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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1211
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 1 239-247
Copyright © 2006 by The Endocrine Society

The Role of CBP/p300 Interactions and Pit-1 Dimerization in the Pathophysiological Mechanism of Combined Pituitary Hormone Deficiency

Ronald N. Cohen, Thierry Brue, Karuna Naik, Christine A. Houlihan, Fredric E. Wondisford and Sally Radovick

Sections of Pediatric and Adult Endocrinology, Department of Pediatrics and Medicine, University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Dr. Ronald Cohen, Section of Endocrinology, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC 1027, Chicago, Illinois 60637. E-mail: roncohen{at}medicine.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Combined pituitary hormone deficiency (CPHD) in humans is caused by mutations of pituitary-specific transcription factors such as Pit-1. Although many patients with CPHD have an autosomal recessive disorder caused by a Pit-1 DNA-binding mutation, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA.

Objective: The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding.

Design: DNA-binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W.

Results: In gel-shift studies using well-characterized DNA-binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes.

Conclusions: From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY-SPECIFIC TRANSCRIPTION factor-1 (Pit-1; also known as GHF-1) is a member of the POU transcription factor family, whose expression is restricted to the anterior pituitary (1). Pit-1 was the first pituitary-specific transcription factor identified, and it binds to AT-rich, cell-specific elements in the prolactin (Prl), GH, TSH ß-subunit, and Pit-1 genes (2, 3, 4). Pit-1 contains two protein domains, termed POU-specific (POUS) and POU-homeo (POUHD), which are both necessary for high-affinity DNA binding on these genes. Pit-1 usually binds to multiple sites on target genes, and dimerization of Pit-1 on DNA elements is important for high-affinity DNA binding (5, 6, 7). The third {alpha}-helices of both POUS and POUHD make the majority of contacts with the major grooves of DNA. Interestingly, Pit-1 forms dimers on DNA by interactions between the POUS domain of one molecule and the C terminus of the POUHD of the other molecule (8).

In addition to its important role in pituitary gene expression, Pit-1 is also essential for the development of certain anterior pituitary cells. Pit-1 protein is detected in somatotrophs and lactotrophs in the mouse preceding GH and Prl gene expression, suggesting that Pit-1 is the major cell-specific activator of hormone expression from these cell types (9). Pit-1 protein is also expressed in caudomedial thyrotrophs, although a second transient population of thyrotrophs is found in the rostral tip and appears to develop in the absence of Pit-1 expression (10).

Naturally occurring mutations in the Pit-1 gene have confirmed the developmental role of Pit-1 in the anterior pituitary. For example, the Jackson dwarf mouse has a gross structural alteration of the Pit-1 gene, and the Snell dwarf mice is homozygous recessive for a Pit-1 DNA binding mutation (11); both mouse models lack somatotroph, lactotroph, and thyrotroph cell populations and are referred to as having combined pituitary hormone deficiency (CPHD). Patients with CPHD caused by Pit-1 gene mutations have also been described (reviewed in Ref.12). The inheritance pattern and phenotypic presentation are quite different among these patients, probably reflecting the location of and functional perturbations caused by the Pit-1 mutation. In many cases, patients with CPHD have an autosomal recessive disorder caused by a truncated Pit-1 molecule that is unable to bind to DNA. However, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA. The R271W mutation is the most common of this type and has been described in several unrelated patients of different ethnic backgrounds (13, 14). The K216E mutation is another example of a Pit-1 molecule with normal or enhanced DNA-binding characteristics (15). Both the R271W and K216E mutations are inherited as autosomal dominant disorders. Several families with autosomal recessive CPHD caused by Pit-1 mutations with normal DNA-binding characteristics have also been described, including those that harbor the A158P (16) and F135C (17) mutations. Finally, the R143Q mutation is reported to cause autosomal recessive CPHD, but the DNA-binding characteristics of this mutant Pit-1 are unknown (18).

Pit-1 is reported to regulate the GH, Prl, TSH ß-subunit, and Pit-1 genes by binding to response elements present in their promoter regions and recruiting coactivator proteins to the transcriptional complex. The cAMP response element-binding protein (CREB)-binding protein (CBP)/p300 coactivator class has been implicated as important in this process (19, 20). We wanted to determine whether defective interactions between CBP/p300 and mutant Pit-1 molecules might be an important mechanism by which naturally occurring mutations of Pit-1 cause CPHD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pit-1 structure and mutations

The Cn3D application was used to generate a three-dimensional structure of Pit-1 dimerized on a Prl-1P element using the Molecular Modeling DataBase (www.ncbi.nlm.nih.gov/Structure/MMDB).

Cell transfections

Luciferase reporter constructs containing the human GH (–195/+5 bp) and Prl (–164/+34 bp) reporters were used in transfection assays. Human mutations of Pit-1 were introduced into the rat Pit-1 cDNA tagged at the 5' end with hemagglutinin. These cDNAs were inserted into pSG5, a mammalian expression vector. Mouse CBP and p300 cDNAs were inserted into pCDNA3.1 for use in expression studies. For CBP/p300 RNA interference, four specific oligonucleotide pairs directed at either CBP or p300 were generated. The oligonucleotide pair consisted of an inverted repeat of a 19-nucleotide-long sequence separated by a 9-bp spacer and a transcription termination signal of 6 thymidines on the 3' end. A BamHI site was included on the 5' end, and a HindIII site on the 3' end (Integrated DNA Technology, Coralville, IA). The annealed oligonucleotides were cloned into the BamHI and HindIII sites of the pSilencer 2.1-U6 hygro vector (Ambion, Austin, TX).

Cell culture and transient transfection assay

GH3 and 293T cells were maintained in DMEM supplemented with L-glutamine, 10% fetal calf serum, 100 µg/ml penicillin, and 0.25 µg/ml streptomycin. Transient transfections were performed in subconfluent six-well plates; 1.0 µg reporter construct with 75 ng expression vector or same amount of pSG5 vector alone were added to each well using Lipofectamine Plus (Invitrogen Life Technologies, Inc., Carlsbad, CA). For studies involving either RNA interference (RNAi) or CBP/p300 expression vectors, 50 ng of the respective vector was also used. After 18 h, cells were harvested and assayed for luciferase activity using a Lumat LB 9507 Luminometer (Berthold Technologies, Oak Ridge, TN).

EMSA

Proteins for EMSAs were obtained by in vitro transcription and translation using the T7 TNT-coupled reticulocyte lysate system (Promega Corp., Madison, WI). Translation using [35S]methionine was followed by SDS-PAGE analysis and visualization after autoradiography to assess the sizes of translated proteins and to verify that they are expressed as full-length proteins. The upper strand oligonucleotides used for EMSAs are as follows: GH-1 (5'-CTATACATTTATTCATGG-3'), GH-1mut (5'-CTGGACATTTATTCATGG-3'), Prl-1P (5'-ATATATATATTCATGA-3'), and Prl-1Pmut (5'-ATGGATATATTCATGA-3') (mutations are underlined). In vitro translated proteins were preincubated with 3 x 106 cpm 32P-labeled probe for 20 min at room temperature in 10 µl binding buffer containing 20% glycerol, 20 mM HEPES (pH 7.6), 50–140 mM KCl, 1 mM dithiothreitol, 1 µg polydeoxyinosinic-polydeoxycytidylic acid, and 0.1 µg salmon sperm DNA. The protein-DNA complexes were resolved on 4% nondenaturing polyacrylamide gels with 0.5x TBE (0.045 M Tris-borate and 0.001 M EDTA) running buffer and visualized after autoradiography.

Pull-down assay

Wild-type (WT) CBP [amino acids (aa) 1–450] or Pit-1 protein was fused in-frame and downstream of glutathione-S-transferase (GST) in pGEX4T2 vector (Pharmacia Biotech, Inc., Piscataway, NJ). Recombinant proteins were synthesized in JM109 bacteria and purified on glutathione-Sepharose resin under nondenaturing conditions. GST proteins were analyzed on SDS-PAGE before use in the assay. 35S-Labeled WT or mutant Pit-1 constructs were generated in an in vitro transcription/translation system (TNT, Promega Corp.) and exposed to GST-CBP (1–450 aa). 35S-Labeled CBP constructs [aa 1–450 (C/H1) or 1458–1891 (C/H3)]were generated in an in vitro transcription/translation system (TNT, Promega Corp.) and exposed to the indicated GST Pit-1 protein. After extensive washing with 150 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 at 4 C, the proteins trapped by the resin were resolved on SDS-PAGE and detected by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutant Pit-1 molecules used for study

Five human Pit-1 molecules were chosen for study based on their predicted or reported normal DNA-binding characteristics. Three of the mutations are located within the POUS domain (F135C, R143Q, and A158P; Fig. 1AGo), and two of the mutations are located within the POUHD domain (K216E and R271W; Fig. 1AGo). Shown in Fig. 1BGo are the locations of these mutations (in green) on the published crystallographic structure of Pit-1 (in purple), complexed to the Prl 1-P element (in black). WT Pit-1 binds as a dimer (the additional Pit-1 is noted in yellow), where the POUS and POUHD of one Pit-1 molecule bind to perpendicular faces of the DNA element (8). None of the mutations affect aa residues, which make direct DNA contacts based on the crystal structure. One of the mutations (R271W), however, would alter an arginine residue predicted to form a hydrogen bond with a glutamine residue in the POUS domain of the second Pit-1 molecule (in yellow).



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  		FIG. 1. Pit-1 mutations used in this study and structure of Pit-1 bound to a Prl-1P element. A, The inheritance pattern and location of the mutation pattern used in this study. AR, Autosomal recessive; AD, autosomal dominant. B, Structure of Pit bound to the Prl-1P element generated using Cn3D and the Molecular Modeling DataBase. One Pit-1 molecule is colored purple, and the other is yellow in the structure. The location of Pit-1 mutations is indicated in green and annotated. POUS1 and POUS2, POU-specific domains of the first and second Pit-1 molecules, respectively. POUHD1 and POUHD2, POU homeo domains of the first and second Pit-1 molecules, respectively.

 
DNA binding characteristics of the mutant Pit-1 molecules

To characterize the DNA binding of these Pit-1 mutants, gel-shift studies were performed with radiolabeled DNA elements from the GH (GH-1) and Prl (Prl-1P) genes. As shown in Fig. 2AGo, WT Pit-1 bound to both elements as either a monomer or a dimer. We formally established the migration positions of these complexes using a mutation of either element that abolished the homeodomain binding site of one of the Pit-1 molecules (GH-1 Mut and Prl-1P Mut). Dimer formation by WT Pit-1 on the GH-1 element was greater than that on the Prl-1P element. Consistent with previous reports (16, 17), the F135C and A158P mutant Pit-1 molecules displayed near-normal DNA-binding characteristics, although dimerization on both elements appeared somewhat reduced relative to that by WT Pit-1. The R143Q mutant had no detectable DNA binding on either element, even though it was synthesized equally with the other Pit-1 mutants in an in vitro transcription/translation system (Fig. 2BGo), suggesting that this mutation must in some way affect the structure of the POUS domain.



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  		FIG. 2. EMSA on Pit-1 bound to GH and Prl response elements. In vitro transcribed and translated WT and mutant Pit-1 were exposed to radiolabeled elements, as shown at the bottom. The locations of Pit-1 monomeric and dimeric binding are indicated to the left. HD, Homeodomain; S, POU-specific domain.

 
Interestingly, the K216E mutant displayed markedly enhanced DNA binding, favoring dimer formation on both elements. The enhanced dimer formation was graphically illustrated on the Prl-1P Mut element, where dimerization occurred even in the presence of homeodomain DNA mutation. K216E, however, was defective in binding to the GH-1 Mut site. In contrast, the R271W mutant bound to both elements only as a monomer, which was confirmed using the mutant elements.

Functional properties of the mutant Pit-1 molecules

The activities of WT and mutant Pit-1 molecules were evaluated using a rat pituitary cell line expressing both Prl and GH (GH3 cells). Expression vectors containing either WT or mutant Pit-1 cDNA were transfected into GH3 cells, and their activities were evaluated on both a GH and a Prl reporter. As shown in Fig. 3AGo, overexpression of WT Pit-1 increased expression approximately 2-fold over either reporter. In contrast, none of the mutant Pit-1 expression vectors was able to activate these reporters, indicating that all five of the mutant cDNAs were inactive in GH3 cells.



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  		FIG. 3. Functional assays of the GH and Prl reporters in a rat pituitary cell line, GH3. GH3 cells were transfected with Pit-1 expression vectors (A) or with CBP/p300 RNAi vectors (B) and the indicated reporter construct. In the RNAi experiment, 100 ng total small interfering RNA (siRNA) was transfected; when both CBP and p300 constructs were cotransfected, 50 ng each was used to keep the total amount of constructs stable. Relative luciferase activity is indicated as the mean ± SEM of at least three separate experiments performed in triplicate. In the lower panels, activity is corrected to 100% for the Scr vector transfection. All mutant Pit-1 exhibit decreased transactivation compared with WT Pit-1 (P < 0.01, by ANOVA). Luciferase activity in the presence of CBP or p300 siRNA was significantly different from that with scrambled siRNA (P < 0.05 by ANOVA), but was not significantly different from that with combined siRNA constructs. Vector, Empty vector transfection.

 
The CBP/p300 class of coactivator proteins is implicated in mediating Pit-1 transactivation (19, 20). To test whether these proteins mediate Pit-1 transactivation of the GH and Prl genes in GH3 cells, RNAi vectors directed at either p300 or CBP were used in transfection assays. These constructs were chosen from four constructs directed at either CBP or p300 based on their ability to significantly decrease either CBP or p300 expression without affecting the expression of the other coactivator (Deplewski, D., and F. Wondisford, submitted for publication). As shown in Fig. 3BGo, either the CBP or p300 RNAi vector reduced WT Pit-1 transactivation in GH3 cells relative to transfection of a scrambled control RNAi vector (Scr). Of note, the function of both CBP and p300 RNAi vectors together was similar to that observed with either vector alone. These data indicate that the CBP/p300 class of proteins plays a crucial role in controlling GH and Prl gene expression in GH3 cells, but they appear to have overlapping effects.

Binding of CBP to mutant Pit-1 molecules

We have previously demonstrated that both the C/H1 and C/H3 domains of CBP bind to Pit-1 (20). These highly conserved domains between CBP and p300 are so named because they are rich in cysteine and histidine. Of these domains, the C/H1 domain displayed the highest avidity toward Pit-1 and for this reason was chosen for use in the protein-binding assay. The aa 1–450 of Pit-1 were fused in-frame and downstream of GST. GST fusion proteins were expressed in Escherichia coli and purified using a glutathione resin. Equal amounts of GST-CBP were then exposed to increasing amounts of 35S-labeled WT and mutant Pit-1 synthesized in a coupled transcription/translation reticulocyte lysate system. Equivalent amounts of WT and mutant Pit-1 were used in these experiments, as indicated by SDS-PAGE (data not shown). As shown in Fig. 4Go, WT Pit-1 bound strongly to GST-CBP (1–450 aa) but not to GST alone demonstrating specificity of binding. Three of the Pit-1 mutants had normal or near normal binding (F135C, R143Q, and R271W) to CBP 1–450. In contrast, both the A158P and K216E mutants displayed decreased binding. The decrease in binding in three separate experiments averaged 55% for the A158P mutant and 87% for the K216E mutant (P < 0.05 for both mutants vs. WT Pit-1).



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  		FIG. 4. GST pull-down assay of WT and mutant Pit-1 molecules. An increasing amount of 35S-labeled Pit-1 (1, 2, and 4 µl) was exposed to GST or GST-CBP (aa 1–450) proteins bound to resin. After extensive washing, bound Pit-1 was eluted and resolved by SDS-PAGE. IP, Input radiolabeled Pit-1 (4 µl).

 
We next performed this assay using GST-Pit-1 fusion proteins and radiolabeled CBP fragments. The N terminal fragment of CBP containing the C/H1 domain (CBP 1–450) and C-terminal fragment containing the C/H3 domain (CBP 1677–2441) were in vitro translated and 35S labeled in a reticulocyte lysate system; both domains are known to bind to Pit-1 (20). They were used in a protein-binding assay using WT and K216E mutant Pit-1 expressed as GST fusion proteins. As shown on Fig. 5AGo, both WT and K216E mutant Pit-1 were equally expressed and bound to the glutathione resin generating a 62-kDa fusion protein. Unlike WT Pit-1, however, the K216E mutant Pit-1 protein was unable to bind to either the C/H1 or C/H3 domain (Fig. 5BGo), confirming results obtained in Fig. 4Go.



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  		FIG. 5. GST pull-down assay of radiolabeled CBP molecules. Either 35S-labeled C/H1 or C/H3 domain of CBP (4 µl) was exposed to GST or GST-Pit-1 (WT and K216E mutant) proteins bound to resin. After extensive washing, bound CBP was eluted and resolved by SDS-PAGE. A, Coomassie blue-stained SDS-PAGE gel showing WT and K216E GST-Pit-1 fusion proteins. B, Eluted radiolabeled CBP proteins. To the left of the gel, radiolabeled input amounts are shown. UP, Unprogrammed in vitro transcription/translation assay.

 
CBP/p300 transactivation of mutant Pit-1 molecules

To characterize the role of CBP and p300 in Pit-1-dependent activation of the GH and Prl gene, transient cotransfection experiments were performed in a Pit-1 deficient cell line (293T cells) using the proximal promoter regions of the human GH and Prl promoters. Both promoters contain well-defined Pit-1 binding elements (3, 21). As shown in Fig. 6AGo, WT and mutant FLAG-tagged Pit-1 molecules were equally expressed in 293T cells after transfection of expression vectors containing Pit-1 cDNAs. Figure 6BGo demonstrates, however, that CBP and p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of the GH reporter gene compared with wild-type Pit-1 cotransfected with either CPB or p300, respectively. This was most prominent for the R143Q mutant (DNA-binding mutant) and least prominent with the F135C mutant. Similar findings were observed on the Prl reporter (Fig. 6CGo), with all mutant Pit-1 constructs exhibiting decreased transcriptional activity in the presence of CBP compared with WT Pit-1. All mutants, except F135C, also exhibited decreased transcriptional activity with p300 on the Prl reporter. In data not shown, CBP and p300 were unable to significantly activate these promoters in the absence of cotransfected WT Pit-1. These functional data suggest that defects in the action of CBP/p300 on Pit-1 may be important in the pathogenesis of CPHD.



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  		FIG. 6. Functional assays of the GH and Prl reporters in a Pit-1-deficient cell line, 293T. A, Western blot for FLAG-tagged Pit-1 proteins showing equivalent expression in 293T cells. The lower 293T gel shows a blot for actin as a loading control. B, The indicated reporter construct and Pit expression vector were transfected with either CBP or p300 expression vector. Relative luciferase activity is indicated as the mean ± SEM fold of at least three separate experiments performed in triplicate compared with activity of the reporter without Pit-1 or CBP/p300 transfection. After cotransfection with CBP or p300, all mutants exhibited statistically decreased levels of transactivation compared with WT Pit-1 (cotransfected with CBP or p300, respectively; P < 0.05) with the exception of the R135C mutant cotransfected with p300 on the Prl reporter. Vector, Empty vector transfection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have demonstrated the importance of both Pit-1 dimerization and CBP/p300 interactions in Pit-1-dependent gene activation. Using Pit-1 mutants defective in either DNA or CBP/p300 binding, we have been able to implicate both in the pathophysiological mechanism of CPHD.

Clearly, DNA binding is essential for Pit-1 action in the pituitary. Mice containing a point mutation in the DNA-binding domain (Snell dwarf) lack somatotrophs, lactotrophs, and thyrotrophs and have the human equivalent of CPHD. The Snell dwarf mutation (W261C) is contained with the third {alpha}-helix of the POUHD, and V260 and C263 surround this mutation and hydrogen bond with POUHD nucleotides on the DNA element (8). As predicted, the Snell mutant is unable to bind to DNA (11). Several human missense mutations of the Pit-1 gene have been described (K145X, R172X, and E250X), resulting in truncated proteins that all lack this critical third {alpha}-helix of the POUHD (12).

Study of the R271W mutation indicates that the nature of Pit-1 complex on DNA in important for its function. The R271W mutation was predicted to affect Pit-1 dimerization, because this residue makes an important POUs domain contact in the dimer structure. We show for the first time that this is indeed the case for a Pit-1 gene mutation. Based on gel-shift studies, however, R271W interacts quite well with either the GH-1 or Prl-1P element, but only as a monomer. This preserved DNA binding may underlie its ability to act as a dominant-negative mutation in humans (13), because it would be predicted to block dimer formation on Pit-1 target gene elements. Given the normal interaction between R271W and CBP in the protein binding assay, but the defective transactivation by CBP in the cotransfection assay, dimer formation on target elements is essential. Interestingly, most of the other mutations have impaired (R143Q) or normal (F135C and A158P) binding to DNA and exhibit an autosomal recessive pattern of inheritance. Surprisingly, the other dominant mutant studied, K216E, exhibits enhanced binding to DNA; this may explain its dominant activity, because K216E would be expected to bind preferentially to DNA over the WT protein in the heterozygous state.

Dimer formation on Pit-1 elements is not sufficient for activation, as shown by the K216E mutant. The mutant displays enhanced dimerization on either the GH-1 or Prl-1P element; it was even able to dimerize on the Prl-1P mut site, suggesting that this mutation must enhance Pit-1 protein-protein interactions. Interestingly, and in contrast to the R271W mutation, the K216E mutation was unable to bind to the GH-1 mut element as a monomer. This finding suggests that K216E may be forming dimer in solution. Even with enhanced DNA binding, however, the K216E mutant was still defective in CBP/p300 transactivation on the GH and Prl reporters. This is probably due to its inability to bind to CBP, as demonstrated in protein-binding assays. Like the K216E mutation, the A158P mutation displayed a reduction in binding to CBP and transactivation of target genes by CBP/p300. Given the enhanced or near-normal DNA-binding characteristics of the K216E and A158P mutants, they are likely to cause CPHD due to their inability to interact with the CBP/p300 coactivator class. Moreover, the enhanced dimer formation by K216E may prevent its interaction with other transcription factors, such as the retinoic acid, as previously described (15). The K216E and A158P mutations begin to define a surface for coactivator interactions with Pit-1 that is above the plane of DNA binding. It should be noted that Pit-1 has also been shown to interact with nuclear corepressors [N-CoR (19), GATA2 (22), and CCAAT/enhancer-binding protein {alpha} (23)]. Whether these proteins interact with Pit-1 in a fashion similar to CBP/p300 or play some role in the pathogenesis of CPHD has yet to be determined.

The R143Q DNA-binding mutant illustrates the importance of Pit-1 itself in the ability of CBP/p300 to activate pituitary target genes. Clearly, the R143Q mutation is unable to bind to DNA. Because this residue does not make direct DNA contacts in the crystal structure (8), it must change the conformation of POUs in some significant way. In support of this finding, a previous study designed to define the DNA-binding domain of Pit-1 created a R143G mutation that abolished Pit-1 DNA binding (24). Although R143Q can bind CBP/p300 in GST interaction assays, cotransfection of either CBP or p300 expression vectors was unable to active either the GH or Prl reporter when R143Q was employed. These results indicate that CBP/p300 binding to Pit-1 is essential for CBP/p300 action on these target genes.

Previous studies have suggested that the F135 mutant exhibits decreased transactivation on Prl and GH genes in HeLa cells (17). Interestingly, our studies show that the F135C mutant has near-normal DNA-binding characteristics and recruits CBP. However, F135C transactivation is partially impaired when CBP is overexpressed in 293T cells. Although these data do not fully explain the F135C defect, they suggest one contributory factor. Interestingly, F135C is fully inactive in GH3 cells. These data suggest that interaction of F135C with a GH3-specific protein may be impaired. Additional studies are necessary to define the full mechanism mediating defective F135C-mediated transactivation.

From these studies, we suggest several important mechanisms important in the pathogenesis of CPHD. 1) Pit-1 dimerization is necessary for activation of Pit-1 target genes, because the dimerization-defective R271W mutant is unable to normally activate target genes even though it binds normally to CBP/p300. 2) Pit-1 dimerization is not sufficient for target gene activation unless CBP/p300 is also bound to Pit-1, because the dimerization-enhanced (K216E) or dimerization-sufficient (A158P) mutant Pit-1 molecules are unable to bind normally to CBP/p300 and activate target genes. 3) CBP/p300 mediate activation of GH and Prl genes by binding to Pit-1 on DNA, as illustrated by the DNA-binding mutant (R143Q) that normally interacts with CBP/p300, but does not transactivate.


   Footnotes
 
Current addresses for T.B.: Laboratory Interactions Cellulaires Neuro-Endocriniennes Unité Mixte de Recherche 6544, Institut Fédératif de Recherche Jean-Roche, 13916 Marseille Cedex 20, France.

Current address for C.A.H.: Studley Road, Heidelberg, 3084 Victoria, Australia.

Current address for F.E.W.: Departments of Pediatrics and Medicine, Johns Hopkins Medical Institutes, Baltimore, Maryland 21287.

Current address for S.R.: Department of Pediatrics, Johns Hopkins Medical Institutes, Baltimore, Maryland 21287.

First Published Online November 1, 2005

Abbreviations: aa, Amino acid; CBP, cAMP response element-binding protein (CREB)-binding protein; CPHD, combined pituitary hormone deficiency; GST, glutathione-S-transferase; Pit-1, pituitary-specific transcription factor-1; Prl, prolactin; RNAi, RNA interference; Scr, scrambled RNAi control vector; WT, wild type.

Received May 31, 2005.

Accepted October 21, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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