©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pit-1 Exhibits a Unique Promoter Spacing Requirement for Activation and Synergism (*)

(Received for publication, November 28, 1994)

Kelly P. Smith(§)(¶) Bing Liu (¶) Clara Scott Z. Dave Sharp (**)

From the Center for Molecular Medicine, University of Texas Institute of Biotechnology, Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78245

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The developmentally regulated Pit-1 transcription factor is involved in the activation of prolactin, growth hormone, and TSHbeta expression. Using templates with spacing mutations to program an in vitro transcription system, the activity of a single Pit-1 proximal binding site within the rat prolactin promoter was shown to have a unique bimodal distance requirement. Transcription activity rapidly decreased with each 5-base pair (bp) addition to the spacing between the binding site and the TATA box. When positioned 20 bp upstream from its normal -36 position in the prolactin promoter, the activity of the Pit-1 binding site is reduced to basal levels. Placement of the site at a position 30 bp upstream resulted in a return of Pit-1-mediated activation. Using transient transfection assays in GH(3) cells, the prime bimodal sites are also a requirement for optimum expression of chimeric prolactin-luciferase reporter constructs. Interestingly, optimal synergism of transcription in vivo by the prolactin distal enhancer, containing four Pit-1 binding sites and an estrogen-responsive element, is also sensitive to the placement of the proximal Pit-1 binding site. These data have important implications for Pit-1 activator function in pituitary cells and for general models of transcription synergism.


INTRODUCTION

Binding site position relative to the transcription initiation site has been shown to be important in defining the activity of numerous transcription factors in a variety of eukaryotic and prokaryotic systems(1, 2, 3, 4) . Many of these studies have shown that the transcription factor binding sites under investigation expressed position-dependent activity in a stereospecific manner. The movement of a binding site away from its native position resulted in the maintenance of activity only when moved in multiples of 10 bases, or one helical turn, in RNA polymerase II (Sp1 binding sites(4, 5) ) and I promoters(6) . It is thought that sterospecificity reflects a requirement of the transcription factor to reside on the same helical face for precise protein-protein interactions to occur. In many eukaryotic class II TATA box-containing promoters, the most proximal transcription factor binding sites appear to allow the interaction of the activator with TFIIB(7) , RNA polymerase(8) , or with one or more components of the TFIID complex(9) . This, in turn, may promote the formation of or increase the stability of the initiation complex and stimulate transcription(9) .

Cell type-specific transcription of the rat prolactin, growth hormone, and mouse TSHbeta genes during development is controlled by a pituitary-specific transcription factor, Pit-1 (10, 11, 12, 13) (also known as PUF-1 (14) and GHF-1(15) ). Pit-1 is a member of the POU domain family of transcription factors, many of which appear to be involved in neural development(16) . At least seven Pit-1 binding sites have been identified within the rat prolactin gene(12) ; however, a single proximal Pit-1 binding site has been found to be sufficient to support cell-specific transcription in vitro(14, 17, 18) . The distal group of four Pit-1 binding sites and an estrogen-responsive element residing at about -1500 comprise the prolactin enhancer. It is believed that the function of the prolactin enhancer is mediated by the transcriptional synergism provided by multiple Pit-1 binding events in the enhancer and promoter regions(19) . DNA-binding and cell-free transcription assays indicated that the phasing of factor binding sites in the prolactin proximal 5`-region may influence cooperative binding and transcriptional efficiency(20) . Transient transfection assays of growth hormone promoters containing insertions of 44 base pairs at -148 (distal to the upstream Pit-1 binding site) and of 23 base pairs at -51 (between the downstream Pit-1 binding site and the TATA box) indicated that response to serum stimulation was impaired by alterations in spacing(21) .

Although much is known concerning the structure and functional domains of the Pit-1 protein(22, 23) , it is not understood how Pit-1 mediates its influence on the transcription machinery. This is an important issue that has a direct bearing on the pivotal role of Pit-1 in prolactin, growth hormone, pit-1 gene regulation, and in pituitary cell differentiation. In this report, we used a cell-free in vitro system and transient transfection assays to examine the basic position requirements for Pit-1 function in the prolactin promoter. The results showed that the proximal Pit-1 binding site of the rat prolactin promoter exhibits a novel bimodal pattern of activity. We also provide the first in vitro and in vivo evidence that the position of the most proximal binding site influences the level of transcriptional synergism mediated by additional Pit-1 binding sites that are located in the promoter or by the sites in the prolactin enhancer.


EXPERIMENTAL PROCEDURES

Construction of Templates

To produce the templates used in the first experiments, a 40-bp (^1)high affinity proximal Pit-1 binding site was inserted into the KpnI site of the Bluescript SK vector (Stratagene). An isolated fragment from plasmids that contained the binding site in the normal(N) and reversed (R) orientation plus the entire multiple cloning region was inserted into the prolactin clone dm36, which contains no binding site(18) . This procedure produced templates with a single Pit-1 binding site in both orientations 147 bp upstream from the transcription start site. The production of the remainder of the templates was accomplished either by deletion of selected portions of the multiple cloning region using restriction cleavage and religation or by addition of HaeIII fragments of X174 into this region.

For in vitro transcription assays in the second set of experiments, all of the prolactin DNA templates used were produced from a construct containing 66 bp of 5`-flanking DNA, the first exon and a portion of the first intron cloned into the plasmid vector pUC12(18) . This deletion mutation in plasmid dm66 has a regulatory region consisting of a TATA box (-23 to -28) and a single proximal Pit-1 binding site (-36 to -63)(18) . A definitive study of the relationship between position and function of the proximal Pit-1 binding site required the addition of 5-bp insertions between the 8 bp separating the TATA box and the Pit-1 binding site. Seven spacing mutations, in which the distance between the TATA box and the proximal Pit-1 binding site was increased by approximately 5-bp intervals, were created using polymerase chain reaction and overlapping primers (one with a non-homologous tail containing several unique restriction sites at 5-base intervals). All template structures, indicated in Fig. 2A as -36 (wild type), -41, -46, -50, -56, -60, -66, and -72, were confirmed by sequencing. For the transient transfection assays in the second set of experiments, plasmids containing Pit-1 binding sites located at -36, -56, and -66 (Fig. 2A) plus pdm36 with no Pit-1 binding site (18) were used to amplify prolactin promoter fragments using primers that introduced Asp718 and XhoI sites at each end. After cleavage with Asp718 and XhoI, DNA fragments were ligated into the pGeneLight basic plasmid (Promega). Each promoter construct (pGLprl) contained one Pit-1 binding site in its normal -36 position, one site with alternative spacing, or no Pit-1 binding site, the TATA box, and the prolactin cap site fused to the luciferase transcription unit (Fig. 2D). All promoter constructions were checked by DNA sequencing.


Figure 2: Effect of 5-bp spacing between the TATA box and Pit-1 binding site on transcription in vitro and in vivo. A, sequences of 5-bp spacing mutants. The Pit-1 binding site footprinted region and the TATA homologies are boxed. B, polyacrylamide gel analysis of primer extension products from spacing mutant transcription assays. Primer extension yields an 83-nucleotide (nt) prolactin product and a 54-nucleotide adenovirus MLP product. C, mean (± S.E.) transcription activity of each template (as a percentage of wild-type (wt) activity). Levels of primer extension products were determined using a Betascope and standardization against MLP counts. Values with** differ from NS (p < 0.01). D, GH(3) cells transfected with the indicated prolactin-luciferase chimeric constructions were harvested, and cellular lysates were assayed for luciferase and beta-galactosidase activities (See ``Experimental Procedures''). Each bar in the graph represents the percentage stimulation below -36 Pit-1 site (wild-type position) from three separate transfections. Means (± S.E.) are shown, with** and * indicating a difference (p < 0.01 and p < 0.05, respectively) from NS.



To produce the templates with multiple Pit-1 binding sites used in the third in vitro transcription experiments, a parent plasmid, pPal3pUC, was used. This plasmid contains three tandem Pit-1 binding regions (corresponding to the footprinted region of the proximal Pit-1 binding site of the rat prolactin gene(10, 14, 17) ) within the SmaI site of pUC19. To produce template 3P, a BamHI fragment containing the prolactin template was cut from plasmid N147 (a Bluescript plasmid containing a prolactin gene sequence with 36 bp of 5`-flanking DNA cloned into the KpnI site) and inserted by blunt-end ligation into the HindIII site of Pal3pUC. Restriction analysis and sequencing allowed identification of the desired clone, 3P (Fig. 4A). Template 3P-36 (Fig. 4A) was constructed by inserting an Asp718-BamHI fragment of template dm66 into the HindIII site of Pal3pUC. Template 3P-46 (Fig. 4A) was produced by first removing a 10-bp HindIII-BamHI fragment from Pal3pUC and blunt-end ligating the plasmid. The plasmid was then recut with BamHI (site was regenerated), and an Asp718-BamHI fragment of plasmid -46 (from the second experiment) was inserted. The structure of all constructs (Fig. 4A) was confirmed by sequencing. Template NS is a rat prolactin Bal31 deletion mutant possessing 36 bp of 5`-flanking DNA (TATA box but no Pit-1 binding site)(18) . Plasmid PRL125 (17) was used in the construction of the prolactin enhancer test plasmids used in the transient transfection assays in the third set of experiments. Primers flanking the enhancer (-1807 to -1498) were used to amplify DNA fragments containing terminal Asp718 sites that were then cloned into the Asp718 sites of the pGLprlNS, -36, -56, and -66 promoter plasmids (Fig. 2D). The prolactin enhancer constructions shown in Fig. 4D were verified by DNA sequencing (Sequenase).


Figure 4: The effect of proximal Pit-1 binding site position on in vitro and in vivo transcription synergism mediated by distal Pit-1 elements. A, structure of the three multiple Pit-1 binding site clones. The NS (no Pit-1 site), -36 (wild type), and -46 templates used in Fig. 2were also assayed in this experiment. B, gel analysis of primer extension products. Prolactin and major late promoter control products are indicated. C, graph indicating the relative transcription activity of each template. Data were combined from three separate experiments. D, GH(3) cells transfected with the indicated prolactin-luciferase chimeric constructions were harvested, and cellular lysates were assayed for luciferase and beta-galactosidase activities (See ``Experimental Procedures''). Each bar in the graph represents levels of luciferase activity as a percentage of prolactin enhancer, Pit-1 -36 construction. Means (± S.E.) are shown, with * indicating a difference (p < 0.05) from enhancer-NS.



DNA Binding Assay

DNase I protection assays were done according to previously reported procedures (14) using GH(3) nuclear extracts(17) . The DNA fragments were prepared by excision from the each of the pGLPrl -36, -56, and -66 promoter plasmids using KpnI and BglII and from pGLPrlNS plasmid using KpnI and HindIII. The resulting fragments were isolated by polyacrylamide gel electrophoresis and elution by the crush and soak method(24) . The 3`-end of the fragments were labeled by Klenow fill-in of the BglII or HindIII sites using [alpha-P]dATP(24) . This strategy results in fragments with the Pit-1 binding at the 5`-end of the fragments, except for pGLPrlNS, which has no binding sites.

In Vitro Transcription and Primer Extension Assay

All DNA templates were assayed in an in vitro transcription reaction by incubating 200 ng (or molar equivalent) of each construct along with 75 ng of control template pHTX-B, a plasmid containing three copies of the adenovirus major late promoter, in 15 µl of GH(3) nuclear extract (prepared by a method modified from Dignam et al.(25) ). GH(3) cells from a rat pituitary-derived cell line that expresses the Pit-1 transcription factor were grown as previously described(14) . Primer extension assays of transcribed RNA were carried out using P-labeled oligonucleotide primers, which produced prolactin and major late promoter (MLP) products of 83 and 54 nucleotides, respectively. These products were separated on 8% polyacrylamide gels, and the resulting bands were quantified using a Betagen Betascope (Fig. 2B and Fig. 4B) or scintillation counting of the excised gel bands (Fig. 1B). Counts from each test template were normalized by adjusting each value according to the percentage of variation of each control template (MLP) from the averaged values of all control templates in the experiment. All test template values were then expressed as a percentage of transcription activity by arbitrarily assigning a value of 100% to the counts obtained from the -36 (wild type) or -66 templates. This allowed the combining of results from multiple experiments and statistical analysis. Transcription activity results were analyzed by one-way ANOVA, and means were separated with Student-Newman-Keuls procedure.


Figure 1: Distance and orientation requirements for function of the proximal Pit-1 binding site. A, gel analysis of in vitro transcription products assayed by primer extension. NS contains no Pit-1 binding site (corresponds to clone 36 in (18) ). Prolactin (83 nucleotides (nt)) and the MLP (54 nucleotides) are shown. B, mean (± S.E.) transcription activity of each template (as a percentage of wild-type (WT) activity). Levels of primer extension products were determined by counting of gel bands and standardization against MLP counts. Values with** and * differ (p < 0.01 and 0.05, respectively) from NS. Orientation does not affect binding site function (two way ANOVA, F = 0.001). Data represent at least four experiments for each sample.



Transient Transfection Assays

GH(3) cells were grown (17) and co-transfected with 5 µg of prolactin luciferase test plasmids and 2 µg of pTK-beta-galactosidase using a lipofectamine procedure (Life Technologies, Inc.). Three separate transfections for each plasmid construct were performed, and, after 45 h, cell lysates were prepared for luciferase (Promega) and beta-galactosidase (Galacto-Light, Tropix) assays. Both activities were measured using a luminometer (AutoLumat LB953, EG&). Luciferase activity was normalized to beta-galactosidase activity to correct for different transfection efficiencies. The mean luciferase activity of each construction was contrasted with NS using Student's t test.


RESULTS

The Proximal Pit-1 Binding Site Exhibits a Unique Bimodal Pattern of Position-dependent Activity

To establish a broad foundation for understanding the role of position and orientation in the in vitro function of Pit-1, an initial series of test templates with binding sites positioned at variable distances from the wild-type position(-36) in its normal(N) and reversed (R) orientations were assayed in GH(3) nuclear extracts. The Pit-1 binding site increased transcription in an orientation-independent manner when placed at 71, 92, or 128 bp from the cap site (Fig. 1, A and B). In this experiment, the level of Pit-1-mediated activation was optimal at -36 and -71 (Fig. 1, A and B). Previous experiments showed that a high affinity Pit-1 binding site is capable of only marginally stimulating transcription when it is located downstream of the cap site(26) . Therefore, a single high affinity Pit-1 binding site appears to function optimally in vitro when it is situated in either orientation at a distance from -36 to -72 on the 5`-side of the transcription initiation site.

There are reports of transcription factor binding sites that exhibit activity in a helical turn-dependent manner; when moved 5 bp away, activity is lost, and when moved another 5 bp, activity returns(4, 5) . Would a similar pattern of activity be exhibited by Pit-1 and its proximal binding site? Using the prolactin promoter region, seven spacing mutations, in which the distance between the TATA box and the proximal Pit-1 binding site was increased by approximately 5-bp intervals (Fig. 2A), were tested in a GH(3) in vitro transcription system. Leaving the entire footprinted region of the proximal Pit-1 site (-36 to -63) intact ensures that the binding of Pit-1 was not disrupted. This footprinted region has been used as a Pit-1 binding site in a variety of constructions and has always bound Pit-1 with equal avidity. Results of an in vitro transcription reaction are shown in the autoradiogram of a typical experiment (Fig. 2B) and the graphic summarization of several experiments in Fig. 2C. The results clearly showed that 5-bp half-helical turn periodicity of the prolactin proximal Pit-1 binding site is not a requirement for activation. Although, relative to NS, the transcription activity of -41, -46, and -50 increases, their Pit-1-mediated activity relative to -36 (wild type) unexpectedly dropped at each 5-bp interval as the site was moved upstream. At -56 (20 bp from and on the same face of the helix as the normal -36 position) and -60, the level of transcription in vitro was not statistically different from the level of transcription observed using templates with no Pit-1 binding site. Interestingly, the activity rebounded when the binding site was moved to positions -66 and -72 (see also Fig. 1, A and B, lanesN71, R71). At this distance, the Pit-1 binding site is capable of producing a 2.5-fold stimulation in the level of transcription in vitro. Beyond this point, the previous experiment showed that the stimulatory capacity of this binding site steadily decreases as the site is moved farther away (Fig. 1, A and B). It is unlikely that the intervening DNA used to create the spacing alterations is responsible for the observed transcriptional effects since each of the constructions had different DNA sequences inserted.

To assess the bimodal pattern of transcriptional activity using a different assay, several of the key promoter constructions were tested using transient transfection. Prolactin promoters containing no Pit-1 binding sites or ones with sites located in its wild-type position (-36), -56, or -66 were transferred to luciferase reporter plasmids (Fig. 2D). 45 h after co-transfection with pTK-beta-galactosidase into GH(3) cells, luciferase and beta-galactosidase activities were measured. One Pit-1 binding site at -36 (its wild-type position in the prolactin promoter) stimulates luciferase activity approximately 19-fold over the activity of no binding site. Consistent with the in vitro transcriptional activity, a Pit-1 binding site at -66, in the context of the prolactin promoter, also stimulates luciferase activity approximately 19-fold (Fig. 2D). Importantly, the level of luciferase activity driven by a Pit-1 binding site at the -56 position is not statistically different from basal levels of activity observed with no Pit-1 binding site (Fig. 2D). Although the fold in vivo activation of the -66 promoter is about twice that observed in vitro, the bimodal pattern is consistent. DNase I footprint assays using GH(3) extracts showed that the Pit-1 binding sites in the -36 wild-type, -56, and -66 promoters are intact (Fig. 3). As expected, the footprint assays also demonstrate no Pit-1 binding to the promoter from the deletion mutant, pGLPrlNS (Fig. 3).


Figure 3: Pit-1 binds proximal sites located at -36, -56, and -66. DNase I footprint assays were performed using the three indicated DNA templates containing Pit-1 binding sites at positions indicated above each group of reactions and a control with no Pit-1 binding site (NS). All of the DNA fragments were obtained from the pGLPrl plasmids used in Fig. 2D. The first lane of each group contained only bovine serum albumin (BSA), the other lanes used 1, 2, or 3 µl of GH(3) extract. The Pit-1 binding site for each of the DNA fragments is located at the 5`-end of fragments (indicated on the rightside of the figure) next to a KpnI site used to excise them from the pGL plasmids. This site abuts pGL vector sequences common to all four of the plasmids used in Fig. 2D.



The Ability of Upstream Pit-1 Binding Sites to Act Synergistically Is Dependent on the Position of the Proximal Pit-1 Binding Site

To initially address the question of how the number and affinity of Pit-1 binding sites influence the level of prolactin transcription, templates were constructed that contained different Pit-1 sites in variable positions and combinations. The data from these experiments (not shown) indicated, not unexpectedly, that additional Pit-1 binding sites in the promoter serve to stimulate the level of transcription in vitro. The unusual feature revealed in these experiments (not shown) was that the binding affinity of proximal binding site appeared to have a key role in this function. Since the position of a single proximal Pit-1 binding site influenced its capacity to stimulate transcription ( Fig. 1and Fig. 2) and the latter experiments (not shown) suggested it also had a key role in transcriptional synergism in vitro, it is important to link these observations in a more rigorous manner by investigating if a proximal Pit-1 binding site must be precisely positioned to permit transcriptional synergism by additional distal elements. This is a biologically significant question since the mechanism to achieve normal pituitary levels of prolactin expression in transgenic mice is thought to involve the synergistic interaction of the seven Pit-1 binding sites in the prolactin gene(19) .

To address this question, we fused selected templates (NS, -36, and -46) used in Fig. 2A to DNA containing three additional Pit-1 binding sites (Fig. 4A) and tested them by in vitro transcription. To observe the synergistic effect in vitro, three high affinity Pit-1 binding sites were positioned at a constant distance from the cap site while the proximal binding site was moved within this region. The assays test the ability of three distal binding sites to act as a synergist of transcription in the context of a functional (-36 position), a disabled (-46 position), and a nonexistent proximal Pit-1 binding site. The results of a typical polyacrylamide gel assay of the primer extension products are shown in Fig. 4B, and a graphic illustration of results from several experiments is shown in Fig. 4C. These results showed that three upstream binding sites at -115 (3P) only marginally increase transcription above basal levels (NS, no Pit-1 sites). A single proximal Pit-1 site in its normal position(-36) increases transcription 6-fold, and addition of three upstream sites (3P-36) increases transcription another 5-fold to 11times basal levels. A proximal site in which the position was moved 10 bp(-46) increases transcription only 2times, and adding 3 sites (3P-46) only increases transcription another 0.6times for a total of a 2.6times stimulation above basal levels. If all sites act independently and their levels of activation are additive, the linkage of three Pit-1 binding sites to the -46 prolactin promoter (3P-46) should result in a 5-fold increase over basal transcription as in the case of 3P-36. However, this did not occur. Rather, the addition of three upstream sites increases the level of transcription by a factor of 1.5 to 2 over that mediated by a single proximal site. Thus, transcription synergism in vitro mediated by upstream Pit-1 binding sites is dependent upon the activity of the proximal site, and they do not appear to act independently.

Positioning of the Pit-1 binding site in the prolactin proximal promoter is important for cell-free, in vitro transcription synergism. Is this the case in an in vivo setting? To address this issue, luciferase reporter plasmids were constructed to test the effect of additional Pit-1 sites on the various prolactin promoters used in the previous in vivo experiments. To increase the biological relevance of the assay, the four Pit-1 binding sites in the prolactin distal enhancer were used instead of the three high affinity proximal sites used in the previous experiments. Thus, each of the prolactin promoters containing a wild-type proximal Pit-1 binding site(-36), a -56, a -66, or no site was joined to a DNA fragment containing the prolactin enhancer. This enhancer has been reported to increase luciferase activity in similar assays 100-fold (12) . A proximal Pit-1 binding site at its wild-type position(-36) and at -66 supports 13-fold stimulation of luciferase activity by the prolactin enhancer in GH(3) cells (Fig. 4D). In contrast, at -56, a Pit-1 site under the influence of the prolactin enhancer supports levels of activity comparable with those obtained with no proximal Pit-1 site (Fig. 4D).


DISCUSSION

To investigate the relationship between recognition element spacing and function of the Pit-1 transcription factor in the rat prolactin gene, a series of promoter-spacing mutations were constructed in which a single proximal Pit-1 binding site was moved away from the TATA box, first in large steps, then in smaller 5-bp increments. Moving the site outward from -72 bp gradually diminishes its ability to simulate transcription in vitro. The investigation using 5-bp additions showed, interestingly, that Pit-1 does not have a requirement for stereospecific alignment between its binding site and the TATA box as has been reported for the multiple Sp1 binding sites in the SV40 promoter (5) and to a lesser degree in the single Sp1 site in the adenovirus 2 E1B promoter(4) . Rather, activity in vitro decreases rapidly with each 5-bp movement until the site is essentially inactive when moved 20 bp upstream. In vitro activity then returns, although at a lower level, when the binding site was moved distally another 10 bp. Transient transfection assays were consistent with the in vitro results with the exception that the -66 positioning of the Pit-1 binding site, in the context of a prolactin promoter, is equal in luciferase activity to the wild-type -36 position. Thus, two different assays of promoter function indicate that Pit-1 possesses an unusual distance requirement for activation of transcription. Based on footprint assays, the movement of the binding site had no effect on DNA binding by Pit-1.

A DNA bending model may be one explanation of these results. In this model, flexibility of the Pit-1 factor allows it to continue to stimulate transcription as it is moved away from its normal position; however, helical turn spacing does not appear to be important. This activity diminishes quickly as the distance becomes too great to bridge but returns when a sufficient amount of DNA lies between the binding site and the TATA box/cap site so that the DNA can bend enough to allow the precise interactions between the Pit-1 factor and the transcription initiation complex to occur. As more distance is placed between the Pit-1 site and the TATA box, the likelihood of this interaction occurring is lower, and the level of transcription stimulation is again diminished with increasing distance. This type of DNA bending model has been suggested to explain enhancer function (27) and interactions between the multiple transcription factor binding sites and the TATA box of the thymidine kinase gene promoter when intervening DNA was introduced(3) .

The bimodal activation observed for Pit-1 is strikingly similar to the cyclic AMP receptor protein that activates transcription in a variety of promoters in E. coli(28) . In this prokaryotic system, promoter architecture determines which subunit of the CRP dimer is functional(29) . Transcription activation by class II promoters (CRP-binding site at -41.5) is mediated through contacts by the promoter-distal subunit of CRP dimer, and activation by class I promoters (binding sites at -61.5 and -72.5) is by polymerase interactions with the promoter-proximal subunit of CRP dimer(29) . Interestingly, kinetic analyses of CRP indicate that protein bound at the different sites activates transcription in similar but distinct ways. At -41.5, the predominate effect of CRP, in addition to increasing binding of RNA polymerase, is to potentiate isomerization to an open complex(30) . In contrast, at -61.5, the predominate effect of CRP is to increase initial binding by RNA polymerase(30) . For eukaryotic promoters, the pattern of loss and re-establishment of Pit-1 binding site function with distance is, thus far, unique. Since Pit-1 occupies DNA as a dimer(22) , the observed distance requirements may reflect a conserved mechanism of transcriptional activation by DNA binding proteins.

It is noteworthy that in the known Pit-1-regulated genes, including the rat, bovine, and human prolactin genes and the Pit-1 gene itself, the proximal Pit-1 binding site always occupies the same position(-36) in relation to the TATA box(12, 17, 31, 32, 33) . This underscores the importance of spacing to Pit-1 function. However, in other examples of Pit-1-regulated genes, growth hormone and TSHbeta, the proximal Pit-1 binding site occupies a conserved position from -66 to -99(12, 13, 34, 35, 36, 37, 38) . Fig. 5graphically illustrates the conservation of the most proximal Pit-1 binding sites in four known target genes. These conserved locations coincide precisely with the finding in this study that Pit-1 activity is restored when the binding site is moved out to -66 (Fig. 2). It is interesting that Pit-1 appears to have acquired narrow distance requirements for optimal function that match precisely the different spacing of its binding elements in each of its target genes. Analogous to the CRP binding sites, we propose that -66 Pit-1 proximal binding sites comprise class I promoters and -36 proximal binding sites comprise class II promoters (Fig. 5).


Figure 5: Schematic illustrating conservation of proximal of Pit-1-binding sites in four target promoters. Stippledboxes are Pit-1 binding sites. The Pit-1 binding sites in prolactin are from (12) and (17) ; for growth hormone (from Refs. 12, 36-38); for TSHb (from (13) ); and for pit-1 (from (31) ). The classification of Pit-1-responsive promoter is based on the conserved optimal locations (-36 and -66) of the proximal Pit-1 binding sites.



The other results presented in this report showed that the position of the proximal Pit-1 binding site greatly influences the degree of transcription stimulation and reporter expression from additional upstream binding sites. It is interesting that, even under the influence of a strong enhancer, the bimodal pattern of promoter activation is evident. This suggests that the most proximal site is actually the point at which the crucial interactions with the transcriptional machinery are made and that more upstream sites are synergistic through, rather than in addition to, the proximal site. If the independent touching model for synergism as proposed for other activators (39, 40) is operative for Pit-1, the additional sites in 3P-46 and the enhancer -56 constructs should have demonstrated a greater level of activity than what was observed. The data for Pit-1 appear to fit the criteria for kinetic models of synergism (41) with Pit-1 facilitating different steps in initiation that are either partially rate limiting or those in branched pathways of preinitiation complex assembly.

Another explanation for these results is that distal binding sites occupied with Pit-1 could serve to increase the local concentration of general transcription initiation factors through transient interactions. This local increase in general factor concentration could, in turn, lead to a greater probability of the assembly of a productive initiation system by, perhaps, stabilizing the protein complex(39, 42) . In this model, the seven Pit-1 binding sites in the prolactin proximal promoter and distal enhancer are synergistic by virtue of their ability to increase the local concentration of yet unidentified Pit-1 target molecules. Alternative explanations for the observed results also include the possibility that only a properly positioned Pit-1 polypeptide can promote the assembly of higher order Pit-1 structures, as conjectured for Sp1 synergism(43) , or that it promotes initiation complexes that are either more stably associated with the promoter or can reinitiate transcription at a greater frequency. Order of addition experiments using immobilized prolactin templates, affinity purified rat Pit-1, and HeLa nuclear extracts clearly show that Pit-1 is an initiation factor. (^2)A preassembled HeLa initiation complex is insensitive to Pit-1 activation.^2 In this model, transcription synergism by one activator is first dependent on the presence of a receptive preinitiation complex and second on additional activators being able to physically interact with the complex by virtue of lower entropic energy requirements(41) . Finally, it can be argued that Pit-1 can, when bound to the -56 position, act as a transcription repressor. That is, when the proximal binding site is moved from -36 to -56, not only is Pit-1 inactive but it prevents activation by other Pit-1 sites present in the promoter/enhancer. This is a testable hypothesis using heterologous promoter Pit-1 cells.

The data in Fig. 4showing that transcription in vitro is lower when the proximal Pit-1 site is at -46 (same face of the helix as upstream sites at -115) are not consistent with results reported by Harvey et al.(20) . In those experiments, it was shown that in vitro transcription rates of prolactin were reduced when distal and proximal LSF-1 binding sites were placed on opposite faces of the DNA helix by intervening deletions or insertions. Since our experiments did not test stereohelical requirements for the function of multiple Pit-1 sites but focused, rather, on the role of the proximal position in relation to the TATA box/cap site, it is unclear why our results differ. The results of this work seem to be in agreement with the data available on the position effects in the growth hormone promoter, although the spacing alterations under test (23 and 44 base pairs) were much larger(21) .

The data presented here showed, in a system regulated by the transcription factor Pit-1, that spacing between the TATA box and the proximal binding site plays a crucial role in transcription activation in vitro and in vivo. The findings also suggest that transcriptional synergism promoted by Pit-1 binding to the promoter and enhancer is sensitive to the placement of the most proximal Pit-1 binding site. Addressing the mechanism underlying these observations will require a determination of the effect of Pit-1 contacts on the closed or open initiation complex at each of its optimal positions, the identification of Pit-1 nuclear protein targets in pituitary cells, and an elucidation of how these contacts influence the class II basal transcription apparatus.


FOOTNOTES

*
This work was supported by Public Health Service Grant DK38546 from the National Institutes of Health, and additional support was received from the University of Texas Institute of Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Microbiology and Medical Genetics, College of Medicine, University of California, Irvine, CA 92717.

Contributed equally to this work.

**
To whom correspondence should be addressed: Center for Molecular Medicine, University of Texas Institute of Biotechnology, Dept. of Cellular and Structural Biology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245. Tel.: 210-567-7225.

(^1)
The abbreviations used are: bp, base pair(s); MLP, major late promoter; ANOVA, analysis of variance; CRP, cyclic AMP receptor protein.

(^2)
Sharp, Z. D.,(1995) Biochem. Biophys. Res. Commun.206, 40-45.


ACKNOWLEDGEMENTS

We thank Sharon Helsel and Kazi Begum for technical assistance and Debbie Twehous and Milton Thomas for a review of this manuscript. Maureen Mancini, Debbie Munoz-Medillin, and Paul Gardner provided invaluable advice and assistance for the transient transfection assays. We also thank Bob Christy for very helpful advice in the DNase I footprint assays.


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