(Received for publication, November 28, 1994)
From the
The developmentally regulated Pit-1 transcription factor is
involved in the activation of prolactin, growth hormone, and TSH
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
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.
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 TSH 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.
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 cells transfected with the
indicated prolactin-luciferase chimeric constructions were harvested,
and cellular lysates were assayed for luciferase and
-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 cells transfected with the indicated
prolactin-luciferase chimeric constructions were harvested, and
cellular lysates were assayed for luciferase and
-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.
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.
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 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--galactosidase into GH
cells, luciferase and
-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
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 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.
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
11 basal levels. A proximal site in which the position was moved
10 bp(-46) increases transcription only 2
, and adding 3
sites (3P-46) only increases transcription another 0.6
for a
total of a 2.6
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 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).
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
TSH, 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. ()A preassembled HeLa initiation
complex is insensitive to Pit-1 activation.
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.