of Medicine, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Arthur Gutierrez-Hartmann, University of Colorado Health Sciences Center, P.O. Box 6511, Mail Stop 8106, Aurora, Colorado 80045. E-mail: a.gutierrez-hartmann{at}uchsc.edu and dawn.duval{at}uchsc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1ß, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1–45), and a basal and Ras-responsive region, R2 (AA46–80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1ßTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50–70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60–80. In contrast, GAL4-Pit-1ßTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the ß-domain, we inserted the ß-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1ß, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, ß-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61–71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1ß TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a ß-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PIT-1/GHF-1 (GH FACTOR 1) is a member of the POU-homeodomain family of transcription factors, and it is a determining factor in the ontogeny of the somatotrope, lactotrope, and thyrotrope lineages. Consequently, Pit-1 is responsible for the regulated cell-specific expression of the respective hormones synthesized and secreted by these cell lines: GH, prolactin (Prl), and the ß-subunit of TSH. To fulfill its role as a cell-specific signal integrator, Pit-1 cooperates with a variety of transcription factors including itself, estrogen receptor (ER), thyroid hormone receptor (TR), c-Jun, Oct-1, GATA-2, P-Lim, Ptx-1, and Ets-1 (1, 2, 3, 4, 5, 6, 7, 8, 9). These activities may also be regulated by specific interactions with transcriptional coactivators and corepressors including cAMP response element binding protein-binding protein (CBP)/p300, p300/CBP-associated factor (p/CAF), mediator 220, mammalian SIN3, nuclear receptor corepressor, receptor-interacting protein 140 (Rip140), steroid receptor coactivator (SRC)-1, and glucocorticoid receptor interacting protein (10, 11, 12, 13, 14, 15).

Pit-1 contains three well-defined functional motifs: 1) an amino terminal transcriptional activation domain (TAD), spanning AA8–80; 2) a POU-specific domain (AA128–198) defined by a region of homology shared by members of the POU-homeodomain family; and 3) the homeodomain (AA214–273). The POU-specific and homeodomains are tethered by a peptide linker that varies in length, sequence, and flexibility among members of this transcription factor family, generating a bipartite DNA binding domain (DBD) (16). Whereas the homeodomain represents the minimal region required for DNA binding, the POU domain contributes to high-affinity binding and recognition-site specificity (2). This bipartite DBD, in combination with the specific DNA binding site, forms an elaborate code dictating the configurations of subdomains and subsequent recruitment of specific coregulators to control transcription. For example, increased spacing between the contact points for the POU-specific and homeodomains from 4-bp in the Prl promoter to 6-bp in the GH promoter alters the structure of Pit-1 sufficiently to convert it from a trans-activating factor to a repressor in pituitary lactotropes (11). Whereas Pit-1 is typically thought to bind to DNA elements as a homodimer, the binding of Pit-1 as a monomer at composite elements may contribute to its ability to synergize with other transcription factors and target signaling pathways to Pit-1-regulated gene promoters. For example, binding of Pit-1 as a monomer at the 1-d site (–1600 to –1586), which is juxtaposed to an estrogen response element in the distal enhancer of the rPRL promoter, dictates use of a Pit-1 synergy domain located in the amino-terminal TAD (5, 17). Interestingly, synergistic activation by thyroid hormone, estrogen, GATA-2, and Ras with Pit-1 all require adjoining sections of the Pit-1 TAD (17, 18, 19, 20). In addition, Pit-1ß, a splice-variant of Pit-1, arises due to the insertion of a highly conserved 26-AA ß-domain at position 48 of the TAD, converting Pit-1ß to a pituitary-specific transcriptional repressor of Prl, GH, and TSHß gene expression (12, 21, 22, 23). This ß-domain contains two sequence-specific hydrophobic patches that recruit histone deacetylase (HDAC) complexes (22, 23). The recruitment of these HDAC complexes by Pit-1ß, primarily acting at the FPI and FPIII sites, inhibits the recruitment of the coactivator CBP to transcriptional complexes on the Prl promoter in GH4T2 cells (12, 24).

Detailed analysis of the molecular mechanisms regulating basal activity of the rat Prl (rPRL) promoter has revealed that, in addition to Pit-1, Ets factors, in particular Ets-1 and GA binding protein, also play critical roles. In fact, oncogenic Ras utilizes a RAS/MAPK kinase/MAPK cascade to impinge upon the composite Ets/Pit-1 binding site (EBS/FPIV; Ets Binding Site/Foot Print IV) located at positions –217 to –190 in the proximal rPRL promoter. Contributing to this tripartite code is the fact that a 75-AA region containing the Pit-1 POU-homeodomain physically interacts with a 68-AA segment in the Ets-1 RIII transcriptional activation domain (TAD) (AA190–257) (3, 20, 25, 26, 27). Although the precise mechanism for Ras activation of the rPRL promoter is unknown, the phosphorylation of Ets-1 (28) and the Ets-1/Pit-1 combination may either act to recruit specific coactivator complexes, or it may provide several sites of interaction with the basal transcription machinery, thus stabilizing the preinitiation complex. In this regard, phosphorylation of Ets-1 has been shown to increase the affinity of Ets-1 for CBP (29, 30, 31). Alternatively, Sur-2, a member of the mediator transcriptional complex, has been reported to be responsible for Ras activation of Elk-1 and may contribute to Ets-1 activation (32, 33). In addition, we have shown that deletion of Pit-1 AA45–80 (Region 2) blocks the ability of transfected Pit-1 to stimulate the Ras responsiveness of the rPRL promoter in GH4 cells, suggesting that this domain binds transcriptional cofactors that regulate Ras signaling (20). In a reconstitution model in Hela cells, deletion of Pit-1 Region 2 stimulates Pit-1 basal activity, but inhibits the synergistic activation of the Prl promoter by Pit-1 and Ets-1 (20). In contrast, deletion of the entire Pit-1TAD completely restores Ets-1 synergy to Pit-1, suggesting that the Pit-1 POU-homeodomain, in cooperation with AA2–40 (Region 1), is responsible for basal transcriptional activity, whereas AA40–80 contribute to cooperative interactions with other transcriptional mediators.

The current studies explore this novel mechanism in which different coactivator and corepressor complexes appear to compete for interaction sites in the Pit-1 TAD and regulate the ability of Pit-1 to serve as a pituitary-specific signaling mediator. We have used a series of TAD deletion and mutation constructs to elucidate the interplay between the three subdomains (Region 1, the ß-domain, and Region 2) to regulate basal activity and Ras responsiveness of the amino-terminal transcriptional activation domain of Pit-1. We show that the Pit-1/Pit-1ß TADs integrate the activities of several subdomains including: the R1 domain (AA1–45), which regulates basal activity and Ets-1 synergy (20); a positive TAD region (AA50–70); an inhibitory segment (AA70–80); in addition to a ß-specific repressor domain. This novel integration mechanism fine-tunes the transcriptional responses of Pit-1 target genes and may dictate the activity of Pit-1 in partnership with other factors. In fact, we have shown that Ras activation through Pit-1 does not require the CBP/p300 coactivator previously associated with transcriptional activation of a dimeric Pit-1 binding Prl-1P reporter (10) but rather is augmented by members of the p160 SRC family. Finally, our association of CBP and SRC-1 with basal transcriptional activity and Ras activation through Pit-1, respectively, is consistent with the localization of a CBP interaction motif centered around Proline 24 in the Region 1 Ets-1 synergy/basal activity domain (20, 34) and our mapping of the Ras-responsive domain to AA70–80, coincident with a domain used by members of the p160 SRC family for ER and TR synergy with Pit-1 (15, 35).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Pit-1 TAD Is Not a MAPK Target
Deletion analysis of Pit-1 indicated an absolute requirement for the Pit-1 TAD to enhance Ras responsiveness of the rPRL promoter because deletion of the region from AA2–80 completely abrogated the ability of Pit-1 to enhance Ras responsiveness (20). The location of this Ras-responsive region has been further refined to AA46–80 because a Pit-1 deletion mutant (Pit-1{Delta} 2–45) retaining AA46–80 of the TAD has full Ras responsiveness (20). Because Ras activation was previously shown to be mediated by a Ras-Raf-MAPK pathway, we wanted to determine whether, like Ets-1, the Pit-1 TAD was a target for phosphorylation by MAPKs (36, 37). We incubated glutathione-S-transferase (GST)-fusions of full-length Ets-1, the Pit-1 TAD (AA2–82), and GST alone with activated ERK1 in the presence of {gamma}–32P-ATP (Fig. 1Go). GST-Ets-1 served as a positive control because it is a bona fide MAPK target (37). Autoradiographic analysis of the fusion proteins revealed no incorporation of radiolabel in either the negative control GST lane or the Pit-1 TAD lane, but intense labeling of Ets-1 as well as several Ets-1 fragments (Fig. 1AGo). Coomassie staining of the gel to control for protein loading indicates that similar levels of the GST-fusion proteins were present in each sample (Fig. 1BGo). Thus, because a similar nanomolar amount of the positive control, Ets-1, was highly labeled under identical assay conditions, the Pit-1 TAD does not appear to represent a target for phosphorylation by ERK1.


Figure 1
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  		Fig. 1. The Pit-1 Transcriptional Activation Domain Is Not a MAPK Target

GST-fusions of the Pit-1 TAD (AA2–80) and p68 c-Ets-1 were incubated with activated Erk1 and 32P-ATP and electrophoresed on an SDS-PAGE gel. A, Autoradiograph; B, Coomassie stained gel. MW, Molecular weight.

 
Pit-1 TAD-GAL4DBD Fusions Identify a Strong Activation Domain Located at AA50–70 of Pit-1, Followed by a Repressor Domain Located in the AA70–80 Region
Our previous studies implicated AA45–80 (R2 region) in the Pit-1 stimulation of Ras responsiveness of the rPRL promoter (20). Having verified that this Ras response was not due to MAPK phosphorylation of the Pit-1 TAD, we next sought to determine whether the Pit-1TAD represented an independent Ras-responsive domain and to further fine-map the domain in the R2 region of Pit-1 responsible for this Ras stimulation. Specifically, is the Pit-1 TAD alone capable of mediating Ras activation of a promoter other than the rPRL promoter, and what is the motif in the TAD that mediates this response?

To answer these questions, we constructed chimeric proteins linking a series of carboxy-terminal extensions from AA40–85 of Pit-1 (full-length TAD) to the DBD of GAL4 (Fig. 2AGo). We transiently cotransfected these constructs into GH4T2 cells with a luciferase reporter construct containing five tandem repeats of the upstream activating sequence (UAS) binding site for GAL4 with (+ Ras, white bars) and without (Basal, black bars) an expression construct for oncogenic V-12 Ras. The UAS promoter construct alone had minimal activity and was stimulated 3.1-fold in response to Ras (Fig. 2AGo). The addition of the GAL4DBD without a Pit-1 TAD fusion (GAL4) slightly stimulated the reporter construct to 3.7 relative light units (RLU), but showed a 13.1-fold response to Ras activation (49 RLU, Fig. 2AGo). The cotransfection of chimeric proteins linking either AA2–40 or AA2–50 (GAL4N40, GAL4N50) of the Pit-1 TAD to the GAL4DBD resulted in a slight stimulation of basal reporter activation (5.3 and 6 RLU, respectively, Fig. 2AGo, black bars) and a corresponding slight decrease in Ras responsiveness (57 and 54 RLU, 10.8 and 9-fold Ras, respectively, Fig. 2AGo), but these differences are not significantly different from the GAL4 control. The progressive addition of Pit-1 AA2–60 (GAL4N60) resulted in significant increases in basal activity to 36.4 RLU, but the fold Ras response was unchanged from previous constructs (7.4-fold Ras; Fig. 2Go). The addition of Pit-1 AA2–70 (GAL4N70) resulted in a construct with significantly higher basal activity at 107.5 RLU (Fig. 2AGo) in comparison to the full-length Pit-1 TAD (AA2–85, GAL4TAD, basal activity 10.3 RLU). Finally, although the total Ras-stimulated activity of GAL4N70 is elevated to 920 RLU, the 8.6-fold Ras response (Fig. 2AGo) is still significantly lower than the 21.4-fold Ras response of GAL4TAD. These data reveal the presence of an inhibitory domain at AA70–85 that blunts the strong activation by the domain located at AA50–70. Interestingly, based on these experiments, this same inhibitory domain appears to contain the Ras-responsive region of Pit-1, suggesting that basal Pit-1 transcriptional activity may compete with Ras activation of Pit-1, rather than complementing it. Western blot analysis for expression of the GAL4 fusion proteins verifies that these differences in transcriptional activity were not due to variations in protein expression among the different constructs (Fig. 2BGo).


Figure 2
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  		Fig. 2. Pit-1 TAD Region 2 Deletions Identify Both Activator and Repressor Domains Target

A, GH4T2 cells were transiently cotransfected with 2 µg of a luciferase promoter construct containing five copies of the UAS GAL4 DNA binding site, 100 ng of phRL-TK, and either 2 µg of pSV-Ras or empty pSG5 control. Where indicated, varying concentrations of the GAL4DBD-Pit-1TAD fusions were added to give equivalent levels of expression: GAL4 6 µg, GAL4{Delta} 40 1.2 µg, GAL4{Delta} 50 1.2 µg, GAL4{Delta} 60 1.65 µg, GAL4{Delta} 70 1.2 µg, and GAL4TAD 3 µg. DNA concentration was held constant with the addition of empty pcDNA3.1. After 24 h, the cells were harvested and assayed for luciferase activity. Bars represent the mean and SD of triplicate samples from a representative transfection. Fold Ras stimulation is expressed as the fold increase in reporter activation by each GAL4 construct in the presence and absence of oncogenic Ras. *, P < 0.05. The transfection was performed three times with triplicate samples. B, Cellular lysates (100 µg total protein) from cells transfected as in panel A, were analyzed by Western blot for GAL4fusion expression using anti-GAL4 DBD (Santa Cruz Antibodies, Santa Cruz, CA), and horseradish peroxidase-conjugated goat antimouse IgG (Bio-Rad).

 
The ß-Domain Inhibits Both Basal Activity and Ras Responsiveness of the TAD
We have previously shown that the Pit-1ß splice isoform, which contains a 26- AA insertion in the TAD, functions as a dominant-negative inhibitor of Ras stimulation of the rPRL promoter (22, 38). Consequently, we wanted to determine whether the ß-domain had a similar effect on TAD activity in this isolated system. Similar to the experiment in Fig. 2Go, we transiently transfected GH4T2 cells with a 5xUAS reporter construct and expression constructs containing the Pit-1 and Pit-1ß TADs fused to the GAL4 DBD, with and without an expression construct for oncogenic V12Ras. In the absence of a GAL4 activator, the luciferase activity of the reporter was very low and was stimulated 2.5-fold by the addition of Ras (Fig. 3Go). The addition of the GAL4DBD alone stimulated reporter activity to 2.2 RLU and increased the Ras response to 20.2-fold. GAL4TAD significantly stimulated basal reporter activity and induced a 34-fold Ras response, similar to Fig. 2AGo. In contrast, GAL4ß TAD, containing the ß-domain inserted at AA48, blocked basal activation by the TAD to 1.5 RLU and reduced the Ras response to a level lower than that of GAL4 alone (10.7-fold Ras, Fig. 3Go). Expression of GAL4TAD and GAL4ß TAD was equivalent in these studies (Fig. 3Go, inset). Thus, the ß-domain inhibits Pit-1 TAD basal activity and Ras responsiveness even in isolation from other Pit-1 domains.


Figure 3
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  		Fig. 3. The ß-Domain Inhibits Both Basal and Ras-Stimulated TAD Activity Target

GH4T2 cells were transiently cotransfected with 2 µg of a luciferase promoter construct containing five copies of the UAS GAL4 DNA binding site, 100 ng of phRL-TK, and either 2 µg of pSV-Ras or empty pSG5 control. Where indicated, varying concentrations of the GAL4DBD-Pit-1TAD fusions were added to give equivalent levels of expression: GAL4 3.2 µg, GAL4TAD 1.04 µg and GAL4ß TAD 4.2 µg. DNA concentration was held constant with the addition of empty pcDNA3.1. After 24 h, the cells were harvested and assayed for luciferase activity. Bars represent the mean and SEM of triplicate samples from three separate transfections. Fold Ras stimulation is expressed as the fold increase in reporter activation by each GAL4 construct in the presence and absence of oncogenic Ras. Inset, Cellular lysates (100 µg total protein) from transfected cells were analyzed by Western blot for GAL4fusion expression using anti-GAL4 DBD (Santa Cruz Antibodies), and horseradish peroxidase-conjugated goat antimouse IgG (Bio-Rad).

 
Myc Substitution Mutagenesis Scanning the R2 Subdomain of the Pit-1TAD
In previous studies, we compared the ability of deletions of Pit-1 AA2–45 and AA2–80 in the context of Pit-1 to mediate Ras stimulation of the rPRL promoter in GH4T2 cells with their ability to activate the rPRL promoter under basal conditions (20). We found that deletion of AA2–45 had no effect on Ras responsiveness, elevated basal activity in the HeLa reconstitution system, and reduced synergy with Ets-1. In contrast, the deletion of AA2–80 eliminated the Ras response and reduced basal activity, but did not affect the ability of Pit-1 to synergize with Ets-1. In the current study, we compared the ability of a series of mutant Pit-1 constructs to activate basal rPRL promoter transcriptional activity and mediate the Ras response. To generate these mutant constructs, we replaced overlapping portions of the Pit-1 TAD AA46–80 with 11 AA from the Myc epitope-tag (Fig. 4AGo, Myc 1–4), to fine-map the Ras-responsive domain in Region 2. This motif was selected because it has been used previously in studies to mutate the ß-domain of Pit-1ß and did not alter the ability of Pit-1ß to function as a transcription factor (22). Because previous studies suggested that the tyrosine residues located at AA53, 60, and 67 were important for Pit-1’s synergy with the ER (17), we also mutated tyrosines 60 and 67 to alanines (Fig. 4AGo, Y60A/Y67A). We transiently cotransfected each of these constructs with a luciferase reporter containing the proximal 425 bp of the rPRL promoter (–425PRL-pA3 Luc), with or without a mammalian expression plasmid for oncogenic V12Ras, into the GH4T2 cell line (Fig. 4AGo). The first panel in Fig. 4Go examines the ability of these constructs to stimulate basal rPRL promoter activity (first panel, white columns). We found that the basal activity of Myc mutations 1 through 3 was equivalent to that of wild-type Pit-1. However, the activity of the Myc-4 mutation, which spans AA70–80, was significantly increased (P < 0.01) compared with the Pit-1 control (2.66- and 1.07-fold, respectively). Similarly, the mutation of tyrosines 60 and 67 to alanines (Y60A/Y67A) also increased the basal transcriptional activity of this mutant Pit-1 construct (1.78-fold, P < 0.05). The second panel in Fig. 4AGo expresses the results of this experiment as the fold Ras response in which the activity of each construct in the presence of Ras is divided by its activity in the absence of Ras. Consistent with previous studies (20, 27, 38), oncogenic Ras stimulates the rPRL promoter approximately 12-fold. The addition of wild-type Pit-1 enhances the Ras response from 12- to 26-fold (Fig. 4AGo). Similar to the comparison of the effects of the Myc mutations on basal activity, Myc mutations 1–3 did not significantly alter the ability of Pit-1 to stimulate the Ras responsiveness of the rPRL promoter; however, a significant decrease in Ras responsiveness of the Myc-4 and Y60A/Y67A mutations from 26-fold to 9- and 15-fold, respectively, is revealed (Fig. 4AGo, P < 0.05). Western blot analysis of the transfected GH4T2 cells illustrates that the differences in activity were not due to differences in expression of the mutant Pit-1 proteins (Fig. 4BGo). Thus, the Myc-4 and Y60A/Y67A mutations stimulated basal activation but appeared to inhibit Ras responsiveness in GH4T2 cells, suggesting that basal activity of Pit-1 is balanced against Ras responsiveness.


Figure 4
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  		Fig. 4. Myc Scanning and Alanine Point Mutations Identify Repressor and Ras-Responsive Regions in the Pit-1 TAD Target

A, GH4T2 cells were transiently cotransfected with 3 µg of –425rPRL-PA3Luc, 100 ng of phRL-TK, and either 2 µg of pSV-Ras or empty pSG5 control. Where indicated, varying concentrations of the pRSV-HA-Pit-1 Myc scanning or alanine point mutations were added to give equivalent levels of expression: pRSV-HA-Pit-1 5 µg, Myc-1 5 µg, Myc-2 5 µg, Myc-3 5 µg, Myc-4 10 µg, and Y60A/Y67A 5 µg. DNA concentration was held constant with the addition of pRSV-ß globin. After 24 h, the cells were harvested and assayed for luciferase activity. Bars represent the mean and SEM of triplicate samples from four separate transfections. B, Cellular lysates (100 µg total protein) from transfected cells were analyzed by western blot for HA-tagged Pit-1 expression using anti-HA (Covance Research Products, Berkeley, CA), and horseradish peroxidase-conjugated goat antimouse IgG (Bio-Rad).

 
Insertion of the ß-Domain at Various Sites in the Pit-1 TAD Dominantly Inhibits TAD Activity and Highlights the Importance of the Region from AA45–80 for Ras Responsiveness
The naturally occurring splice isoform of Pit-1, Pit-1ß, appears to negatively regulate the rPRL promoter in GH4T2 cells by recruiting a specific histone deacetylase-containing, corepressor complex to the Pit-1 TAD (12, 22, 23). Mutation of either select hydrophobic patches or the entire 26-AA ß-domain can switch Pit-1ß from a repressor to a Pit-1-like activator (22, 23), suggesting that simply inserting 26AA in the Pit-1 TAD is not a critical factor in its repressive activity, but rather it is the precise AA sequence that is important. To test the interplay of this repressor domain with other subdomains of the Pit-1TAD, we generated a series of hemaglutinin (HA)-tagged Pit-1 mutant plasmids in which the ß-domain was shifted from its natural position at AA48 to other sites within the TAD at 10-AA intervals (ß10TAD, ß20TAD, ß30TAD, ß40TAD, ß60TAD, and ß70TAD; Fig. 5AGo). We have previously shown that both Pit-1 and Pit-1ß activate the rPRL promoter in HeLa cells indicating that the ß-domain repressor activity is pituitary cell specific (22, 23 ; and Jonsen, M., K. Brodsky, P. Murapa, S. Diamond, and A. Gutierrez-Hartmann, in preparation). Consequently, we tested the Pit-1 ß-shuffle mutants for activity in HeLa cells to verify their functional integrity (Fig. 5BGo), before assessing their ability to repress rPRL promoter activity in transient transfections of GH4T2 cells (Fig. 5CGo). Finally, we verified their expression levels in the transfected cells by Western blot analysis with an antibody directed against the HA tag (Fig. 5CGo, blot). Each of the ß-shuffles (striped bars) activated the rPRL promoter in HeLa cells to levels similar to those of wild-type Pit-1 and Pit-1ß (black bars), verifying the functional integrity of each mutant Pit-1 construct. However, transcriptional activity of ß70TAD was slightly lower than that of Pit-1 and Pit-1ß, consistent with ß70TAD’s interruption of the strong AA50–70 activation domain identified in Fig. 2Go. Interestingly, the ß30TAD construct had nearly twice the activity of either Pit-1 or Pit-1ß, suggesting that this ß-shuffle either disrupted a domain inhibiting basal activation in HeLa cells or generated a new activation domain. We next assessed the ability of these constructs to repress Ras activation of rPRL in the GH4T2 cell line (Fig. 5CGo). Cotransfection of Ras resulted in a 9-fold activation of the rPRL promoter (Fig. 5BGo, white bars), and inclusion of Pit-1 enhanced this Ras response to 33-fold (Fig. 5BGo, black bar), essentially as previously described (22, 23). In contrast, Pit-1ß not only failed to enhance the Ras response, but it actually inhibited activation of the rPRL promoter to 40% of the Ras-only response (Fig. 5BGo, black bar), verifying its ability to dominantly repress the rPRL promoter despite the strong Ras activation signal as previously described (22, 23). Although each of the ß-shuffles repressed the Ras stimulation of rPRL, ß-shuffles inserted in the TAD from AA2–40 (ß10TAD, ß20TAD, ß30TAD, ß40TAD, striped bars) were less repressive of Ras stimulation than Pit-1ß. In contrast, ß-domain inserts in the TAD from AA47–80 (ß60TAD and ß70TAD, striped bars) were more repressive than Pit-1ß. Indeed, ß70TAD completely blocked Ras activation of rPRL. Western blot analysis reveals equal expression of these Pit-1 constructs, verifying that the differences in transcriptional activity were not due to variations in protein expression. An asterisk in the Western blot at ß40TAD denotes that a mutation in the HA tag of this construct was identified by sequencing (Fig. 5CGo). We subsequently verified the expression of this construct in HeLa cells by Western blot analysis using a Pit-1 antibody (data not shown). Finally, the activity of ß40TAD was consistent with the activity of Pit-1/Pit-1ß in transient transfections of HeLa cells (Fig. 5BGo), verifying that this construct was expressed and is functional, but failed to bind to the HA antibody. Taken together, these profiles of transcriptional and Ras repressive activity are consistent with the previous deletion and mutation studies that localized an activation domain to AA50–70 and the Ras-responsive region to AA60–85.


Figure 5
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  		Fig. 5. ß-Domain Shuffle Mutations Differentially Repress R1 and R2 of the Pit-1 TAD Target

A, Schematic of Pit-1ß. Arrows illustrate the alternate insertion sites for the ß-domain within the TAD. B, HeLa nonpituitary cells were transfected by electroporation with pRSV-HAPit-1 (4 µg), pRSV-HAPit-1ß (15 µg) or pRSV-HAShuffle (15 µg) expression constructs and 3 µg of the –425rPRL-PA3Luc reporter construct. Total amount of transfected DNA was kept constant by addition of pRSV-ßglobin expression construct. Activity is expressed as fold stimulation of the reporter in the presence of the Pit-1 constructs. C, GH4T2 cells were electroporated with 3 µg of –425rPRL-PA3Luc and either pRSV-HA Pit-1, Pit1ß, and ß-shuffle constructs at the same amounts as in B. Where indicated, cells were cotransfected with 5 µg pSV-Ras. Activity is graphed relative to reporter cotransfected with pRSV ß-globin expression construct, whose activity is set to 1. Values are presented as fold induction relative to the Prl reporter in the absence of oncogenic Ras. Repression is defined as activity less than Prl reporter + ß-globin and oncogenic Ras. Below each labeled ba,r cellular lysates (100 µg total protein) from transfected cells were analyzed by Western blot for HA-tagged Pit-1 expression using anti-HA (Covance Research Products, Berkeley, CA), and horseradish peroxidase-conjugated goat antimouse IgG (Bio-Rad). *, Designates protein in which HA tag was mutated. Protein expression was verified in HeLa cells using anti-Pit-1 antibody (data not shown).

 
Because the R1 and R2 subdomains encode transcriptional inhibitory and activating regulatory regions within the TAD, respectively (20), we hypothesized that the enhanced repression displayed by ß70TAD was due to disruption of the Ras-responsive region in combination with the ß-mediated repressor effect. We previously reported that Pit-1ß-mediated repression of the rPRL promoter is completely reversed in the presence of HDAC inhibitors [trichostatin A (TSA) or sodium butyrate] (23). However, because TSA will only reverse active, HDAC-mediated repression, but not passive inhibition due to physical disruption of the Ras-responsive region, addition of TSA should distinguish between active and passive repression. We transfected a ß-shuffle in which the ß-domain was centered in subdomain R1 (ß30TAD) or the strongly repressing ß70TAD, as well as Pit-1, and Pit-1ß, with the –425rPRL promoter in GH4T2 cells and treated them with 0.5 µM TSA. TSA enhances the Pit-1 response of the rPRL promoter from 1.2- to approximately 2.5-fold, whereas TSA fully reverses the repressor effects of ß30TAD from approximately 2-fold repression to a 4.5-fold activation and wild-type Pit-1 ß from approximately 4-fold repression to a 3.5-fold activation (Fig. 6Go). Thus, TSA completely reversed the repressive effects of ß30TAD and Pit-1ß making them as active as Pit-1. In contrast, whereas TSA did partially reverse the repressive effects of ß70TAD, it failed to enhance ß70TAD activity to the levels mediated by Pit-1ß or ß30TAD in the presence of TSA (Fig. 6Go). These TSA data are consistent with the notion that insertion of the ß-domain into the Pit-1 R2 region resulted in stronger repression due to inactivation of the positive R2 TAD segment plus inclusion of the HDAC-dependent ß-domain repressor motif.


Figure 6
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  		Fig. 6. TSA Treatment of ß-Domain Shuffle Mutations Further Differentiate ß-Domain Repression of Pit-1 TAD Target

GH4T2 cells were cotransfected by electroporation with pRSV-HAPit-1 (4 µg), pRSV-HAPit-1ß (15 µg) or pRSV-HAShuffle (15 µg) expression constructs and 3 µg of –425rPRL-PA3Luc. Total amount of transfected DNA was kept constant by addition of pRSV-ßglobin expression construct. Six hours after transfection, cells were treated with 0.5 µM TSA in ethanol (+) or ethanol alone (–) for 18 h before harvesting. Values are presented as fold induction relative to the rPRL promoter whose activity both with and without TSA was set to one to isolate the effects of TSA on the transfected Pit-1 constructs.

 
E1A Inhibition of CBP Reduces Basal Activation But Not Ras Responsiveness of the –425 rPRL Promoter in GH4 Pituitary Cells
Because transcriptional activation of a Prl-1P response element in response to forskolin and insulin has been shown to be mediated through Pit-1 homodimers interacting with a coactivator complex containing CBP/p-CAF (10), and CBP interactions have also been associated with Ras activation of Ets-1 (29), we wanted to determine whether binding and inhibition of CBP/p300 by the adenoviral E1A protein (40) would be able to inhibit the Ras activation of the rPRL promoter through Pit-1 and Ets-1 (Fig. 7Go). We transiently transfected GH4T2 cells with the –425 rPRL-luciferase reporter construct both without (top panel) and with (bottom panel) oncogenic V12Ras and increasing amounts of an expression construct for adenoviral E1A and the deletion mutant of E1A {Delta} 2–36, which lacks the CBP-binding domain. We found that E1A significantly inhibited basal activation of the rPRL promoter in both GH4T2 and HeLa cells (data not shown) without reducing the Ras response. The negative control, E1Adel, had no effect on either basal activation or Ras responsiveness at equivalent concentrations. These data reveal that CBP mediates basal rPRL promoter activity in GH4 cells but is not involved in Ras stimulation of the Ets-1/Pit-1 composite element. To verify the specificity and activity of the E1A constructs, we tested their ability to inhibit basal and Ras-stimulated activation of a luciferase reporter construct by Gal4DBD fusion constructs of the Ets factors, Ets-1, and Elk-1 in transient transfections of Hela cells (Fig. 8Go). Because prior data have implicated CBP and Sur-2 in the Ras activation of Ets-1 and Elk-1, respectively (29, 33), these constructs provide both putative positive (Ets-1Gal4DBD) and negative (Elk-1Gal4DBD) controls for the involvement of CBP in the Ras response. Similar to its effect on the –425rPRL promoter, E1A inhibited the basal activity of an Ets-1Gal4DBD fusion construct (Fig. 8AGo) but did not block its ability to respond to Ras activation (Fig. 8BGo). Elk-1, an Ets factor reported to be activated by Ras through a Mediator complex including Sur-2, was not inhibited by E1A in the presence of Ras but did exhibit reduced basal activity in the presence of E1A, although this reduction did not achieve significance (Fig. 8Go, A and B, right side).


Figure 7
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  		Fig. 7. E1A Inhibits Basal Activation by CBP, But Not Ras Activation of the Prl Promoter in GH4 Pituitary Cells Target

GH4T2 cells were transiently cotransfected in 96-well plates with 75 ng of –425rPRL-pA3-Luc promoter and 1 ng of phRL-TK. Twenty-five nanograms of pSV-Ras and increasing amounts of E1A and E1Adel2–36 constructs were added where indicated. After 24 h, the cells were assayed for luciferase activity. Bars represent mean ± SEM of triplicate samples in three transfections. *, Significantly different from control using Dunnett’s method; P < 0.05.

 

Figure 8
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  		Fig. 8. E1A Does Not Inhibit Ets-1 or Elk-1-Mediated Ras Activation of a Heterologous Promoter Target

GH4T2 cells were transiently cotransfected in 96-well plates with 75 ng L8G5-Luc promoter and 1 ng of phRL-TK. Fifty nanograms of Ets-1GAL4DBD or Elk-1GAL4DBD, pSV-Ras (25 ng) and E1A and E1Adel2–36 (1 ng) constructs were added where indicated. After 24 h, the cells were assayed for luciferase activity. A, Basal transcriptional activity of reporter construct in the absence of Ras. Bars represent mean ± SEM of triplicate samples. B, Fold Ras activation is expressed as the activity of a construct in the presence of Ras divided by the activity in the absence of Ras. Bars represent mean ± SEM of triplicate samples.

 
SRC-1 Stimulates Pit-Mediated Ras Activation of the rPRL Promoter in GH4 Pituitary Cells
Within Pit-1, Ras responsiveness has been localized to AA70–85, an amino-terminal transcriptional activation domain important for synergy of Pit-1 with ER, TR, and GATA-2 (6, 7, 8, 9). Because previous studies have implicated the p160 SRCs in the synergistic activation of the Prl promoter by ER and Pit-1 (15), we tested the ability of p160 SRC-1, -2, and -3 to stimulate Pit-1-mediated Ras activation of the –425 rPRL promoter in transient transfections of GH4T2 cells (Fig. 9Go). Although expression of the SRC constructs did not significantly alter basal transcriptional activity of the –425 rPRL promoter (Fig. 9Go, white bars), their expression did increase the Ras stimulation of the rPRL promoter. In fact, we found that SRC-1 increased the activity of the –425rPRL promoter in the presence of Pit-1 and Ras from 29 to 57 RLU (Fig. 9Go, black bars). Significantly, the addition of SRC-1 increased the fold Ras response of the rPRL promoter from 30- to 39-fold (P < 0.05, Fig. 9Go, right column), implicating p160 SRC-1 as a coactivator in the complex mediating Ras activation of the rPRL promoter.


Figure 9
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  		Fig. 9. SRCs Augment Pit-1-Mediated Ras Responsiveness of the rPRl Promoter Target

GH4T2 cells were transiently cotransfected in 96-well plates with 75 ng –425rPRL-pA3-Luc promoter and 1 ng of phRL-TK. Fifty nanograms of pRSV-HA-Pit-1, pSV-Ras (25 ng) and 25 ng of the SRC-1, -2, and -3 constructs were added where indicated. After 24 h, the cells were assayed for luciferase activity. Bars represent mean ± SEM of triplicate samples in three transfections. Fold Ras activation is expressed as the activity of a construct in the presence of Ras divided by the activity in the absence of Ras. *, Values significantly different from control P < 0.05 by Tukey’s posttest analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have used gain and loss of function analysis in both the context of chimeric Pit-1TAD Gal4DBD fusion proteins and full-length Pit-1 proteins to examine the regulatory interplay between Pit-1 TAD subdomains in basal transcriptional activity and Ras responsiveness in the physiologically relevant GH4 cell model. These interactions are summarized in Fig. 10Go. Previously, we found that deletion of R1 (AA2–45, gray shading) doubled basal Pit-1 activity in HeLa cells but inhibited its ability to synergize with Ets-1. Region 2 (AA46–80, white shading) contained the Ras-responsive elements of Pit-1 (20). In the current studies, we show that the Pit-1 TAD functions autonomously to mediate the Ras response through AA70–85. This same region inhibits basal TAD activity, apparently by blocking an activation domain localized at AA50–70. Insertion of the repressive ß-domain blocks activity of these subdomains by attracting HDAC containing corepressor complexes. Thus, the activity of the Pit-1 TAD may be dictated by the regulated attraction of corepressor and coactivator complexes. One such coactivator, CBP, has been associated with the activation of Pit-1-responsive gene promoters (10). Our studies indicate that CBP is required for basal activation of the rPRL promoter by Pit-1, but not Ras responsiveness. Consistent with this, Proline 24 in R1, a region we have associated with the regulation of basal activity, is implicated in CBP interactions with Pit-1 (34). In R2, the Ras-responsive region, Pit-1 AA70–85, has also been implicated in the regulation of synergy with TR and ER through RIP140 and p160 SRCs (17, 18, 19, 35). Consistent with this localization, we have found that SRC-1 augments the Pit-1-mediated Ras response of the rPRL promoter. Thus, we have determined that different coactivators may cooperate with specific subdomains of the Pit-1 TAD to regulate basal activity and Ras responsiveness of the rPRL promoter.


Figure 10
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  		Fig. 10. Summary of Pit-1 TAD Subdomain Activities Target

R1 (gray shading) is AA2–45, R2 (white) is AA45–85. The ß-domain is inserted at AA48 and the {gamma}-insert is at AA72. The 10-AA carboxy-terminal extensions of the Pit-1 TAD linked to GAL4 are indicated in R2. The locations of Tyrosines 60 and 67 are indicated with Ys. Identified cofactor interaction or synergy domains are indicated below the model.

 
We used a GAL4 fusion system to examine the subdomains of the Pit-1 TAD that regulate basal and Ras stimulated activity in isolation from other Pit-1 domains and DNA binding sites. Several early studies established the ability of the amino terminal AA2–70 of Pit-1 to function as an independent TAD (2, 42). Although we did not detect independent transcriptional activity for AA2–40 or AA2–50 of Pit-1 when fused to the GAL4DBD (Fig. 2Go), the sequential addition of 10-AA segments provided gradually increasing basal transcriptional activity through AA70, identifying a strong activation domain located at AA50–70. Surprisingly, addition of AA70–85 (GAL4TAD) reduced activity approximately 10-fold compared with GAL4N70, identifying a strong inhibitory domain in the AA70–80 region. When we assessed stimulation by oncogenic Ras, we found that, consistent with basal activation, the highest relative promoter activation in the presence of Ras occurred with the GALN70 construct. However, when Ras responsiveness was calculated as fold stimulation of each construct ± Ras, stimulation of the GAL4N70 construct was unchanged from the GAL4N40, 50, or 60 constructs, whereas Ras activation of the full-length Pit-1TAD was approximately doubled. Thus, the addition of the inhibitory domain correlated with a gain in fold Ras responsiveness. These data were consistent with our previous assignment of the Ras-responsive domain in Pit-1 to Region 2 (AA45–80) (20). Other investigators have also identified motifs in this region that are critical for synergistic interactions with transcriptional partners. Specifically, a motif containing three tyrosine residues separated by six AA (Y6Y6Y), located between AA48 and 72, is critical for synergistic activation of the Prl promoter with ER-{alpha} (17). Other studies have extended the ER synergy domain on either side of this motif into R1 (AA2–45) and AA72–100 (35) and have also associated these domains with Pit-1’s synergy with TR and GATA-2 on the GH and TSHß gene promoters, respectively (18, 19). In general, deletion of residues 2–72 reduced both basal transcriptional activity and synergistic activation of Pit-1 target gene promoters. In contrast, the domain(s) between AA72 and 125 appeared to be synergy-specific because deletion of these domains had minimal effects on basal activity but inhibited synergistic interactions with transcriptional partners (17, 19, 41). Similarly, the gain in Ras responsiveness is due to increased inhibition of basal activity upon addition of the 15 residues between AA70 and 85.

We also assessed the activity of the Pit-1 TAD in the context of full-length Pit-1. There are several key differences between these studies and those employing the GAL4 fusions. First, a complex, multi-Pit-1 target site promoter is used compared with the simple 5xUAS promoter used in the GAL4 assay. Second, both cofactors that bind to the POU-homeodomain (nuclear receptor corepressor 1/mSin3A and CBP/pCAF) and the TAD (CBP and RIP140) of Pit-1 are now involved (10, 34, 41). Finally, the binding and activities of endogenous Pit-1, Ets-1, GA binding protein, and other transcription factors that target the Prl promoter contribute to activity, whereas the GAL4 system is specifically designed to isolate binding of the GAL4-fusions. However, despite these differences, substitution of AA70–80 in Myc-4 increased basal transcriptional activity of Pit-1 in GH4T2 cells corroborating that these 10 AA are inhibitory to basal transcriptional activity. In contrast, the fold Ras response of Myc-4 is reduced compared with wild-type Pit-1 (Fig. 4Go), again confirming the assignment of the Ras-responsive domain to AA70–85 based upon the Ras responsiveness of the GAL4-Pit-1TAD fusion constructs. Similarly, mutation of tyrosines 60 and 67 to alanines also enhanced basal transcriptional activation of the rPRL promoter and reduced the Ras responsiveness of Pit-1, suggesting that the Y6Y6Y motif required for ER synergy with Pit-1 may also play a role in Ras responsiveness. Thus, two distinct analysis have suggested that the subdomains of the Pit-1 TAD that mediate Ras stimulation are distinct from those that regulate basal transcriptional activation of the –425rPRL promoter.

Adding to the complex nature of the Pit-1 TAD, the Pit-1ß splice isoform inserts a 26-AA repressor domain between exons one and two at AA48 in the amino-terminal TAD of Pit-1. Interestingly, the Pit-1ß isoform is the primary isoform expressed in nonmammalian vertebrates and cooperates with a second splice insertion in the TAD referred to as the {gamma}-insert located at AA72, disrupting the inhibitory/Ras-responsive domain identified in the current study (43). Thus, evolutionary compression of the Pit-1 TAD may contribute to differential regulation of Pit-1 target genes in mammals. Using fusions of the GAL4DBD linked to the Pit-1 and Pit-1ßTADs, we confirmed that the ß-domain functioned as a repressor of basal and Ras-stimulated activity, independent of other motifs in the Pit-1 protein or Pit-1 DNA binding sites. We then took advantage of this naturally occurring repressor domain to map the Pit-1 TAD by inserting the ß-domain at 10-AA intervals throughout the Pit-1 TAD. In support of the independent nature of the ß-domain repressor activity, we found that insertion of the ß-domain at any point in the TAD completely blocked the ability of Pit-1 to stimulate the Ras responsiveness of the rPRL promoter. However, ß-domain insertions in R1, which is not important for the Ras response (20), were less inhibitory of Ras activation than ß-domain insertions in R2, including Pit-1ß, ß60TAD, and ß70TAD. The R2 insertions were progressively more repressive of the Ras response, with ß70TAD exhibiting the greatest inhibition. The ß70TAD construct may have the greatest effect because its location disrupts the activation domain located at AA50–70 and/or the Ras-responsive domain in the region from 70–80. In contrast, recent studies from our lab have shown that inserting the ß-domain in Pit-1 at various locations outside of the TAD consistently repressed basal activity in GH4T2 cells but failed to repress the Ras response, confirming the importance of the Pit-1TAD in Ras signaling (Jonsen, M., K. Brodsky, P. Murapa, S. Diamond, and A. Gutierrez-Hartmann, in preparation). Furthermore, the inability of the HDAC inhibitor, TSA, to restore full activity to ß70TAD, supports the hypothesis that it disrupts an important activator domain and further suggests that the normal location of the ß-domain at AA48, near the strong activation domain, may contribute at least partially to its repressor function.

Cofactor interactions with the Pit-1 TAD may regulate shifts in the balance of corepressor and coactivator complexes that are recruited to Pit-1 target gene promoters in response to different signaling pathways. For example, cAMP signaling to a Pit-1-responsive minimal promoter activates the histone acetyl transferase (HAT) activity of CBP, but insulin activates the HAT activity of pCAF (10). Our studies using E1a to inhibit CBP, suggest that CBP is a crucial coactivator for basal transcriptional activity of the rPRL promoter in GH4 cells. In addition to the Pit-1 homeodomain, CBP binding has been localized to Proline 24 in R1 of the Pit-1 TAD (34). This is consistent with our functional mapping, which localizes basal transcriptional activity to the R1 subdomain. Recent studies have suggested that the homodimerization of Pit-1 is an important factor for its interactions with CBP/p300 (13). This suggests that, at monomeric Pit-1 binding sites where Pit-1 partners with heterologous transcription factors, like the Ras response element and the ERE/1d site in the distal rPRL promoter, different coactivator interactions may be important. In support of this hypothesis, we have found that SRC-1 stimulates Pit-1 mediated Ras responsiveness of the rPRL promoter (10). Similarly, Pit-1 AA72–80, were also required for optimal Pit-1/GATA-2 synergy mediated by mediator 220/TR associated protein 220 (14, 19) and for synergy with TR and ER and the associated repressive/activating effects of RIP140/SRCs (17, 18, 19, 41). In fact, SRC-1 phosphorylation by MAPK increases its transcriptional activity by activating the HAT or increasing affinity for transcriptional cofactors (44). SRCs also bind and activate the Ets transcription factors through the highly conserved Ets DBD in response to epidermal growth factor activation (45, 46). Thus, both Ras response element factors, Pit-1 and Ets-1, are activated by p160 SRCs. In contrast, SRC-1 had no effect on the activation of dimeric Pit-1 1P binding sites from the Prl promoter. Instead, this response is mediated by the protein kinase A (PKA)-dependent phosphorylation of CBP (10). Interestingly, the Ras and PKA pathways are mutually antagonistic in their regulation of the rrPRL promoter (47). Although PKA phosphorylation inhibits Raf activation (48), these pathways may also compete for the assembly of active coactivator complexes. In fact, a mutation in CBP that increases its HAT activity antagonizes the function of activated Ras in Caenorhabditis elegans (49). Ras activation of through Pit-1/Ets-1 may also be regulated by the loss of corepressor and gain in coactivator binding. In this regard, MAPK activation of the Ets factor, Elk-1, involves the reversal of small ubiquitin-related modifier-mediated binding of the corepressor HDAC-2 by protein inhibitor of activated STAT (50).

In the current study, the inhibition of Pit-1 basal activity with the addition of AA71–85 is countered by increased Ras responsiveness and may represent a shift in the coactivator/corepressor complexes recruited to Pit-1 to more tightly regulate transcriptional activity. This mechanism is common in biological systems where the dynamic range of a stimulated response like Ras activation is governed not only by the maximal response, but, more importantly, by tightly and negatively regulating basal activity. Thus, the Pit-1 TAD represents a complex regulatory domain with multiple overlapping sites for interaction with both activating and repressing transcriptional cofactors. Taken together, our data provide novel insights into the mechanisms by which the binding of Pit-1 at specific DNA binding sites (monomeric vs. dimeric), interactions with transcription factor partners (Pit-1 vs. heterologous partners), and activation of unique signaling cascades (basal vs. Ras) may contribute to the regulation of the Pit-1 TAD through these overlapping regulatory sites.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
The wild-type pGex-Pit-1 (AA2–80) expression vector (26) contains the amino-terminal TAD of rat Pit-1 fused in frame to GST in pGexDFGK, a modified pGex 2TK vector, incorporating the multiple cloning site derived from pCGN2, constructed and provided by Dr. David Gordon, University of Colorado Health Sciences Center. The GST-Ets-1 fusion construct, pGex-Ets-1 p68 full length was provided by Dr. Bohdan Wasylyk (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Illkirch Cedex, France). For the GAL-fusion experiments, the L8G5-luc reporter construct containing eight LexA operator sites and five UAS/GAL4 binding sites upstream of the luciferase gene (39, 51), was kindly provided by Dr. Saadi Khochbin (Institute Albert Bonniot, La Tronche Cedex, France). The wild-type E1A expression vector and the 2–36 deletion mutant were provided by Drs. Andrew Kraft and Joseph Biggs (Medical University of South Carolina, Charleston, SC). SRC-1, -2, and -3 constructs in the pCR3.1 expression vector were provided by Drs. B. O’Malley and Carolyn Smith (Baylor College of Medicine, Houston, TX).

The GAL4 fusion genes were first constructed in the plasmid, pSG424 (generously provided by M. Ptashne). First, TAD coding regions from Pit-1 (AA2–85) and Pit-1ß (AA2–111) were generated by PCR with the following primers: 5'GAL4TAD-GGGTACCTGAGTTGCCAACC and 3'TADstop-TCTAGAGGGTGTGGTCTGG, cloned into pCR2.1 for verification by sequencing, and then isolated using the restriction enzymes KpnI and XbaI. TAD extension constructs were similarly produced using the primer 5'GAL4TAD (described above) and a series of 3' primers with successive 10-AA additions of the carboxyl terminus of the TAD: 3'GALTAD40-TCTAGACTAGTGGTTGGAGGC, 3'GALTAD50-TCTAGACTATCCTGTCGCTGTGG, 3'GALTAD60-TCTAGACTAATAATGACAGGAAGG, and 3'GALTAD70-TCTAGACTACATCACTCCGTAGG. These products were inserted in-frame, downstream of the GAL4 coding region at the KpnI restriction site. GAL4 DBD fusion proteins were isolated from these pSG424-based plasmids by digestion with HindIII and KpnI and cloned into the pCDNA3.1 (Invitrogen, Carlsbad, CA) plasmid for expression. Plasmid –425PRL-pA3Luc, containing the proximal 425 bp of 5'flanking sequence from the rPRL promoter in the pA3Luc reporter, has been previously described (47). Plasmid pSV-Ras contains the T24 bladder carcinoma Harvey Ras valine 12 mutant oncogene (V-12 Ras) (27). Influenza HA epitope-tagged Pit-1 or Pit-1ß and ß-globin control were under the control of the Rous sarcoma virus (RSV) promoter in the pRSV plasmids (23). Myc 1–4 are scanning mutations of the region 2 TAD, which were generated by replacing overlapping segments of 11 AA with the Myc epitope (EQKLISEEDLL). Point mutations of tyrosine 60 and 67 to alanines (Y60A/Y67A) were generated in the context of pRSV-HA-Pit-1 using mutant internal primers and primers specific to the 5' and 3' ends of HA-tagged Pit-1, with dual rounds of PCR in overlap extension strategies. The mutant PCR primers are listed below, with the sequences homologous to Pit-1 in uppercase letters and sequence of the Myc epitope in lower case: Myc1-S-ctgatcagcgaggaagacctgttgTCCTGTCATTATGGAAACC; Myc1-AS-gtcttcctcgctgatcagtttctgctgctcCATCACGTTGGTGGCGTG; Myc2-S-ctgatcagcgaggaagacctgttgTCCACCTACGGAGTGATG; Myc2-AS-gtcttcctcgctgatcagtttctgctcATAATGAAGTCCTGTCGCTG; Myc3-S-ctgatcagcgaggaagacctgttgACTTTAACCCCTTGTCTTTAC; Myc3-AS-gtcttcctcgctgatcagtttctgctcTCCATAATGACAGGAAGGC; Myc4-S-ctgatcagcgaggaagacctgttgTTTCCAGACCACACCCTG; Myc4-AS-gtcttcctcgctgatcagtttctgctcCACTCCGTAGGTGGATGG; Y60A/Y67A-S-gcgGGAAACCAGCCATCCACCgcgGGAGTG; and Y60A/Y67A-AS-cgcGGTGGATGGCTGGTTTCCcgcATGACA. Primers directed against the 5' and 3' ends of the HA-Pit-1 cDNA were HA-Pit-1 5'-aaaaagcaagcttccatggggtacccatacgatgttccggattacgctAGTTGCAACCTTTC and Pit-1 3'-gcggccgcTTATCTGCACTCAAGATGCTC. Lowercase letters in these primers indicate restriction sites or epitopes added to aid in cloning. The PCR products were cloned into PCR 2.1 before subcloning into pRSV-HA-Pit-1.

The same overlap extension protocol was used to insert the 26-AA ß-domain at 10-AA intervals in the amino-terminal Pit-1 TAD. In the first round of amplification the following chimeric-mutagenic primers were paired with primers specific to the pRSV-HA-Pit-1 vector. Sequences homologous to the Pit-1 coding region are represented with uppercase letters, and sequences homologous to the ß-domain are presented in lowercase letters: Pit1ß2-S-atccaaactcctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaTGCCAACCTT TCACCTC; Pit1ß2-AS-cgagaaatatgtgtgcaaacatttaggagtttggatcaaagacaaaatagacgggacACTAGCGTAATCCGGAAC; Pit-1ß 10-S-ctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaACCTTTATACCTCTGAATTCTG; Pit-1ß 10-AS-gtgtgcaaacatttaggtgtttggatcaaagacaaaatagacgggacATCAGCCGAGGTGAAAG; Pit1ß20-S-ctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaGCTGCCCTGCCTCTGAGAATG; Pit1ß20-AS-gtgtgcaaacatttaggtgtttggatcaaagacaaaatagacgggacAGAAGCGTCAGAATTC; Pit1ß30-S-ctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaGCCGCTGAGGGTCTC; Pit1ß30-AS-gtgtgcaaacatttaggtgtttggatcaaagacaaaatagacgggacACTGTGGTGCATTCTC; Pit1ß40-S-ctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaGCCACCAACGTGATGT; Pit1ß40-AS-gtgtgcaaacatttaggtgtttggatcaaagacaaaatagacgggacGTGGTTGGAGGCTG; Pit1ß60-S-ctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaGGAAACCAGCCATCCA; Pit1ß60-AS-gtgtgcaaacatttaggtgtttggatcaaagacaaaatagacgggacATAATGACAGGAAGGCAC; Pit1ß70-S-ctaaatgtttgcacacatatttctcgatgacaacgatgggaaatacaGCAGGCACTTTAACCCCTTG; and Pit1ß70-AS-gtgtgcaaacatttaggtgtttggatcaaagacaaaatagacgggacCATCACTCCGTAGGTG. The overlapping products from the first round were extended and then amplified using the same primers specific to the pRSV-HA-Pit-1 vector: HA-GHF1/48-S-AAAAAGCAAGCTTCCATGGGGTACCCATACGATGTTCCGGATTACGCTAGTTGCCAACCTTTC and haGHF1/-24-AS-CCATTTGACCATTCACCACA.

GST Fusion Protein Preparation
Recombinant fusion proteins were purified from bacterial extracts by binding glutathione-Sepharose CL-4B (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (41). Protein concentration was measured by the Bio-Rad assay (Bio-Rad, Hercules, CA). Bound protein was analyzed for intactness and mass by SDS-PAGE in parallel with known amounts of BSA and stained with Coomassie Blue.

ERK Phosphorylation Assay
GST-fusion proteins (10 µg) bound to glutathione-Sepharose beads (~25 µl) were washed twice with Buffer A [20 mM HEPES (pH 7.6), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, and 0.4 mM phenylmethylsulfonyl fluoride]. The beads were resuspended in 50 µl of kinase buffer [20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM glycerophosphate (pH 7.0), 0.5 mM NaF, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol, 10 nM okadaic acid] with 20 µM ATP, 1.25 µCi {gamma}-P32-ATP (3000 Ci/mmol), and 0.8 µg ppERK1 (specific activity 200 nmol/min·mg in a kinase assay of myelin basic protein: graciously provided by Dr. Rebecca Schweppe, University of Colorado at Boulder). The samples were incubated with rotating at 30 C for 20 min. To stop the reaction, samples were centrifuged at 3000 rpm in a microcentrifuge for 5 min, the reaction buffer was removed, and 25 µl of 2x Laemmli sample buffer was added to each sample. The samples were heated in a boiling water bath for 5 min and electrophoresed on a 10% SDS-PAGE gel, stained with Coomassie Blue, destained, dried on filter paper, and exposed to autoradiography film for 2 h with amplification screens.

Cell Culture
HeLa and GH4T2 cells were maintained in DMEM (Invitrogen) supplemented with 15% horse serum and 2.5% fetal calf serum (Invitrogen. Cells were grown at 37 C in 5% CO2. Media was changed 4–16 h before transfection.

Electroporation-Mediated Transient Transfection
Cells were harvested in 0.05% trypsin with 0.5 mM EDTA and resuspended in culture medium. Aliquots of approximately 2–4 x 106 cells in 200 µl of media were added to plasmid DNA as described in figure legends and transfected by electroporation at 220 V and 500 µF using a GeneZapper 450/2200 (IBI/Kodak, Rochester, NY) with 4-mm gap cuvettes. After transfection, cells were plated on 60-mm tissue culture plates in culture media and incubated for 24 h. All electroporations included 100 ng of phRL-TK as an internal control for transfection efficiency. Total DNA was kept constant and nonspecific effects of viral promoters were controlled for by transfecting appropriate control vectors. At 24 h after electroporation, the cells were harvested and firefly luciferase and Renilla luciferase activity was determined on a Dynex microtiter plate luminometer using the Dual luciferase reporter assay system (Promega, Madison, WI). Briefly, the cells were harvested with 1x PBS containing 3 mM EDTA, pelleted, and lysed by adding 40 µl of Passive lysis buffer (Promega) and incubating at room temperature with shaking for 15 min. The cell lysate was transferred to an opaque 96-well plate and firefly luciferase and Renilla luciferase activity was measured for 10-sec intervals after addition of either 50 µl luciferase reagent or Stop-N-Glo reagent.


   ACKNOWLEDGMENTS
 
We would like to acknowledge Dr. David Gordon for generously supplying plasmid constructs used in these studies. We also thank Drs. Andy Bradford and David Gordon for critical reading and discussions of this manuscript.


   FOOTNOTES
 
This work was supported by R01 grants from National Institutes of Health through the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (to A.G.H), NIDDK K01 DK02946 (to D.L.D), and grants from the American Cancer Society RSG TBE-105036 and NIH /NIDDK K01 DK02752 (to S.E.D.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 4, 2006

Abbreviations: AA, Amino acid; CBP, cAMP response element binding protein-binding protein; DBD, DNA binding domain; ER, estrogen receptor; GHF-1 GH factor 1; GST, glutathione-S-transferase; HA, hemaglutinin; HAT, histone acetyl transferase; HDAC, histone deacetylase; p/CAF, p300/CBP-associated factor; phRL-TK, plasmid humanized Renilla luciferase-thymidine kinase; PKA, protein kinase A; POU, Pit-1, Oct-1, Unc 86; Prl, prolactin; rPRL, rat Prl; Rip140, receptor-interacting protein 140; RLU, relative light units; RSV, Rous sarcoma virus; SRC, steroid receptor coactivator; TAD, transcription activation domain; TK, thymidine kinase; TR, thyroid hormone receptor; TSA, trichostatin A; UAS, upstream activating sequence.

Received for publication June 12, 2006. Accepted for publication September 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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