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Previous Article | Next Article 
Molecular and Cellular Biology, September 2006, p. 6535-6546, Vol. 26, No. 17
0270-7306/06/$08.00+0 doi:10.1128/MCB.00267-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Yugong Ho,1
Stephen A. Liebhaber,1,2 and
Nancy E. Cooke1,2*
Departments of Genetics,1 Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania 191042
Received 13 February 2006/ Returned for modification 22 March 2006/ Accepted 20 June 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH promoters from all species studied contain a pair of conserved Pit-1 binding sites within the first 200 bp of 5'-flanking sequence (5, 22). It was therefore remarkable to observe that the promoter of the hGH-N gene, containing the conserved pair of Pit-1 binding sites, was not sufficient to activate hGH-N transgene expression in the mouse pituitary. An hGH-N transgene with as much as 7.5 kb of 5'-flanking sequence was either not expressed or expressed only at very low levels with loss of somatotrope specificity (16). These data suggested that remote regulatory determinants are required to establish a transcriptionally active chromatin domain at the hGH locus during pituitary ontogeny. A search for distal regulatory elements revealed a set of four DNase I-hypersensitive sites (HS) located –14.5 to –32 kb relative to the hGH-N gene promoter in pituitary chromatin (16). The full set of HS is required for the robust tissue-specific, position-independent, copy number-dependent expression of the genes of the hGH cluster in transgenic mice, establishing these determinants as the hGH locus control region (LCR) (16, 37). Further analysis of the hGH LCR identified the pituitary-specific HSI at –14.5 kb (Fig. 1A) as the major determinant of pituitary hGH-N transgene activation in vivo. Linkage of the hGH-N gene with its 500-bp proximal promoter to a 404-bp LCR subfragment encompassing HSI demonstrated high-level, position-independent expression in transgenic mouse somatotropes (4, 34). A search for functional trans-factor binding sites in this region identified an array of three clustered Pit-1 binding sites that mediate HSI action in vivo (34). Thus, the three Pit-1 binding sites at HSI were proposed to play a central and essential role in hGH LCR function.
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The basis for the distinct functions of the Pit-1 binding sites at HSI and at the hGH-N promoter remains unclear. However, a special characteristic of Pit-1 is its ability to bind to a range of DNA sequences, resulting in a relatively loose binding site consensus sequence. Pit-1 accommodates these different binding sequences by altering its conformation in order to make contacts between DNA-binding domain amino acids and specific bases (14, 32). This structural flexibility provides a mechanism for allosteric regulation of Pit-1 by its DNA binding site. In support of this model, changes in Pit-1 topology, with selective recruitment of specific cofactors by different Pit-1-DNA complexes, appear to play a role in defining the selective activation of GH and Prl by Pit-1 in somatotropes and lactotropes, respectively (32). In the present report, we identify a single base that is distinct between promoter-associated Pit-1 sites and the three Pit-1 sites within HSI. We test whether this divergent base dictates the locus-activating function of the hGH LCR in vivo. The data presented support this model and complement a growing body of evidence for the allosteric control of transcription factor function by DNA recognition sequences.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methylation interference assay.
The sequences of synthetic oligonucleotides used as probes in methylation interference assays are shown in Fig. 2C. To generate double-stranded probes, one strand was 5' end-labeled with [
-32P]ATP and T4 polynucleotide kinase and annealed to the unlabeled complementary strand. Assays were performed as described previously (3) with recombinant rPit-1. Conditions for the binding reactions with methylated probes and the gel separation of Pit-1-bound and unbound probe fractions were as described for the electrophoretic mobility shift assay (EMSA) (below).
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-32P]ATP and T4 polynucleotide kinase. Binding reactions (20 µl) each contained 200 ng (8 pmol) of labeled DNA probe in buffer composed of 10 mM HEPES (pH 7.9), 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT), and 10% glycerol. Approximately 200 ng (4 pmol) of purified recombinant rPit-1 was added last, and the mixture was incubated for 30 min at room temperature. One microliter each of a protease dilution series was added to the replicate binding reactions to final concentrations of 0.2, 1, 5, 25, and 125 ng/µl. Proteases were purchased from Sigma. Proteolysis reactions were incubated for 2 min at room temperature and stopped with 2 µl of 1-g/liter soybean protease inhibitor (Sigma). Samples were resolved on a 5% nondenaturing polyacrylamide (19:1 acrylamide/bisacrylamide) gel in 2x Tris-glycine buffer, dried, and visualized by autoradiography.
Electrophoretic mobility shift assay (EMSA).
For EMSA, double-stranded oligonucleotide probes were 5' end labeled with [-32P]ATP and T4 polynucleotide kinase. Binding reactions (20 µl) contained 20 ng of DNA probe in buffer composed of 10 mM HEPES, pH 7.9, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, and 0.3 g/liter bovine serum albumin (BSA). Purified recombinant rPit-1 was added last, and the mixture was incubated for 20 min at room temperature. Samples were resolved on a 5% nondenaturing polyacrylamide (19:1 acrylamide/bisacrylamide) gel in 2x Tris-glycine buffer, dried, and visualized by autoradiography.
Construction of recombinant plasmids. The 3GH1(T/A)-hGH and PitABC(A/T)-hGH plasmid constructs were prepared by replacing the wild-type 3GH1 and PitABC Pit-1 site trimer arrays in the previously described constructs 3GH1-hGH and PitABC-hGH (33) with duplex synthetic oligonucleotides comprised of the 3GH1(T/A) and PitABC(A/T) Pit-1 site trimer arrays containing single base changes in the Pit-1 elements (see Fig. 5A). The modified arrays maintain the same orientation of the Pit-1 binding sites relative to the hGH-N gene as the original constructs.
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Generation of transgenic mouse lines.
The 3GH1(T/A)-hGH and PitABC (A/T)-hGH constructs were released from vector sequences of cesium chloride gradient-purified plasmid DNA with BssHII and purified through an Elutip (Schleicher and Schuell). The DNA was diluted to 2 ng/µl in 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA and was microinjected into the pronucleus of C57Bl/6J x SJL mouse zygotes. Founders in litters yielded from implanted embryos were identified by dot blot analysis of tail DNA using as probe a 1.37-kb SmaI fragment from hGH-N (–494 to +876 relative to the initiation site of hGH-N transcription). The presence of intact transgenes in the resulting mouse lines was confirmed by Southern blot analysis of genomic DNA prepared from tail biopsies of F1 animals, digested with HindIII using standard methods and the above hGH-N fragment probe. The single HindIII site within the transgene resulted in detection of a 
2.7-kb fragment yielded by digestion of the tandem array of transgene copies. After stripping the hGH-N probe from the membrane, a mouse zeta-globin fragment probe was hybridized to control for DNA loading.
RT-PCR assay for hGH-N and mGH mRNAs. Pituitary RNA was prepared from F1 animals with Trizol reagent (LifeTechnologies). Levels of hGH-N and mGH mRNAs were determined by coamplification of hGH-N and mGH mRNAs by reverse transcription-PCR (RT-PCR) using primers complementary to both sequences as described previously (16). The amplified mGH and hGH-N cDNAs were distinguished by unique restriction sites, resolved on a 5% nondenaturing polyacrylamide (19:1 acrylamide/bisacrylamide) gel, and visualized by autoradiography.
Immunofluorescent staining. A monoclonal antibody specific to hGH (MAb9) has been described already (4). Monkey anti-rGH, which cross-reacts with mouse GH (mGH) but not hGH, was obtained from the National Hormone and Pituitary Program. Sections (4 to 5 µm) of paraffin-embedded adult pituitaries were dewaxed and rehydrated, followed by microwave antigen retrieval in 10 mM citric acid monohydrate, pH 6.0, and washing in phosphate-buffered saline (PBS). Sections were blocked with protein blocking reagent (Vector Laboratories) and incubated with the primary anti-hGH and anti-mGH antibodies (1:2,000 in PBS containing 10% BSA and 10% Triton X-100) simultaneously at 4°C overnight, followed by washing in PBS. Sections were next incubated with Cy2-conjugated goat anti-mouse immunoglobulin G and Cy3-conjugated goat anti-human immunoglobulin G (Jackson ImmunoResearch) secondary antibodies simultaneously at room temperature for 2 h (1:200 in phosphate-buffered saline containing 10% BSA and 10% Triton X-100), followed by washing in PBS. Slides were mounted in 50 mM Tris, pH 7.5, 80% glycerol and visualized by fluorescence microscopy with the appropriate filters. Images were captured with a color charge-coupled device camera and Photoshop (Adobe) software.
Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assays were performed as previously described (11). The antibody specific for CBP (sc-369) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Normal rabbit serum (Santa Cruz) was used as control for the ChIP assays. The input and bound DNA fractions were amplified by PCR. The sequences of the primers are the following: HSV, 5'-CTTGCCCAGTCCTCACACTT-3' and 5'-CTGAGGCTTCTGTCCTCCTT-3'; HSI, 5'-CCAAGCCTTTCCCAGTTATAC-3' and 5'-GATCTTGGCCTAGGCCTCGGA-3'; hGH-N promoter, 5'-CAGGGCTATGGGAGGAAGAGCTT-3' and 5'-CTTCTCTCCCACTGTTGCCC-3'; mGH promoter, 5'-GTGGGATGTTGCATAACC-3' and 5'-GGTCGACCCCAAGGCTCC. PCR products were resolved on 1.5% agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled probes. The probes were generated by PCR using the same primers. The signals were quantified by PhosphorImager analysis. A series of dilutions of the chromatin input fraction were used to determine the linear region for the PCR amplification at each site as described previously (11). The signals of bound fractions were normalized to the signals of 0.03% of the input and corrected for background by comparison with the ratio obtained with normal rabbit serum.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methylation interference assays were performed to determine whether the A specific to the HSI Pit-1 binding sites makes contact with Pit-1. The assays were carried out on DNA fragments encompassing a representative promoter Pit-1 site (rGH1) and on each of the individual HSI Pit-1 elements (Pit-1A, Pit-1B, and Pit-1C) (Fig. 2A and B). The results of mapping both strands are summarized, with critical purines in boldface (Fig. 2C). This analysis demonstrated that methylation of the HSI-specific A decreased Pit-1 binding to all three of the HSI Pit-1 sites: Pit-1A, Pit-1B, and Pit-1C (Fig. 2A). The data also demonstrated that each binding site has a unique pattern of Pit-1-purine contacts. This diversity reflects the unusual level of sequence variation among functional Pit-1 binding sites (1). The extent of DNA contacts was, however, similar among the four binding sites, suggesting that Pit-1 is binding as a dimer to the HSI Pit-1 elements as has been described previously for the promoter GH1 element (13, 32). Of note, methylation of the A in the GH1 element that pairs with the promoter consensus-specific T did not affect Pit-1 binding. This is consistent with the crystallographic data for the Pit-1/GH1 element complex, which showed that this adenine does not contact Pit-1 (32). It should be noted that only purines are methylated in this assay, and thus additional protein-base interactions remain indeterminate. These observations suggest that the unique sequences of the hGH LCR HSI Pit-1 binding sites may directly affect the conformation of the cognate Pit-1 homodimer complexes.
Protease analysis of Pit-1 complexes indicates that Pit-1 adopts different conformations when bound to GH promoter and HSI binding sites. The possibility that Pit-1/DNA complexes formed on promoter-associated and HSI-associated Pit-1 sites differ in their conformations was explored by a proteolytic clipping bandshift assay (PCBA). Limited protease digestion of Pit-1 complexed with 32P-labeled DNA was followed by resolution of digestion intermediates retaining DNA binding by electrophoretic mobility on a native gel. Differences in the pattern and concentrations of digestion products reflect distinct configurations of the protein bound to corresponding DNA binding sites. PCBA has been used successfully in prior studies to distinguish complexes containing different octamer sequence binding (Oct) proteins in B cells (31). It has also been applied to distinguish the conformations of functionally distinct PRMT1 complexes in yeast (38) and to reveal different conformations of the transcription factor AP1 when bound to functionally distinct binding sites in the rat malic enzyme and collagenase-1 gene promoters (2, 36).
PCBA was performed on complexes of Pit-1 bound to the promoter-associated and HSI-associated Pit-1 binding sites using three proteases with distinct cleavage site specificities (Fig. 3). Clipping of the GH1 Pit-1 complex with proteinase K generated six major digestion intermediates (Fig. 3A, lanes 1 to 7). Parallel analysis of the Pit-1 A complex (Fig. 3A, lanes 8 to 14) generated intermediates 1 and 2, but it did so at a lower intensity than that of the GH1 complex (compare lane 11 with lane 4). Intermediate 3 was not produced at all, intermediate 4 was faint (compare lane 12 with lane 5), and products 5 and 6, seen clearly with the GH1 Pit-1 complex, were replaced by smeared products (compare lanes 12 through 14 with lanes 5 through 7). Clipping of the HSI Pit-1B element failed to generate intermediates 1 through 4. Complexes 5 and 6 were replaced by diffuse smears similar to those generated from the HSI Pit-1A complex. Clipping of the HSI Pit-1C complex yielded a pattern of digestion intermediates nearly identical to that shown by the GH1 sequence, with the exception of the strong smears corresponding to complexes 5 and 6 as seen with HSI Pit-1A and Pit-1B complexes. These proteinase K data revealed that each of the studied Pit-1 complexes demonstrates a unique proteolytic clipping pattern. Such variation is consistent with allosteric effects of the divergent binding sites on the bound Pit-1 protein.
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A thymine-to-adenine mutation within the promoter Pit-1 element alters the conformation of bound Pit-1. The impact of the T/A difference between the promoter-associated and HSI-associated Pit-1 binding sites was directly addressed by introducing a single-base T-to-A alteration in the GH1 sequence. PCBA mapping was carried out to determine whether this substitution had an impact on the conformation of the Pit-1 complex. The analysis revealed that the T-to-A transversion within the promoter GH1 Pit-1 element altered the chymotrypsin digestion pattern of the new Pit-1-DNA complex compared to that of the original complex (Fig. 4A). Most notably, the relative concentration of intermediate 5 was markedly increased in the mutant complex compared to that of the wild-type complex (compare lanes 11 to 13 with lanes 4 to 6). The T-to-A alteration also affected the pronase E digestion profile of the GH1 element-Pit-1 complex (Fig. 4B). Intermediate product 5 produced in the wild-type GH1 complex (lane 5) was absent from the digestion profile of the mutant GH1 complex (lane 12). Digestion product 6 was observed only in the mutant complex (compare lane 12 to lane 5). The assays were repeated to evaluate the reproducibility of the results, which were consistent between experiments. These data suggest that the T-to-A mutation in the promoter-associated Pit-1 site alters the conformation of the Pit-1 complex, as reflected by the changes in protease accessibility in the bound Pit-1.
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The function of the substituted Pit-1 trimer arrays was next assessed in a conventional cell transfection study. The 3GH1(T/A) and PitABC(A/T) arrays were linked to the hGH-N gene with its contiguous 500 bp of 5'-flanking promoter sequences [constructs 3GH1(T/A)-hGH and PitABC(A/T)-hGH; Fig. 6A ]. Each of these recombinant genes was analyzed for hGH-N expression in transfected GHFT1 presomatotrope cells, which contain substantial levels of Pit-1 (20; our unpublished data). The two substituted Pit-1 arrays conferred an equivalent and modest enhancement of expression from the linked –0.5hGH gene. These effects were identical to the activities of the wild-type 3GH1 and PitABC arrays in this assay (Fig. 6B). Thus, the single A/T base substitutions within the GH promoter and LCR Pit-1 element arrays have no effect on their in vitro binding properties as assessed by EMSA, nor do they impact on the enhanced expression of a linked hGH-N gene as assessed in transiently transfected cells.
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The model showing that different coactivators are recruited to the Pit-1 complexes at the hGH promoter and HSI was tested by chromatin immunoprecipitation (ChIP) assays for CBP. The initial analysis was done on pituitary chromatin isolated from mice carrying an 86-kb human genomic transgene that encompasses the entire hGH LCR and hGH-N gene (hGH/P1 transgene [37]) (Fig. 9, top). Accurate expression of this hGH/P1 transgene has been previously validated (37). ChIP assays in two mouse lines carrying this transgene (lines 811D and 809F [37]) revealed robust CBP occupancy at the hGH-N promoter and at the endogenous mGH promoter (Fig. 9). In marked contrast, there was no evidence for CBP occupancy at HSI (Fig. 9). A second set of CBP ChIP assays was performed on pituitary chromatin from mice carrying the hGH/P1 transgene specifically lacking two essential Pit-1 binding sites at HSI [hGH/P1(
HSI) transgene; lines 960G and 969E] (11). This Pit-1 binding site deletion has been previously shown to result in loss of HSI formation, loss of histone acetylation throughout the LCR, loss of Pit-1 occupancy at the hGH-N promoter, and a dramatic repression of hGH-N expression (11). ChIP analysis revealed that HSI inactivation markedly repressed CBP occupancy at the hGH-N promoter. Because this loss of CBP occupancy was concomitant with the loss of hGH-N promoter Pit-1 occupancy (11), we conclude that CBP is selectively recruited to the hGH-N promoter Pit-1 complexes. In contrast, HSI-mediated LCR function is dependent on a distinct and as-yet unidentified Pit-1 coactivator complex (see Discussion).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hGH-N activation mechanism is specifically dependent upon the array of Pit-1 complexes at HSI. Three copies of the promoter GH1 Pit-1 recognition sequence are not able to recapitulate the consistent activation of an hGH-N transgene in pituitary somatotropes that is mediated by a synthetic array of the three HSI Pit-1 elements. This distinction in in vivo activity of HSI and GH promoter sequences was observed despite identical Pit-1 binding affinities and enhancer activities in transiently transfected cells (33). The activity of isolated HSI Pit-1 binding sites in these constructs strongly suggested that the observed selectivity relates to specific conformations of the Pit-1 complexes adopted upon binding to the unique recognition sequences at HSI.
Here we show that the nucleotide sequences of the HSI Pit-1 binding sites differ from the GH promoter Pit-1 sequences. Most notably, an adenine common to all three HSI elements contrasts with a thymine conserved at the corresponding position in the promoter Pit-1 elements (Fig. 1B). The methylation interference studies indicated that DNA contacts differed among the promoter and HSI Pit-1 complexes. Of interest, the adenine unique to the HSI Pit-1 elements is involved in the interaction with Pit-1 (Fig. 2). Results from the PCBA indicated that these recognition sequence differences can alter the conformation of the Pit-1/DNA complex. Thus, the functional distinction between the promoter and HSI Pit-1 elements may be related to critical differences in Pit-1 complex topology (Fig. 3). In support of this model, reciprocal exchange of the GH promoter-specific thymine and HSI-specific adenine interconverts their activities in a transgenic mouse model (Fig. 6, 7, and 8). This functional interchange is accompanied by a change in the conformation of the Pit-1/DNA complex in vitro (Fig. 4). Together these observations support a model in which the sequence of the Pit-1 binding site mediates allosteric specification of Pit-1 complex activity. The adenine-containing Pit-1 complex conformation appears to significantly contribute to the unique activities of the Pit-1 elements at HSI.
It should be noted that the proteolysis patterns obtained by PCBA were not identical among the HSI Pit-1 complexes. This lack of uniformity most likely reflects differences in the sequences of these sites beyond the conserved adenine. Such differences in overall Pit-1 conformation of the three HSI-derived site complexes is consistent with their differences in relative potency assessed in transgenic models (33, 34). The involvement of additional bases in the specificity of HSI Pit-1 complex activities is further suggested by the transgenic analyses. The HSI-specific adenine is not fully sufficient for characteristic HSI activity, as two of the six 3GH1(T/A)-hGH lines did not express the transgene. The reciprocal observation that the activity of the HSI Pit-1 element trimer is not completely abrogated by the A-to-T alteration also suggests the involvement of additional base pairs in the specificity mechanism. However, no additional sequences within the aligned binding sites perfectly segregate between the HSI and promoter Pit-1 elements, masking any obvious candidates. We conclude that the ability to alter the activity of Pit-1 elements in a transgenic system by a single base substitution, without affecting Pit-1 affinity, supports an allosteric model for the specificity of the hGH LCR HSI Pit-1 complexes.
The ability of Pit-1 to be allosterically controlled by DNA sequence is supported by crystallographic analyses of POU domain/DNA complexes (14, 18, 32) and can be attributed to the unique structural nature of the POU class of proteins. Pit-1 is a member of a family of transcription factors defined by a region of homology, the POU domain (for "Pit-1, Oct-1/2, Unc-86"). The POU domain is a bipartite DNA binding domain comprised of an amino-terminal 75-amino-acid POU-specific domain (POUS) tethered by a disordered linker of variable length and composition to a carboxy-terminal 60-amino-acid homeodomain (POUH) (reviewed in reference 30). The POUS domain consists of a cluster of four alpha helices. The central two helices, 
2 and 3, form a helix-turn-helix DNA binding domain similar to the DNA binding domains of the phage 434 and 
repressors. The POUH domain is comprised of three alpha helices; the carboxy-terminal helices 2 and 3 form an helix-turn-helix motif (14). Crystallographic studies of the Pit-1 and Oct-1 POU domains bound to DNA have revealed that the flexibility of the disordered linker region allows the subdomains to bind DNA in multiple orientations. In addition, the alpha-helix side chains of the POU domain are involved in DNA binding through a diversity of potential interactions with multiple bases and with the phosphate backbone. Thus, Pit-1 can bind to a range of DNA sequences with variable conformations dictated by the underlying nucleotide sequence (14, 18). An additional layer of complexity results from the fact that Pit-1 typically binds to DNA as a cooperative dimer (13). The nature of the interaction of one Pit-1 subunit with the POU domain of its dimerizing partner is also facilitated by the loose binding specificity and linker flexibility and is dictated by the structure of the DNA binding site (14). The resulting differences in subunit interactions allow for further structural distinctions between Pit-1 dimer complexes assembled at distinct binding sites.
The contacts between amino acids in the POU domain and DNA bases revealed by structural analysis of Pit-1 POU domain/DNA complexes support the specificity of the HSI Pit-1 sites. In complexes containing either a synthetic promoter-type Pit-1 element (13) or the natural GH promoter GH1 or Prl promoter Prl-1P Pit-1 elements (32), specificity for the conserved thymine in the DNA sequence (Fig. 1B) is conferred by a major groove hydrogen bond linking this base to the threonine at position 45 (Thr-45) of the POUS 3 helix. Specificity for this base is further conferred by van der Waals contacts between its methyl group and the Cß methylene groups of Thr-45 and Ser-43 of the POUS domain (14). The thymine at this position in the Prl-1P element (32), and its paired adenine in the synthetic Pit-1 element (14), can also contribute to the conformation of the POU domain dimer by forming a minor groove hydrogen bond with Arg-5 of the POUH 1 helix in the partner POU domain monomer. Thus, one would predict that the presence of an adenine in place of a thymine in this position, as is seen in the HSI Pit-1 elements (Fig. 1B), would result in distinct contacts with both POU domains of the Pit-1 dimer and the potential for a concomitant adjustment in the topology of the complex.
The unique flexibility of the POU domain-DNA interactions is postulated to modulate and specify interactions of bound POU domain proteins with cofactors (14, 26, 30). In this manner, a POU domain protein can display distinct context-specific biological activities when bound to different DNA elements (14). Direct evidence in support of this model has been gathering in recent years. A study of two different Oct-1 binding sites revealed that both binding sites have equal affinities for Oct-1, but only one of the complexes can recruit the B-cell coactivator OBF-1 (OCA-B, Bob-1) (39). This specificity reflects an allosteric induction of the required OBF-1 interaction surface on the Oct-1 dimer by the underlying DNA element (28). Allosteric specification has also been observed with Pit-1. In this context, an additional two base pairs between the POUS and POUH interaction sites in the prolactin gene Prl-1P Pit-1 element prevents the recruitment of the nuclear receptor corepressor N-CoR to the complex, unlike the GH gene GH1 Pit-1 element. In contrast, the more compact Pit-1 dimer complex conferred by the GH-1 element is able to interact with N-CoR (32). This paradigm for the allosteric control of cofactor recruitment by nucleotide sequence has recently extended beyond the POU domain proteins. Like Pit-1, the dimeric transcription factor NF-
B recognizes a particularly loose consensus sequence. Investigation of the mechanistic basis for a functional distinction among different NF-B binding sites revealed that a single nucleotide difference is able to determine which cofactor is recruited by the NF-B dimer (19).
The results of our studies, along with a body of precedents in the literature, lead us to conclude that the HSI Pit-1 complexes recruit cofactors critical to LCR functions and that the nature of these cofactors is specified by a DNA-mediated allosteric mechanism. Studies by others have documented an interaction between Pit-1 and the HAT coactivator CBP (7, 40). Thus, CBP was an attractive candidate for the HAT recruited by HSI and was responsible for the acetylation of the hGH locus in the pituitary. ChIP assays revealed that CBP is in fact recruited to the hGH-N promoter but appears not to be targeted to HSI. Interestingly, the recruitment of CBP to the hGH-N promoter is itself dependent upon HSI action (Fig. 9) and parallels the HSI-dependent Pit-1 occupancy at the hGH-N promoter (11). This suggests that CBP is recruited to the hGH-N promoter by the promoter Pit-1 complexes, the assembly of which is dependent upon global hGH locus histone acetylation. These data support our hypothesis that the distinct Pit-1 element sequences at HSI and the hGH-N promoter result in differential cofactor recruitment. ChIP analysis with antibodies to several other HATs (p300, TAF-250, PCAF, and GCN5) failed to detect the HSI region (data not shown), indicating that they are unlikely to be involved in Pit-1-mediated hGH locus acetylation. Thus, the HAT activity(ies) recruited by HSI involved in the initial LCR action at the hGH locus remains unknown. Future studies will address the characterization of such pioneer HAT cofactor complexes. The eventual goal is to correlate high-resolution structural analysis of the Pit-1 complex and cofactor interactions at HSI and at the hGH-N promoter and to define the order and timing of their respective functions in pituitary organogenesis and hGH gene activation.
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ACKNOWLEDGMENTS |
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The work was supported by NIH K01 DK62164 (B.M.S.), NIH K01 DK064011 (Y.H.), and R01 HD25147 (N.E.C. and S.A.L.).
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FOOTNOTES |
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Present address: Department of Biochemistry and Molecular Biology, East Carolina University School of Medicine, Greenville, NC 27834.
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REFERENCES |
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2. Ayala, J. E., R. S. Streeper, C. A. Svitek, J. K. Goldman, J. K. Oeser, and R. M. O'Brien. 2002. Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs. J. Biol. Chem. 277:27935-27944.
3. Baldwin, A. S., M. Oettinger, and K. Struhl. 1996. Methylation and uracil interference assays for analysis of protein-DNA interactions, p. 12.3.1-12.3.7. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. John Wiley & Sons, Inc., Hoboken, N.J.
4. Bennani-Baiti, I. M., S. L. Asa, D. Song, R. Iratni, S. A. Liebhaber, and N. E. Cooke. 1998. DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression. Proc. Natl. Acad. Sci. USA 95:10655-10660.
5. Castrillo, J.-L., L. E. Theill, and M. Karin. 1991. Function of the homeodomain protein GHF1 in pituitary cell proliferation. Science 253:197-199.
6. Chen, R. P., H. A. Ingraham, M. N. Treacy, V. R. Albert, L. Wilson, and M. G. Rosenfeld. 1990. Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements. Nature 346:583-586.[CrossRef][Medline]
7. Cohen, L. E., Y. Hashimoto, K. Zanger, F. Wondisford, and S. Radovick. 1999. CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression. J. Clin. Investig. 104:1123-1130.[Medline]
8. Cohen, L. E., F. E. Wondisford, A. Salvatoni, M. Maghnie, F. Brucker-Davis, B. D. Weintraub, and S. Radovick. 1995. A "hot spot" in the Pit-1 gene responsible for combined pituitary hormone deficiency: clinical and molecular correlates. J. Clin. Endocrinol. Metab. 80:679-684.[Abstract]
9. Cohen, L. E., K. Zanger, T. Brue, F. E. Wondisford, and S. Radovick. 1999. Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency. Mol. Endocrinol. 13:476-484.
10. Elefant, F., N. E. Cooke, and S. A. Liebhaber. 2000. Targeted recruitment of histone acetyltransferase activity to a locus control region. J. Biol. Chem. 275:13827-13834.
11. Ho, Y., F. Elefant, N. Cooke, and S. Liebhaber. 2002. A defined locus control region determinant links chromatin domain acetylation with long-range gene activation. Mol. Cell 9:291-302.[CrossRef][Medline]
12. Ingraham, H. A., R. P. Chen, H. J. Mangalam, H. P. Elsholtz, S. E. Flynn, C. R. Lin, D. M. Simmons, L. Swanson, and M. G. Rosenfeld. 1988. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519-529.[CrossRef][Medline]
13. Ingraham, H. A., S. E. Flynn, J. W. Voss, V. R. Albert, M. S. Kapiloff, L. Wilson, and M. G. Rosenfeld. 1990. The POU-specific domain of Pit-1 is essential for sequence specific, high affinity binding and DNA dependent Pit1-Pit1 interactions. Cell 61:1021-1033.[CrossRef][Medline]
14. Jacobson, E. M., P. Li, A. Leon-del-Rio, M. G. Rosenfeld, and A. K. Aggarwal. 1997. Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected arrangement and flexibility. Genes Dev. 11:198-212.
15. Ji, X., P. Zhang, R. N. Armstrong, and G. L. Gilliland. 1992. The three-dimensional structure of a glutathione S-transferase from the mu gene class: structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. Biochemistry 31:10169-10184.[CrossRef][Medline]
16. Jones, B. K., B. R. Monks, S. A. Liebhaber, and N. E. Cooke. 1995. The human growth hormone gene is regulated by a multicomponent locus control region. Mol. Cell. Biol. 15:7010-7021.[Abstract]
17. Kingston, R. E., C. A. Chen, H. Okayama, and J. K. Rose. 1996. Transfection of DNA into eukaryotic cells, p. 9.1.1-9.1.11. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 1. John Wiley & Sons, Inc., Hoboken, N.J.
18. Klemm, J. D., M. A. Rould, R. Aurora, W. Herr, and C. O. Pabo. 1994. Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77:21-32.[CrossRef][Medline]
19. Leung, T. H., A. Hoffmann, and D. Baltimore. 2004. One nucleotide in a B site can determine cofactor specificity for NF-B dimers. Cell 118:453-464.[CrossRef][Medline]
20. Lew, D., H. Brady, K. Klausing, K. Yaginuma, L. E. Theill, C. Stauber, M. Karin, and P. L. Mellon. 1993. GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice. Genes Dev. 7:683-693.
21. Li, S., E. B. Crenshaw III, E. J. Rawson, D. M. Simmons, L. W. Swanson, and M. G. Rosenfeld. 1990. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347:528-533.[CrossRef][Medline]
22. Mangalam, H. J., V. R. Albert, H. A. Ingraham, M. Kapiloff, L. Wilson, C. Nelson, H. Elsholtz, and M. G. Rosenfeld. 1989. A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally. Genes Dev. 3:946-958.
23. McCormick, A., H. Brady, L. E. Theill, and M. Karin. 1990. Regulation of the pituitary-specific homeobox gene GHF1 by cell-autonomous and environmental cues. Nature 345:829-832.[CrossRef][Medline]
24. Parker, M. W., M. Lo Bello, and G. Frederici. 1990. Crystallization of glutathione S-transferase from human placenta. J. Mol. Biol. 213:221-222.[CrossRef][Medline]
25. Pfaffle, R. W., G. E. DiMattia, J. S. Parks, M. R. Brown, J. M. Wit, M. Jansen, H. Van der Nat, J. L. Van den Brande, and M. G. Rosenfeld. 1992. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 257:1118-1121.
26. Phillips, K., and B. Luisi. 2000. The virtuoso of versatility: POU proteins that flex to fit. J. Mol. Biol. 302:1023-1039.[CrossRef][Medline]
27. Radovick, S., M. Nations, Y. Du, L. A. Berg, B. D. Weintraub, and F. E. Wondisford. 1992. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 257:1115-1118.
28. Remenyi, A., A. Tomilin, E. Pohl, K. Lins, A. Philippsen, R. Reinbold, H. R. Scholer, and M. Wilmanns. 2001. Differential dimer activities of the transcription factor Oct-1 by DNA-induced interface swapping. Mol. Cell 8:569-580.[CrossRef][Medline]
29. Rhodes, S. J., R. Chen, G. E. DiMattia, K. M. Scully, K. A. Kalla, S. C. Lin, V. C. Yu, and M. G. Rosenfeld. 1993. A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene. Genes Dev. 7:913-932.
30. Ryan, A. K., and M. G. Rosenfeld. 1997. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev. 11:1207-1225.
31. Schreiber, E., P. Matthias, M. M. Muller, and W. Schaffner. 1988. Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA). EMBO J. 7:4221-4229.[Medline]
32. Scully, K. M., E. M. Jacobson, K. Jepsen, V. Lunyak, H. Viadiu, C. Carriere, D. W. Rose, F. Hooshmand, A. K. Aggarwal, and M. G. Rosenfeld. 2000. Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification. Science 290:1127-1131.
33. Shewchuk, B. M., S. A. Liebhaber, and N. E. Cooke. 2002. Specification of unique activity in the hGH locus control region. Proc. Natl. Acad. Sci. USA 99:11784-11789.
34. Shewchuk, B. M., S. L. Asa, N. E. Cooke, and S. A. Liebhaber. 1999. Pit-1 binding sites at the somatotrope-specific DNase I hypersensitive sites I, II of the human growth hormone locus control region are essential for in vivo hGH-N gene activation. J. Biol. Chem. 274:35725-35733.
35. Simmons, D. M., J. W. Voss, H. A. Ingraham, J. M. Holloway, R. S. Broide, M. G. Rosenfeld, and L. W. Swanson. 1990. Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors. Genes Dev. 4:695-711.
36. Streeper, R. S., S. C. Chapman, J. E. Ayala, C. A. Svitek, J. K. Goldman, A. Cave, and R. M. O'Brien. 1998. A phorbol ester-insensitive AP-1 motif mediates the stimulatory effect of insulin on rat malic enzyme gene transcription. Mol. Endocrinol. 12:1778-1791.
37. Su, Y., S. A. Liebhaber, and N. E. Cooke. 2000. The human growth hormone gene cluster locus control region supports position-independent pituitary- and placenta-specific expression in the transgenic mouse. J. Biol. Chem. 275:7902-7909.
38. Tan, S., and T. J. Richmond. 1990. DNA binding-induced conformational change of the yeast transcriptional activator PRTF. Cell 62:367-377.[CrossRef][Medline]
39. Tomilin, A., A. Remenyi, K. Lins, H. Bak, S. Leidel, G. Vriend, M. Wilmanns, and H. R. Scholer. 2000. Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration. Cell 103:853-864.[CrossRef][Medline]
40. Xu, L., R. M. Lavinsky, J. S. Dasen, S. E. Flynn, E. M. McInerny, T. M. Mullen, T. Heinzel, D. Szeto, E. Korzus, R. Kurokawa, A. K. Aggarwal, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1998. Signal-specific coactivator domain requirements for Pit-1 activation. Nature 395:301-306.[CrossRef][Medline]
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-globin confirm the presence of the hGH-N transgenes compared with an endogenous control. The hGH-N hybridization signal is proportional to the hGH-N transgene copy number. (C) The frequency of hGH-N transgene activation in the mouse pituitary is responsive to the sequence of the linked Pit-1 trimer array. The bar in each case represents the total number of lines generated with each indicated transgene, and the shaded region indicates the number of those lines that express the hGH-N transgene in the pituitary. The 3GH1 and PitABC results indicated by an asterisk (extreme right and left bars, respectively) have been previously reported (
