Activities in Pit-1 Determine Whether Receptor Interacting Protein 140 Activates or Inhibits Pit-1/Nuclear Receptor Transcriptional Synergy

F. Max Chuang, Brian L. West, John D. Baxter and Fred Schaufele

Metabolic Research Unit University of California, San Francisco, California 94143-0540


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary-specific transcription of the evolutionarily related rat (r) GH and PRL genes involves synergistic interactions between Pit-1 and other promoter-binding factors including nuclear receptors. We show that Pit-1/thyroid hormone receptor (TR) and Pit-1/estrogen receptor (ER) synergistic activation of the rGH and rPRL promoters are globally similar. Both synergies depend upon the same activation functions in Pit-1 and also require activation function-2 conserved in TR and ER. The activation function-2 binding protein, RIP140, previously thought to be a nuclear receptor coactivator, strongly inhibits both Pit-1/TR and Pit-1/ER synergy. RIP140 inhibition is profoundly influenced, in a promoter-specific fashion, by a synergism-selective function in Pit-1: deletion of Pit-1 amino acids 72–100 switches RIP140 to an activator of Pit-1/ER and Pit-1/TR synergy at the rPRL promoter but not at the rGH promoter. Pit-1 amino acids 101–125 are required for RIP140 inhibition or activation again only at the rPRL promoter. Therefore, functions within one factor can determine the activity of a coactivator binding to its synergistic partner. This promoter context-specific synergistic interplay between transcription factors and coactivators is likely an essential determinant of cell-specific transcriptional regulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The rat (r) GH and PRL genes are transcribed specifically in distinct cell types within the anterior pituitary gland (1, 2, 3). Pituitary-specific expression of both genes requires a pituitary cell-specific transcription factor Pit-1 that activates the rGH and rPRL promoters. However, Pit-1, by itself, does not explain why GH and PRL are expressed in different, Pit-1-containing pituitary cell types. Differential GH and PRL promoter activity arises from synergistic interactions between Pit-1 and numerous other promoter-specific transcription factors including thyroid hormone receptor (TR) (4, 5), CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) (6), Zn15 (7), estrogen receptor (ER) (8, 9, 10), Ets-1 (11), c-jun (12), and pituitary LIM homeodomain factor (P-LIM) (13). Some of these transcriptional synergies are strongly dependent upon protein kinase activation (4, 5, 11) and, in those synergies involving TR or ER, hormone concentration (4, 8, 9, 10). Thus, pituitary cell-specific transcription is mediated by environment-sensitive synergies between transcription factors, most of which are present in a wide variety of nonpituitary cells in which they do not influence GH or PRL transcription.

Most other promoters are also activated cell specifically by factors of a comparatively broad tissue distribution. Although isolated transcription factor activities seem to be secondary to synergistic activities in the formation of expression patterns that determine cell identity, the mechanisms by which the synergistic partners affect each other’s activities remain largely unknown. Of the few studies of transcriptional synergy, the synergistic activations of the rGH promoter by Pit-1 and TR and of the rPRL promoter by Pit-1 and ER are possibly the best characterized. Because the rGH and rPRL genes (14), as well as TR and ER (15, 16, 17), are evolutionarily related and because GH and PRL are expressed in developmentally related anterior pituitary cell types (8, 18, 19, 20, 21), comparing Pit-1/TR synergy at the rGH promoter (4, 5) with Pit-1/ER synergy at the rPRL promoter (8, 9, 10) will allow us to delimit common and unique mechanisms involved in transcriptional synergy. Previous studies of Pit-1/TR synergy at the rGH promoter (5) and Pit-1/ER synergy at the rPRL promoter (10) suggested that both synergies depend upon overlapping but clearly unique activities in Pit-1,but this conclusion is limited by the differences in the experimental and cellular systems used in these separate studies. Nothing is yet known of the effects of any of the recently identified nuclear receptor coactivators on these, or any other, synergies.

Here, we demonstrate that identical activities in Pit-1 are required for Pit-1/TR and Pit-1/ER synergy under identical experimental conditions. Both synergies also depended upon activation function-2 (AF-2), conserved in TR and ER. Expression of an AF-2 interacting protein RIP140 (22) selectively inhibited both Pit-1/TR synergy at the rGH promoter and Pit-1/ER synergy at the rPRL promoter. Inhibition by RIP140 was profoundly affected by activities within Pit-1 in a promoter-specific fashion. Deletion of Pit-1 of amino acids (aa) 101–125 eliminated RIP140 inhibition of Pit-1/ER synergy at the rPRL promoter but had no effect on RIP140 inhibition of Pit-1/TR synergy at the rGH promoter. Intriguingly, RIP140 expression caused the otherwise synergy-deficient {Delta}72–100 deletion of Pit-1 to synergize with ER at the rPRL promoter. Deleting Pit-1 aa 72–100 similarly activated a latent, RIP140-dependent, Pit-1/TR synergy at the rPRL promoter but not at the rGH promoter. Thus, activities within Pit-1 dictate whether a factor binding to a nuclear receptor inhibits, has no effect on, or activates synergy. This interplay of transcription factors and coactivators is influenced by promoter-specific elements or factors and is likely crucial to the determination and maintenance of cell-specific expression patterns.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pit-1/ER Synergy at the rPRL Promoter in Pituitary Progenitor Cells
We initially characterized Pit-1/ER synergy at the rPRL promoter in mouse pituitary progenitor GHFT1–5 cells to compare Pit-1/ER synergy at the rPRL promoter with our previously characterized (5) Pit-1/TR synergy at the rGH promoter in the same cells. GHFT1–5 cells are an ideal cellular model in which to study the similarities and differences in GH and PRL gene activation because they express low levels of Pit-1 but are not yet committed to either the GH or PRL synthetic pathways (23). GHFT1–5 cells were cotransfected with a luciferase reporter construct under the control of 3 kb of the rPRL promoter/enhancer and with expression vectors containing the rat Pit-1 and human (h) ER cDNAs. Luciferase assays conducted with cytoplasmic extracts of the transfected cells showed the rPRL promoter to be markedly more active when Pit-1 and ER were coexpressed than when Pit-1 and ER were separately expressed (Fig. 1AGo). Pit-1 and ER Western blots conducted on extracts subsequently prepared from the nuclear pellet proved this activation to be genuinely synergistic and not simply due to enhanced expression of either Pit-1 or ER (Fig. 1Go, B and C). For these Westerns, Pit-1 was tagged at its amino terminus with the FLAG epitope to distinguish transiently expressed Pit-1 (Fig. 1BGo) from endogenous Pit-1. An antibody that preferentially, although not exclusively, recognized hER was used to distinguish transiently expressed hER (Fig. 1CGo) from the endogenous mouse ER that we observed to be present in GHFT1–5 cells.



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Figure 1. Pit-1 and ER Synergistically Activate the 3-kb rPRL Promoter/Enhancer in GHFT1–5 Pituitary Progenitor Cells

A, Representative experiment showing much greater than additive luciferase expression from the 3-kb rPRL promoter/enhancer when FLAG-Pit-1 and hER expression vectors are cotransfected than when they are transfected separately. Western blots demonstrate that increased luciferase activity is not due to enhanced FLAG-Pit-1 expression (blotted with anti-FLAG antibody in panel B) or hER expression (blotted with anti-hER antibody in panel C) when hER and FLAG-Pit-1 are coexpressed.

 
No Pit-1/ER synergy was observed in the absence of estradiol (data not shown). The quite strong activation of the rPRL promoter/enhancer by Pit-1 alone (Fig. 1AGo) also was mostly estradiol-dependent (data not shown) demonstrating that Pit-1 synergizes with endogenous ER present in GHFT1–5 cells as well as with coexpressed ER. Pit-1/ER synergy was observed in the absence of the protein kinase A and protein kinase C activators, forskolin and phorbol 12-myristate 13-acetate (PMA), which activated synergy only 1.6-fold on average (data not shown). This contrasts with Pit-1/TR synergy at the rGH promoter, which was wholly dependent upon incubating transfected GHFT1–5 cells with forskolin and PMA (5). To permit further direct comparisons between Pit-1/ER synergy at the rPRL promoter with Pit-1/TR synergy at the rGH promoter under the same conditions, all subsequent experiments were performed in GHFT1–5 cells incubated with forskolin and PMA. Thus, Pit-1 and ER synergistically activate the rPRL promoter in an excellent pituitary cell culture model, confirming that Pit-1/ER synergy observed in some nonpituitary cell types (8, 9, 10) is likely relevant to pituitary biology.

Pit-1 Activities Required for Pit-1/ER Synergy at the rPRL Promoter
The same set of Pit-1 mutations used to determine the activities in Pit-1 necessary for Pit-1/TR synergy at the rGH promoter in GHFT1–5 cells (5) was used to determine that grossly similar activities in Pit-1 were required for Pit-1/ER synergy at the rPRL promoter (Fig. 2Go). Pit-1 deletions within a previously described (5, 24) activation function residing within aa 2–73 supported neither activation by Pit-1 alone nor synergy with ER (Fig. 2AGo). Therefore, Pit-1/ER synergy, like Pit-1/TR (5) synergy, required active participation by Pit-1.



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Figure 2. Deletions Affecting the Pit-1 Activation Domain (A) or Pit-1 Synergism-Selective Activity (B) Show That Both Are Necessary for Pit-1/ER Synergy

wt, Wild-type Pit-1. Pit-1 mutations are labeled by the inclusive amino acid positions deleted. Three (A) or five (B) independent experiments were normalized to the expression level of the wild type Pit-1-activated promoter to facilitate comparisons of independent and synergistic induction.

 
Previously, we showed in GHFT1–5 cells that strong synergistic activation of the rGH promoter by wild type Pit-1 and TR was abolished by the deletion of Pit-1 aa 72–125 whereas, in the absence of coexpressed TR, wild type and the {Delta}72–125 mutant of Pit-1 were equally capable of activating a minimal promoter to which only Pit-1-binding sites were appended (5). At the rPRL promoter, we observed a 2.2-fold activation, on average, by Pit-1{Delta}72–125 in the absence of coexpressed ER that was similar to the 2.8-fold activation by wild type Pit-1 alone in the absence of estradiol (data not shown). By contrast, in the presence of estradiol and in synergy with endogenous ER (Fig. 2BGo), the 2.2-fold activation by Pit-1{Delta}72–125 was significantly less than the 4.8-fold enhancement by wild type Pit-1 (Fig. 2BGo). This suggested that Pit-1/ER synergy at the rPRL promoter in GHFT1–5 cells was much more sensitive to the deletion of Pit-1 aa 72–125 than was activation by Pit-1 alone, which was confirmed by the finding that Pit-1{Delta}72–125 did not act in synergy with coexpressed hER (Fig. 2BGo). We refer to this region as SynAF-1 to reflect its selective participation in synergy with both ER (Fig. 2BGo) and TR (5).

As with Pit-1/TR synergy at the rGH promoter (5), the SynAF-1 requirement for Pit-1/ER synergy at the rPRL promoter mapped to Pit-1 aa 72–100 (Fig. 2BGo). Pit-1, with aa 101–125 alone removed, behaved as wild-type Pit-1 in the Pit-1/ER synergistic activation of the rPRL promoter whereas Pit-1, with aa 72–100 removed, like Pit-1{Delta}72–125, did not synergize with ER. Western blots of FLAG-tagged {Delta}72–100 and {Delta}101–125 Pit-1 mutants showed that both were efficiently expressed in GHFT1–5 cells (Fig. 3Go; activation of rPRL promoter activity by expression of each Pit-1 alone is presented for the experiment from which the representative Western blot was taken). The efficient expression of these Pit-1 mutants agrees with our previous findings that the Pit-1{Delta}72–100 and Pit-1{Delta}101–125 mutants activated a minimal, Pit-1-sensitive promoter as effectively as wild type Pit-1 under identical assay conditions (5). Thus, a series of activities in Pit-1 including activation functions residing between aa 2 and 73 and a synergism-selective activity dependent upon aa 72–100 are required for both Pit-1/ER and Pit-1/TR synergies at the rPRL and rGH promoters.



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Figure 3. The Pit-1 {Delta}72–100 and {Delta}101–125 Mutations Are Efficiently Expressed in GHFT1–5 Cells

Anti-FLAG Western blot of FLAG-tagged wild type and mutant Pit-1 expressed in GHFT1–5 cells show that the decreased activation by the {Delta}72–100 mutation is not a reflection of decreased Pit-1 amount. In fact, the Pit-1{Delta}72–100 and {Delta}72–125 (not shown) proteins were more efficiently expressed than the wild type and {Delta}101–125 proteins: transfecting 1.5 µg of Pit-1{Delta}72–100 expression vector was sufficient to yield the same amount of Pit-1 protein as 5 µg of the wild type and{Delta}101–125 vectors in this representative experiment.

 
Pit-1/TR and Pit-1/ER Synergy at the rGH and rPRL Promoters Depends upon Nuclear Receptor AF-2
We previously demonstrated that Pit-1/TR synergistic activation of the rGH promoter required an intact TR ligand-binding domain (5). A sequence at the carboxyl-terminal end of the TR and ER conserved in the ligand-binding domain of most nuclear receptors is necessary for the ligand-dependent transcriptional activation by nuclear receptors and binds in a ligand-dependent fashion to a number of factors termed coactivators of nuclear receptors (15, 16, 17). Consistent with the apparently conserved nature of Pit-1/nuclear receptor synergies at the evolutionarily related rGH and rPRL promoters, both synergies were completely inhibited by point mutations within AF-2 of the cognate receptor (Fig. 4Go). Thus, in addition to similarly requiring independent and synergism-selective activation functions within Pit-1, both Pit-1/TR and Pit-1/ER synergistic activation require the conserved nuclear receptor AF-2.



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Figure 4. Point Mutations in AF-2 Disrupt Pit-1/ER (A) or Pit-1/TR (B) Synergy at the 3-kb rPRL and 245-bp rGH Promoters

AF-2 mut, L543A/L544A double mutant in ER, E451K mutant in TR. Four (panel A) or two (panel B) independent experiments were normalized to the synergistically activated rPRL or rGH promoters (100% with wild type receptor) and plotted as the mean ± SD or range, respectively.

 
The AF-2-Interacting Factor RIP140 Inhibits Pit-1/ER Synergy at the rPRL Promoter
The dependence of both Pit-1/nuclear receptor synergies on AF-2 suggested that any of the known AF-2-interacting proteins (22, 25, 26, 27, 28, 29, 30, 31) might participate directly in synergistic activation and that their overexpression might affect synergy. Coexpression of the AF-2-interacting protein RIP140 (22) abolished Pit-1/ER synergy at the rPRL promoter (Fig. 5AGo). RIP140 also reduced expression from the rPRL promoter activated by Pit-1 "alone" probably by affecting Pit-1 synergy with low levels of endogenous ER. A trivial explanation for RIP140 inhibition of synergy might be that RIP140 inhibited ER and/or Pit-1 synthesis from their expression vectors. Western blots indicated that RIP140 had no effect on the expression of hER (detected with the hER antibody, Fig. 6BGo), FLAG-Pit-1, or endogenous Pit-1 (both detected with a Pit-1 antibody and distinguished by the larger size of FLAG-Pit-1, Fig. 6CGo), or endogenous ER (Fig. 6DGo, detected with a rodent-specific ER antibody). Thus, RIP140 directly abolished Pit-1/ER synergistic activation of the rPRL promoter.



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Figure 5. The AF-2-Interacting Protein RIP140 Inhibits Pit-1/ER Synergistic Activation of the rPRL Promoter (A) but not C/EBP{alpha} Activation of the Same Promoter (B)

Seven (A) or four (B) independent experiments were normalized to the Pit-1/ER- or C/EBP{alpha}-activated rPRL promoter set as 100%.

 


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Figure 6. RIP140 Inhibits Pit-1/Synergy at the rPRL Promoter Without Affecting Pit-1 or ER Expression

rPRL promoter activity (panel A) and associated Western blots (panels B–D) from a representative experiment. Transfected hER (arrow) was detected by Western blot with an anti-human ER antibody (panel B), transfected FLAG-Pit-1 (arrow) or endogenous Pit-1 (bracket) was detected with an anti-Pit-1 antibody (panel C) (the FLAG epitope results in transfected FLAG-Pit-1 being larger than endogenous Pit-1) and endogenous mouse ER (arrow) was detected with an anti-rodent ER antibody (panel D). GC, Equivalent amount of identically prepared extract from rat pituitary GC cells serves as a blotting control and indicator of the expression level achieved.

 
RIP140 expression did not affect the strong activation of the rPRL promoter by C/EBP{alpha} (Fig. 5BGo) demonstrating that RIP140 inhibition was selective for Pit-1/ER synergy. Other nuclear receptor-interacting proteins including TIF1 (26), SRC-1 (29), GRIP1 (31), and CBP (32) did not inhibit Pit-1/ER synergy although CBP enhanced C/EBP{alpha} activation of both the rGH and rPRL promoters in parallel experiments (our unpublished data). RIP140 inhibition of synergy was retained when both RIP140 and CBP were coexpressed (data not shown). RIP140 expression similarly inhibited TR synergy with wild type Pit-1 at the rGH promoter in GHFT1–5 cells (Fig. 7AGo) without affecting C/EBP{alpha} activation of the rGH promoter (our unpublished data). Thus, Pit-1/nuclear receptor synergistic activation is acutely sensitive to RIP140, which presumably interferes with AF-2 function. These results strongly suggest that we should rethink the assumption that any factor that binds to AF-2 in a ligand-dependent fashion always functions as a coactivator.



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Figure 7. Effect of RIP140 Expression on Pit-1 Synergy with TR at the rGH Promoter (A, C, and E) or with ER at the rPRL Promoter (B, D, and F)

The effect of RIP140 on Pit-1/ER synergy depends upon whether wild type Pit-1 (A and B) or the {Delta}101–125 (C and D) or {Delta}72–100 (E and F) mutants of Pit-1 are used. Three (A, C, and E) or five (B, D, and F) independent experiments were normalized to the level of rGH or rPRL promoter activity in the presence of coexpressed nuclear receptor and Pit-1 mutant (100%). The effects of RIP140 expression on the rGH or rPRL promoters themselves or activated by Pit-1 or nuclear receptor alone were mostly negligible except where discussed within the text (data not shown).

 
Promoter-Specific Relief of RIP140 Inhibition by the Pit-1{Delta}101–125 Mutation
Our results indicated that two activities function preferentially in Pit-1/nuclear receptor synergy: 1) SynAF-1, selectively required for Pit-1 synergy with ER (Fig. 2BGo) and TR (5), but not with C/EBP{alpha} (6), and 2) RIP140, which selectively inhibited Pit-1/ER (Figs. 5Go, 6Go) and Pit-1/TR (Fig. 7AGo) synergy without affecting C/EBP{alpha} activation of the rGH or rPRL promoters. To determine whether these two synergism-selective effects were related, we studied the effects of the {Delta}72–100 and {Delta}101–125 Pit-1 mutants on RIP140 inhibition of Pit-1/TR and Pit-1/ER synergy at the rGH and rPRL promoters (Fig. 7Go). For simplicity, only shown are the effects of RIP140 expression on rGH and rPRL promoter activity when Pit-1 or mutants thereof are coexpressed with the indicated nuclear receptor.

Pit-1 with aa 101–125 deleted still synergized with ectopically expressed ER (Fig. 2BGo) and TR (5). However, ER synergy with Pit-1{Delta}101–125 at the rPRL promoter was no longer or, at best poorly, inhibited by RIP140 expression (Fig. 7DGo). This contrasts with Pit-1{Delta}101–125/TR synergy at the rGH promoter, which was still inhibited by RIP140 expression (Fig. 7CGo). Similarly, RIP140 inhibition of Pit-1 synergy with endogenous ER was eliminated by deleting Pit-1 aa 101–125: in synergy with endogenous ER, Pit-1{Delta}101–125 activation of the rPRL promoter was not inhibited by RIP140 expression (89.0 ± 20.3% as active as the Pit-1{Delta}101–125-activated promoter in the absence of RIP140) whereas wild type Pit-1 was inhibited to 58.6 ± 4.8% the activity in parallel experiments (n = 5). Thus, an activity dependent upon Pit-1 aa 101–125 is required for the RIP140 inhibition of Pit-1/ER synergy at the rPRL promoter. This demonstrates that activities within one transcription factor influence the transcriptional effect of a coactivator interacting with its synergistic partner. This influence is somehow altered by other elements specific to the rGH or rPRL promoters or to the TR and ER themselves.

A SynAF-1 Mutant of Pit-1 that Switches RIP140 from an Inhibitor to an Activator of Pit-1/ER Synergy
Surprisingly, coexpression of the synergy-defective Pit-1{Delta}72–100 mutant with ER resulted in RIP140-dependent activation, not inhibition, of the rPRL promoter (Fig. 7FGo). Again, this effect was rPRL-specific as the Pit-1{Delta}72–100 mutant did not synergize with TR at the rGH promoter (Fig. 7EGo). This activation represented bona fide RIP140-dependent synergy because it was observed only when all three factors (Pit-1{Delta}72–100, ER, and RIP140) were coexpressed (Fig. 8AGo). Thus, when deleted of aa 72–100, Pit-1 is defective in synergy with ER unless RIP140 is added. This activation of Pit-1{Delta}72–100 synergy by RIP140 contrasts starkly with the inhibition of wild type Pit-1/ER synergy by RIP140. In contrast, the similarly synergy-defective Pit-1{Delta}72–125 mutant did not synergize with RIP140 and ER (data not shown), demonstrating that Pit-1 aa 101–125 were required for the RIP140-dependent Pit-1{Delta}72–100/ER synergy. Like RIP140 inhibition of wild type Pit-1/ER synergy, RIP140 activation of Pit-1{Delta}72–100/ER synergy therefore requires Pit-1 aa 101–125.



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Figure 8. Pit-1{Delta}72–100 Activates the rPRL Promoter only in Synergy with Both ER and RIP140 (A) or TR and RIP140 (B)

Five (A) or three (B) independent experiments were normalized to the level of rPRL promoter activity in the presence of coexpressed TR and Pit-1 mutant (100%) and plotted as the mean fold activation ±SD.

 
Pit-1/TR Synergistic Activation of the rPRL Promoter Is RIP140-Dependent SynAF-1-Sensitive
The influence of SynAF-1 on Pit-1/nuclear receptor synergies at the rGH and rPRL promoters is clearly different and may represent promoter-specific influences on synergy or mechanistic differences in TR and ER themselves. The rPRL promoter/enhancer is regulated by thyroid hormone via a site immediately downstream of the estrogen response element (33). In GHFT1–5 cells, TR expression neither activated the rPRL promoter nor synergized with wild type Pit-1 or with Pit-1 missing aa 101–125 (data not shown) or aa 72–100 (Fig. 8BGo). RIP140 expression did not change the absence of TR synergy with wild type Pit-1 or Pit-1{Delta}101–125 (data not shown). In contrast, deletion of Pit-1 aa 72–100 resulted in a RIP140-dependent, Pit-1/TR synergy at the rPRL promoter (Fig. 8BGo). Therefore, as with Pit-1/ER synergy at the same rPRL promoter (Fig. 8AGo), the deletion of Pit-1 aa 72–100 activated a latent synergy with TR and with a coactivator that interacts directly with TR. This result suggests that the differences in SynAF-1 effects on RIP140 modulation of Pit-1/nuclear receptor synergies are related to promoter-specific activities other than simple differences in TR and ER.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As with most cell-specific transcription control sequences, the promoters of the rGH and rPRL genes are regulated by factors of a comparatively broad cellular distribution (2, 34, 35). This paradox can be resolved by considering transcription as a function of principally interdependent promoter-binding cofactors. For instance, thyroid hormone and estrogen activation of the rGH and rPRL genes is pituitary-specific because of the mutual dependence of their broadly distributed receptors on the pituitary-specific transcription factor Pit-1 (4, 5, 8, 9, 10). Pit-1 also synergizes with a number of other rGH and rPRL promoter-binding factors (6, 7, 11, 12, 13), and the sum total of these synergies, and even synergies between different independent synergies (our unpublished data), likely underlie the physiological, developmental, age-related, and gender-specific variations in pituitary hormone synthesis.

Very little is known of the molecular events of such transcriptional synergies despite their undoubted importance to gene expression. We compared activation of the phyllogenetically and ontologically related rGH and rPRL genes to define activities required for Pit-1/nuclear receptor synergies. Both synergies required activation functions (Fig. 9Go, AF) in both Pit-1 and in the nuclear receptors (Ref. 5 , Figs. 2AGo and 4Go) indicating that both partners actively participated in activation. Both synergies were inhibited by the expression of RIP140 ( Figs. 5–7GoGoGo), which likely occurred by RIP140 interference with AF-2, possibly by competing for AF-2 binding with a hypothetical strong, endogenous coactivator (Fig. 9Go). An equally viable alternative not depicted in Fig. 9Go is that RIP140 actively inhibited synergy. We currently prefer the scheme outlined in Fig. 9Go only because it is the simplest explanation for all of our data regarding the ability of RIP140 to both activate and repress synergy.



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Figure 9. Interrelationship of Factors and Activities That Participate in the SynAF-1/AF-2 Synergy Switch

Some of the current data may also be interpreted according to modifications of this model of the activities that regulate SynAF-1 modulation of AF-2- interacting proteins in a promoter-specific fashion.

 
Both synergies also required SynAF-1, a synergism-selective activity dependent upon Pit-1 aa 72–100 (Ref. 5 and Fig. 2BGo). The latter results differed somewhat from those reported for Pit-1/ER synergistic activation of the rPRL promoter in CV-1 cells (10) in which synergism-selective effects were observed by the deletion of aa 45–72 (compare with our Pit-1{Delta}48–73 which disrupts both activation by Pit-1 alone and synergy with nuclear receptors, Fig. 2AGo) and in which the analogous deletion of Pit-1 aa 73–128 Pit-1 did not prevent Pit-1/ER synergy. These cell-specific observations are likely related to cell-specific differences in the distribution of other transcription factors and highlight the necessity of comparing Pit-1/TR and Pit-1/ER synergies at the rGH and rPRL promoters under identical experimental conditions.

Although Pit-1/nuclear receptor synergies at the rGH and rPRL promoters were grossly similar, the synergy was influenced in a promoter-specific fashion by other factors or events. Mutations within SynAF-1 determined whether RIP140 inhibited or activated Pit-1 synergy with ER or TR specifically at the rPRL promoter (Figs. 7Go and 8Go). The regulatory properties of this SynAF-1/AF-2 synergy switch were subtly different between synergy itself and RIP140 modulation thereof: Pit-1 aa 101–125 were not required for raw synergy but were required for RIP140 regulation (both activation and inhibition) at the rPRL promoter (Fig. 9Go); Pit-1 aa 72–100 were required for raw synergy and therefore either prevented synergy in the presence of RIP140 (Fig. 9Go) or actively supported inhibition by RIP140 (not depicted). The differential rGH (Fig. 7EGo) and rPRL (Fig. 8BGo) promoter response under identical experimental conditions suggested that elements or factors specific to the rGH or rPRL promoters could modulate the SynAF-1/AF-2 synergy switch and dramatically alter gene transcription. Candidates for the promoter-specific activity include promoter-specific binding factors, differences in Pit-1/nuclear receptor binding site orientation and spacing, or the known difference in dimeric status of Pit-1 or TR bound to certain sites (10, 17).

Thus, a molecular ménage-à-trois involving Pit-1, nuclear receptors, RIP140, and probably other AF-2-interacting proteins governs synergistic transcriptional activation of the rGH and rPRL promoters in GHFT1–5 pituitary progenitor cells and is differentially influenced by promoter structure. It will be very important to characterize this synergy switch involving SynAF-1, its interdependence with AF-2-binding coactivators and with transcriptional activation functions in both Pit-1 and nuclear receptors, and to determine how components of this switch are altered in different physiological and developmental expression states (8, 20, 36, 37, 38, 39, 40). The finding that coactivator action can be influenced by specific combinations of transcription factor activities can be explained by numerous models, but it would appear that the activities that participate in Pit-1/nuclear receptor synergy, possibly including an endogenous GHFT1–5 cell AF-2-interacting protein, are skewed or usurped by the AF-2-interacting protein, RIP140. The complexity and interrelatedness of these activities demonstrate the necessity of ultimately viewing transcriptional events at complete, natural promoters to understand gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transfection and Analysis
GHFT1–5 cells were grown in DME-H21 supplemented with 10% FCS. Estradiol response was determined in cells that had been grown for 24 h before transfection in media containing 5% FCS and 5% newborn calf serum stripped of steroid and thyroid hormones by charcoal binding and batch AG-1X column chromatography. Cells were treated with 10-8 M estradiol, 10-5 M forskolin, 10-7 M PMA, and/or delivery vehicles 1 day after transfection and collected the following day. Transfection was by electroporation under the conditions previously described (6). Choramphenicol acetyltransferase and luciferase activities were determined as previously described (4, 5). Collected cells were lysed in reporter lysis buffer (Promega, Madison, WI) and choramphenicol acetyltransferase and luciferase activities in these extracts were determined as previously described (4, 5, 6). Data from multiple independent experiments were normalized to specific reference points (see figure legends for n and points) and the mean ± SD was determined for each.

For Westerns, the cell pellet posttreatment with reporter lysis buffer was resuspended in 2-(N-morpholino)ethanesulfonic acid-Tris (pH 7.8), 1 mM dithiothreitol, and 0.1% Triton X-100, pelleted, resuspended in the same buffer, and pelleted again. The resulting crude nuclei were resuspended three times with 20 µl of 20 mM HEPES (pH 7.9), 300 mM KCl, 200 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 15% glycerol and the extracts were pooled. Equivalent amounts of extract protein (5–20 µg depending on the experiment) were loaded onto SDS acrylamide gels and probed with either the FLAG {alpha}M2 antibody (ICI-Kodak, Rochester, NY), the Pit-1 214–230 antibody (Berkeley Antibody Company, Berkeley, CA), the human-specific ER HC-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or the rodent-specific ER MC-20 antibody (Santa Cruz Biotechnology). Rat PRL promoter activity normalized to cytoplasmic extract protein is shown in Figs. 1Go, 3Go and 6 for direct comparison with Pit-1/ER expression level determined by Western blots on nuclear extracts.

Plasmids
Two and five-tenths micrograms of the -237/+8 rGH promoter (where +1 is the transcription start site) cloned in front of the chloramphenicol acetyltransferase gene (4) or 2.5 µg of the -3 kb rPRL promoter/enhancer cloned in front of the luciferase gene (41) were transfected in each experiment shown. Each promoter was cotransfected with 10 µg of the indicated TR or ER expression vectors or cognate expression vectors not containing an inserted cDNA. The hTR (6) or hER (42, 43) expression vectors have been previously described. FLAG-tagged Pit-1 was constructed by inserting an oligonucleotide encoding the amino acids MDYKDDDDKDYA with an optimal Kozak sequence into an NcoI site overlapping the initiator Met of Pit-1. Five micrograms of FLAG-tagged Pit-1, cloned behind the cytomegalovirus (CMV) promoter in the vector Rc/CMV (Invitrogen, San Diego, CA), were transfected (Figs. 1Go, 3Go, and 6Go) whereas 10 µg of the unmodified Pit-1 expressed from the previously described (4) Rous sarcoma virus promoter were transfected in the remaining experiments. Equivalent amounts of cognate expression vectors not containing an inserted cDNA were substituted for transfections in which Pit-1 was not expressed. In experiments in which the amounts of FLAG-tagged wild type and mutant Pit-1 were varied (Fig. 3Go and data not shown), the decreased amount of FLAG-tagged vector was supplemented with Rc/CMV to 5 µg.

The Pit-1 mutants were identical to those used previously (5, 24). The point mutation in AF-2 of TR was constructed by oligonucleotide-directed mutagenesis in which the glutamic acid at aa 451, conserved in most AF-2 sequences, was changed to lysine. The L543A/L544A AF-2 double-point mutation in AF-2 of mouse ER was previously described (44), and the cognate mouse wild type ER expression vector was used as the control in Fig. 4AGo. The RIP140 expression vector was previously described (2), and maximal inhibition of synergy was observed with the 10 µg of expression vector used in all of the experiments presented here.


    ACKNOWLEDGMENTS
 
We wish to thank Malcolm Parker for providing us with the RIP140 expression vector and the wild type and L543A/L544A mouse ERs.


    FOOTNOTES
 
Address requests for reprints to: Fred Schaufele, Metabolic Research Unit, University of California, San Francisco, California 94143-0540.

This work was supported by Grant BE-195 from the American Cancer Society (to F.S.)

Received for publication February 13, 1997. Revision received May 14, 1997. Accepted for publication May 16, 1997.


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 MATERIALS AND METHODS
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