(Received for publication, July 24, 1995)
From the
To identify elements of the human subunit gene necessary
for cell-specific expression, we generated an array of block mutations
spanning approximately 400 base pairs (bp) of promoter proximal region
and examined them using transient transfection analysis in pituitary
(
T3) and placental (BeWo) cell lines. Comparison of promoter
activity in the two cell types revealed both common and unique elements
required for transcription in pituitary and placenta. Two strong
elements, the cyclic AMP response element (CRE) and the upstream
regulatory element (URE), regulate expression of the
subunit gene
in BeWo cells. In contrast, promoter activity in
T3 cells requires
an array of weaker elements. These include the CREs, the URE, as well
as two previously described elements, pituitary glycoprotein hormone
basal element (PGBE) and gonadotrope-specific element (GSE), and two
new elements we designated as the
basal elements 1 and 2
(
BE1 and
BE2). These new elements reside between -316
and -302 bp (
BE1) and -296 and -285 bp
(
BE2) of the human
subunit promoter and bind distinct
proteins designated
BP1 and
BP2, respectively. Southwestern
blot analysis revealed that
BE1 specifically binds 54- and 56-kDa
proteins. Additional studies disclosed several potential interactions
between proteins that bind the CRE and proteins that occupy PGBE,
BE1, and
BE2, suggesting that gonadotrope-specific expression
occurs through a unique composite regulatory element that includes
components of the placenta-specific enhancer.
The glycoprotein hormones luteinizing hormone,
follicle-stimulating hormone, thyroid-stimulating hormone, and
chorionic gonadotropin regulate a variety of key biological functions,
including reproduction, pregnancy, and metabolism. These hormones
belong to a family of heterodimeric proteins composed of a common
subunit and a specific
subunit(1) . The
subunit is
expressed in the pituitaries of all mammals as the product of a
single-copy gene (2) . In this tissue, expression occurs in two
distinct cell types: gonadotropes, where it is a subunit of both
follicle-stimulating hormone and luteinizing hormone, and thyrotropes,
where it is a subunit of thyroid-stimulating hormone(3) . In
addition, the
subunit gene is expressed in the placenta of
primates and horses(4, 5) . Here, it is expressed in
trophoblast cells as a subunit of chorionic gonadotropin in primates or
as pregnant mare serum gonadotropin in horses. Because the human
subunit gene is expressed in both placenta and pituitary, examination
of its promoter allows a comparative analysis of the mechanisms
involved in directing expression of the same gene to two separate
tissues. Such studies have been aided by the availability of both
placenta and pituitary cell lines that express the
subunit gene.
Analysis of the human subunit promoter in placental cell lines
(BeWo or JEG3) has revealed the presence of a composite enhancer
located within the first 180 bp (
)of the
promoter(6, 7, 8, 9, 10, 11, 12, 13) .
This enhancer confers placenta-specific expression and consists of an
upstream regulatory element (URE) and two juxtaposed (tandem) cyclic
AMP response elements (CREs). Many earlier studies revealed a
synergistic relationship between the URE and CREs in placenta,
suggesting a functional interaction between the proteins that bind
these elements(7, 9) . Additional elements are also
required for optimal expression in placenta cells and include the
junctional response element (JRE) (14) and the CCAAT
box(15) .
The emerging model for gonadotrope-specific expression builds on the current model of placenta-specific expression. Although incomplete at this stage, transfection studies with mouse and human promoters containing deletion and clustered mutations have revealed the location of several elements important for expression in gonadotropes(16, 17, 18) . These elements reside within the first 435 bp of the human promoter and 507 bp of the mouse promoter(17, 18) .
In the human promoter,
several elements have been identified as important for promoter
activity in the pituitary gonadotrope cell line T3. These include
the gonadotrope-specific element (GSE)(18) , the CREs, and the
ACT element(17, 19) . An additional element in
the mouse
promoter, the pituitary glycoprotein hormone basal
element (PGBE), has been identified as an important regulator of basal
transcription in gonadotropes(17, 20) . The
significance of the PGBE in the human promoter is not known.
Although these studies add to our understanding of subunit
expression in gonadotropes, a more extensive analysis of the promoter
is clearly needed to help identify the regions important for regulation
of this gene. We therefore utilized an array of single and double block
mutations spanning nucleotides -430 to -95 of the human
subunit promoter to further define the regulatory elements
necessary for expression. These studies unveiled several potential
interactions that collectively define a new composite regulatory
element that controls pituitary-specific expression and established the
presence of two new elements,
BE1 and
BE2, important for
expression in gonadotropes.
Generation of each of the mutant constructs was
accomplished by bidirectional PCR using Deep Vent DNA polymerase (New
England Biolabs, Beverly, MA). The reactions were performed using the
buffer supplied by the manufacturer in the presence of 300 µM each dNTP, 100 pmol of each primer, 2 ng of H(-1500)CAT
template, and 1.5 units of Deep Vent polymerase per 100-µl
reaction. For each clone, two independent PCR reactions were performed:
an upstream PCR reaction using a standard 5`-primer plus a 3`-mutant
primer and a downstream PCR reaction using a 5`-mutant primer and a
3`-standard primer. The primer set for each mutant contained
overlapping sequence within the mutation in which there was a common
restriction enzyme site. The reaction mixtures were precipitated with
ethanol, and the DNA was redissolved in the appropriate restriction
enzyme buffer and digested with restriction enzymes compatible with
both the H
(-845)Luc cloning vector and the restriction
enzyme common to both mutant primers. The products of the digestion
were separated by electrophoresis on an 6-8% polyacrylamide gel
in TBE and 5% glycerol. After staining, the bands of the appropriate
size were excised and isolated. The new clones were generated by a
triple ligation with each digested PCR product and a
H
(-845)Luc vector that was digested with either NsiI/SnaBI or SnaBI/PstI.
To
generate the H(-1500)Luc mutants, the upstream fragment,
which extends from the BglII site at -845 to the HindIII site at approximately -1500, was subcloned into
each mutant using the BglII site and an appropriate site
within the multiple cloning site. All clones were confirmed by
sequencing using the Sequenase 2.0 sequencing kit from U. S.
Biochemical Corp.
Double mutants were made by subcloning DNA
fragments containing one mutation into a vector having the second
mutation. The double mutant H(-1500)µ7/µ14 was made
by subcloning the 600-bp SnaBI-BglII fragment of
µ7 into the analogous site in H
(-1500)µ14.
H
(-1500)µ8/µ14 contains the SnaBI-BglII fragment of µ8 inserted in the
analogous position in H
(-1500)µ14.
H
(-1500)µ11/µ14 contains the 846-bp SauI
fragment of H
(-1500)µ14 cloned into the analogous site
in H
(-1500)µ11. H
(-1500)µ7/µ11
contains the 600-bp SnaBI-BglII fragment of µ7
into the analogous site in µ11. H
(-1500)µ8/µ11
contains the 600-bp SnaBI-BglII fragment of µ8
cloned into the analogous site of µ11. The µ7/µ8 clone
required an additional PCR reaction. The PCR reactions for this mutant
were the same as with the original µ8 clone, except that the
3`-primer of the upstream PCR reaction was changed to
5`-CGTAAGAGCTCGGTACCTCCCGGGCTCGGTACCTGTACCTGATATTTAC-3`, and the
template for the PCR reaction was H
(-1500)µ7. The clone
was generated as described above. The mutated sequence within region 8
is altered slightly from the original µ8 mutation in that it has an
8-bp deletion. Removal of bases from region 8 is unlikely to alter the
interpretation of the results, as the binding sites for the proteins in
question are still fully disrupted, and there are no upstream elements
that would be affected by the spacing change.
Luciferase assays were
performed using 10-20 µl of lysate and 100 µl of
luciferase assay reagent (Promega). Emitted light was quantified as
residual light units in a Berthold Lumat LB9501 luminometer (WALLAC
Inc., Gaithersburg, MD). The light units emitted for the human
wild-type subunit promoter were in the range of 1-3
10
residual light units.
-Galactosidase activity was
quantified by luminescence as above using the Galacto-light assay
system (Tropix, Bedford, MA). The light units emitted for
-galactosidase activity were in the range of 2-6
10
residual light units. The linear range of both assays
was determined, and all data presented fell within those ranges. For
each transfection, the luciferase/
-galactosidase activity of each
construct was normalized to the luciferase/
-galactosidase activity
of the wild-type promoter. The values were then averaged over a minimum
of three independent experiments.
Figure 1:
Sequence of the human
subunit gene promoter from -445 to -95. The sequence of the
human
subunit promoter from bases -445 to -95 is
given on the top line in upper case letters. The boxed regions indicate the numbers and positions of each
mutation with the mutant nucleotides shown below the wild-type
sequence in lower case letters. Also shown are the names of
relevant response elements positioned next to the mutation number.
These include PGBE(34) , GSE(18) ,
URE(7, 9, 10, 31, 35) ,
CRE(6, 11, 12) , and
JRE(14) .
Results from transfection in BeWo cells confirmed that activity of this promoter is regulated by three main elements that lie within the regions disrupted by µ13 (URE), µ14 (tandem CREs), and µ15 (JRE) (Fig. 2A). All of these reside within the first 182 nucleotides upstream of the transcriptional start site. Mutations in either the URE or the CREs had a dramatic effect on promoter activity, whereas the effect of the JRE mutation was less pronounced. These elements have been identified previously as important for placenta-specific expression(6, 7, 8, 9, 10, 11, 12, 13, 14) .
Figure 2:
Transfection analysis of the human
subunit promoter in BeWo and
T3 cells. Mutations indicated in Fig. 1were inserted into 1500 bp of the human
subunit
promoter and used to drive expression of the luciferase reporter gene.
Human
subunit promoter constructs were cotransfected with Rous
sarcoma virus-
-galactosidase into either
T3 or BeWo cells.
The luciferase activity of each construct was determined and normalized
to
-galactosidase activity to control for transfection efficiency.
The data represent the luciferase/
-galactosidase activity of each
mutant or truncated promoter normalized to the
luciferase/
-galactosidase activity of the wild-type construct.
Corresponding mutations are indicated below the bars. A, results of transient transfection analysis in BeWo cells. B, results of transient transfection analysis in
T3
cells. At the top of both A and B are
schematic diagrams showing the relative positions of each mutation. Shading of the elements represents the relative impact on
promoter function, where darker shading indicates a greater
effect on function. Transfections were done a minimum of three times,
and the error bars represent standard error of the mean. Numbers above bars indicate the relative activity of each
construct.
In contrast to BeWo cells, expression in T3 cells requires a
more complex array of elements that lie within regions disrupted by
µ7, µ8, µ11, µ13, µ14, and to a lesser extent
µ9 (Fig. 2B). Elements disrupted by µ11,
µ13, and µ14 have previously been described for their role in
regulating expression of the human
subunit gene in gonadotropes
and correspond to the GSE, the URE, and the CREs,
respectively(17, 18, 19) . µ7 is a
mutation through the PGBE, an element that was described previously
only in the mouse promoter(17, 20) . This study
therefore establishes a role for the PGBE in transcriptional regulation
of the human promoter as well. More importantly, the presence of a new
element within the bases altered by µ8 has been revealed. Further
analysis of this element is presented below.
An important attribute of the block replacement approach is the ability to compare the relative contributions that each element makes to promoter function while maintaining the normal sequence environment. In the placental cell line, the tandem CREs are clearly the dominant basal element. Mutation of this element dramatically reduced promoter activity to 2% of wild type (Fig. 2A). Mutation of the URE reduced activity to 15% of wild type, while mutation of the JRE only reduced activity to 32%. In contrast, the main elements involved in gonadotrope-specific expression had less dramatic effects on promoter activity, with the majority of the mutations reducing promoter activity to between 30 and 50% of wild type (Fig. 2B). Although not as clearly dominant as in BeWo cells, the CREs did have the strongest effect on promoter activity in gonadotropes. Thus, while expression in the placenta appears to be controlled by two strong elements, expression in the pituitary is regulated by an array of weaker elements. Furthermore, these results show that distinct arrays of regulatory elements direct expression to placenta and pituitary.
Figure 3:
Transfection analysis of smaller mutations
within region 8 of the subunit promoter. A, sequence of
region 8 (shaded area) and adjoining regions are indicated in upper case letters. The bracketed areas indicate the
numbers and positions of each mutation with the mutant nucleotides
shown in lower case letters either below the
wild-type sequence (µ8.1, µ8.2, µ8.4, µ9.1) or above the wild-type sequence (µ8.3). B, results
of transient transfections in
T3 cells. The indicated mutations
were placed in the context of -845 bp of the human
subunit
promoter and used to drive expression of the luciferase reporter gene.
The transfections and reported results are as described in the legend
of Fig. 2, except the wild-type construct is
H
(-845)Luc.
Sequence analysis of this region disclosed the presence of a potential Ets protein-binding site spanning positions -301 to -298. This family of transcription factors interacts specifically with sequences containing a common core tetranucleotide, GGAA, where both guanines are required for binding(24, 25, 26, 27, 28, 29) . A mutation that alters the essential guanines of this site (µ8.3) had little influence on promoter activity, suggesting that under basal conditions the transcription factor involved in activation of this promoter via this new element is not a member of the Ets family.
Figure 4:
Electrophoretic mobility shift analysis of
proteins binding to region 8 of the human subunit promoter. A, radiolabeled probe corresponding to bases within region 8 (wt, 5`-TGTCTCTTGTTTATAGGAAAGTGTCAGCT-3`) of the human
subunit promoter was used in an electrophoretic mobility shift assay
with
T3 nuclear extracts. Unlabeled competitors were added to the
indicated assays and include wild type (wt, homologous
competitor added in increments of 50
, 100
, 200
,
500
, 1000
), µ8 (oligonucleotide containing the
sequence to which region 8 was mutated as shown in Fig. 1,
1000
), and nonspecific competitor (NS, 1000
). B, electrophoretic mobility shift assay using the radiolabeled
wild-type oligonucleotide as probe and
T3 nuclear extracts.
Unlabeled competitors were added to the indicated assays and include
wild type (200
), PGBE (5`-ATATCAGGTACTTAGCTAATTAAATGTCT-3`,
200
), and nonspecific competitor (NS,
200
).
EMSA in the presence of competitor oligonucleotides that
contain mutations within the µ8 region was used to identify the
bases important for binding of BP1 (Fig. 5).
Oligonucleotides µ2 through µ5, which contained mutations
between bases -313 and -302, failed to compete the
BP1
signal, indicating that bases within this region are important for
formation of the
BP1 complex. In contrast, oligonucleotides
containing mutations downstream of base -302 (µ6 and µ7)
effectively competed for
BP1 binding, while an oligonucleotide
containing mutations upstream of -312, µ1, was capable of
only partial competition. This indicates that the bases important for
BP1 binding lie between -316 and -302, defining the
basal element one (
BE1).
Figure 5:
Electrophoretic mobility shift analysis of
BP1 using wild-type and mutant competitors. A, sequence
of oligonucleotides used in electrophoretic mobility shift analysis.
The top shows the wild-type promoter sequence from -319
to -277. The boxed area indicates the sequence of the
oligonucleotide radiolabeled and used as a probe (wt). The
base mutations are shown for each oligonucleotide. A dash indicates no change from wild type. B, a radiolabeled
probe corresponding to bases -316 to -288 (wt) was
used in an electrophoretic mobility shift assay with
T3 nuclear
extracts. Competitors were added at a 100-fold molar excess. NS corresponds to an oligonucleotide having a nonspecific
sequence.
Figure 6:
Electrophoretic mobility shift analysis of
proteins binding to -306 to -277 of the human subunit
promoter. A, radiolabeled probe corresponding to bases
-306 to -277 bp (wt2, 5`-TTATAGGAAAGTGTCAGCTTTCAGGATGTT-3`)
of the human
subunit promoter was used in an electrophoretic
mobility shift assay with
T3 nuclear extracts. Unlabeled
competitors were added to the indicated assays and include homologous
competitor (wt2) added in increments of 50
, 100
,
and 200
, PGBE (200
), and nonspecific competitor (NS, 200
).
EMSA in the presence of
unlabeled competitor oligonucleotides containing mutations that span
bases -306 to -285 further defined the region of BP2
binding (Fig. 7). Mutations within µ3, which disrupted the
binding of
BP1, had little or no effect on the binding of
BP2, as indicated by the ability of the µ3 oligonucleotide to
compete effectively. However, mutations in µ7, which had no effect
on the binding of
BP1, interfered with the ability of the
BP2
complex to bind DNA. Thus, the bases required for
BP2 binding are
clearly distinct from those of
BP1. This conclusion is also
supported by the observation that the wild-type oligonucleotide used as
the probe to reveal
BP1 binding is not effective as a competitor
for
BP2 binding, and conversely the oligonucleotide used to reveal
BP2 binding (wt2) does not compete for the binding of
BP1 (Fig. 5). Thus a second element, distinct from
BE1, resides
between bases -296 and -285 and defines the
basal
element two,
BE2.
Figure 7:
Electrophoretic mobility shift analysis of
BP2 using wild-type and mutant competitors. A, sequence
of oligonucleotides used in electrophoretic mobility shift analysis.
The top shows the wild-type promoter sequence between
-306 to -277 and indicates the sequence of the
oligonucleotide radiolabeled and used as a probe (wt2). The
base mutations are shown for each oligonucleotide. A dash indicates no change from wild type. Note that oligonucleotide
µ8 is not the same as the previous µ8 which has the entire
sequence of the region 8 mutated. B, a radiolabeled probe
corresponding to bases -306 to -277 (wt2) was used
in an electrophoretic mobility shift assay with
T3 nuclear
extracts. Competitors were added at a 100-fold molar excess. wt corresponds to an oligonucleotide with the sequence given in Fig. 5. NS corresponds to an oligonucleotide having a
nonspecific sequence.
Figure 8:
Southwestern blot analysis of BE1
binding proteins. 40 µg of
T3 nuclear extracts were resolved
on an SDS-polyacrylamide gel with a 4% stacking gel and a 10% resolving
gel and transferred to nitrocellulose membrane. Membrane-bound proteins
were renatured, blocked, and probed with a radiolabeled probe
corresponding to
BE1 (-316 to -288). Competitors, when
included, were at a 200-fold molar excess to that of the probe.
Unlabeled competitors include wild type (homologous), µ8
(corresponding mutation through region 8 as indicated in Fig. 1), and µ2, µ3, and µ7 (sequences of µ2,
µ3, and µ7 are given in Fig. 5). The results shown from
Southwestern blots in the presence of oligonucleotides µ2, µ3,
and µ7 were from a different experiment than those done in the
absence of competitor, wild-type competitor, or µ8. In this case,
the control blot in the absence of added competitor and presence of
wild-type competitor was nearly identical to the one shown here and
therefore was not included. Migrations of molecular weight markers are
indicated on the right.
Figure 9:
Functional analysis of mutations in
multiple elements of the human subunit promoter. Mutations in
both single elements and pairs of elements were used to drive
expression of the luciferase reporter gene. The relative activities of
each of the promoters observed in the
T3 cell transient
transfection are as described in the legend of Fig. 2. Numbers at the top of the bars give the
activity of each promoter relative to wild type. The names of the
elements mutated as well as the mutation numbers as given in Fig. 1are indicated to the left of each
construct.
Assessment of promoters with paired mutations in
PGBE and GSE (µ7/µ11), BE(1+2) and GSE
(µ8/µ11), and GSE and CRE (µ11/µ14) indicated that
mutations through the GSE had the same relative impact on promoter
activity in the presence or absence of PGBE,
BE(1+2), or CRE.
Thus, a mutation through the GSE decreased promoter activity by
approximately 2.5-fold when mutated in the intact promoter or promoters
with an additional mutation in PGBE,
BE(1+2), or CRE. In
contrast, a paired mutation through PGBE/
BE (µ7/µ8) had no
further impact on promoter activity than either of the individual
mutations, suggesting that both sites must be present for either to be
functional. Interestingly, paired mutations in PGBE/CRE
(µ7/µ14) or
BE/CRE (µ8/µ14) resulted in a greater
loss in promoter activity than the independent contributions of the
individual elements. For example, in the intact promoter, a mutation
through the CREs decreased promoter activity by approximately 6-fold,
while in the absence of either PGBE or
BE(1+2), a second
mutation through the CRE decreased promoter activity by an additional
50-fold. Moreover, in the intact promoter, a mutation through PGBE or
BE(1+2) decreased promoter activity by approximately 3-fold,
while in the absence of the CRE an additional mutation in either of
these elements decreased promoter activity by approximately 20-fold.
Thus, mutations in multiple elements revealed that the transcription
factors binding the PGBE, the
BE, and the CRE influence the
transactivation abilities of each other, while the activity of the
transcription factor binding the GSE does not appear to be influenced
by the presence of the activators binding the other three elements.
A key objective of this study was to address how the
subunit gene achieves its highly restrictive pattern of expression. Our
comparative analysis in BeWo and
T3 cells has revealed a number of
differences as well as similarities between the regulatory codes that
direct expression to placenta and pituitary. While placenta expression
is predominantly controlled by two very strong elements, the URE and
the CREs, pituitary expression is regulated by an array of weaker
elements. This suggests that no single element is absolutely essential
for
subunit gene expression in pituitary. Instead, mutational
analysis of multiple elements indicated that there are a number of key
interactions that are important for gonadotrope-specific expression.
It is clear from our studies and others that the CREs of the human
subunit promoter play an important role in the expression of this
gene in both placenta and
pituitary(6, 17, 19, 30) . In
placental cells, the CREs bind homodimers of the ubiquitous protein
CREB. We have preliminary data indicating that CREB binds the CREs in
T3 cells as well (data not shown). The URE also plays a role in
both placenta and pituitary expression of the human
subunit gene.
In placenta, the URE is actually a composite of several elements; the
ACT, the trophoblast-specific element (TSE), and the
URE1(31) . Here, the
ACT element binds to hGATA-2 and
hGATA-3(19) , while the TSE/URE1 forms an overlapping element
that may bind two functionally interchangeable proteins, TSEB and
UREB(31).
In T3 cells, mutation of the URE had a considerably
less dramatic effect on promoter activity than in BeWo cells,
suggesting that it is less critical for pituitary expression than for
placenta. Steger et al.(19) have shown that the
activity associated with the URE in
T3 cells is due at least in
part to the
ACT element. This element both binds to and is
activated by the transcription factor hGATA-2.
Gonadotrope-specific
expression of the subunit gene requires four additional elements
that are not needed for expression in placenta: the GSE, the PGBE, the
BE1, and the
BE2. The GSE was described previously for its
role in regulating the human promoter and binds the orphan nuclear
receptor steroidogenic factor-1
(SF-1)(18, 32, 33) . The PGBE has only been
described for it role in regulating activity of the mouse promoter, and
one of the proteins that binds this element has been identified as the
Lim-homeodomain protein, LH2(17, 20, 34) .
Our studies show that PGBE is involved in the regulation of the human
promoter as well. More importantly, we have shown through transfection
analysis and binding studies the presence of two new elements,
BE1
and
BE2. These elements bind two different transcription factors,
BP1 and
BP2, respectively, both of which contribute to the
function of the
subunit promoter. The binding of multiple
transcription factors to this portion of the promoter is consistent
with an earlier report indicating that these regions as well as PGBE
are protected from digestion by DNase I(18) .
Southwestern
blot analysis revealed that BE1 binds two proteins of similar
molecular weights, 54,000 and 56,000. The binding profile of these
proteins mimics that of the
BP1 complex observed by EMSA ( Fig. 5and Fig. 8), indicating that the 54- and 56-kDa
proteins are components of the
BP1 complex. The presence of two
proteins by Southwestern blot analysis as opposed to the single
BP1 band observed by EMSA suggests that the 54- and 56-kDa
proteins may comigrate as a single band on an EMSA gel or that
BP1
is a higher order complex consisting of both proteins. The relationship
of the two proteins is not yet known, but it is possible that they are
different forms of the same protein. Attempts to reveal
BP2 by
Southwestern blot analysis were unsuccessful, suggesting that this
protein may be a heterodimer incapable of binding
BE2 when
resolved on a denaturing gel. It may also be that the conditions for
Southwestern blot analysis are too harsh to reconstitute
BP2
binding activity.
Mutational analysis of multiple elements
was used to identify potential interactions between regulatory elements
and their cognate transcription factors that play a role in
cell-specific expression. This analysis disclosed three types of
interactions: independent, dependent, and compensatory (Fig. 9).
The studies indicated that the GSE activates the promoter in a manner
that is independent from PGBE, BE(1+2), or CRE. That
is, when a mutation is present in PGBE (µ7),
BE(1+2)(µ8), or the CREs (µ14), a second mutation in
the GSE(µ11) has the same relative effect on promoter activity as
it does when the rest of the promoter is intact. This implies that
SF-1, the GSE binding protein, can activate transcription to the same
degree in the presence or absence of the proteins binding the PGBE, the
BE(1+2), or the CREs.
This contrasts to what was observed
when mutations were made in both PGBE(µ7) and
BE(1+2)(µ8). In this case, no further impact on promoter
activity was observed with the double mutation (µ7/µ8) when
compared to either mutation alone. This suggests that both sites must
be present (and occupied) to get promoter activation, revealing a dependent or synergistic relationship between the proteins
that bind these elements.
A third type of interaction was implicated
by the results of the double mutations through the
PGBE/CRE(µ7/µ14) and the BE/CRE(µ8/µ14). In the
absence of either PGBE(µ7) or
BE(1+2)(µ8), a mutation
in the CRE(µ14) had a much more dramatic effect on promoter
activity than in the presence of these elements. The reverse is also
true. That is, in the absence of the CRE, a mutation in either PGBE or
BE had a much more dramatic effect on promoter activity than in
the presence of the CRE. This effect suggests that PGBE and
BE
become more important in the absence of a functional CRE and vice
versa. It appears that the PGBE/
BE-binding proteins can
partially compensate for the loss of the CRE-binding proteins
and that the CRE-binding protein(s) can partially compensate for the
loss of the PGBE and
BE-binding proteins. The fact that the
binding of one protein can make up for the loss of the other suggests
that the activation pathways of these proteins converge at some point
to stimulate transcription and suggests the possibility that the
proteins binding these elements share a common coactivator or member of
the general transcription machinery.
A different way of interpreting
these results is that proteins binding the CREs partially interfere
with the ability of the proteins binding PGBE and BE(1+2) to
activate transcription and vice versa. Thus, in the absence of
one protein the other is a much stronger activator. Again, this
suggests that proteins binding these elements interact with each other
either directly or indirectly.
The double replacement mutagenesis
strategy has disclosed several potential interactions between proteins
that bind the CRE, PGBE, BE1, and
BE2. This suggests that
gonadotrope-specific expression occurs through a unique composite
element that, like the placenta-specific enhancer, includes the CRE.
This provides incentive for future studies to identify the biochemical
nature of these interactions.