(Received for publication, October 3, 1995)
From the
Thyrotropin (TSH) is a subunit of TSH, the expression of
which is limited to the thyrotrope cells of the anterior pituitary
gland. We have utilized the thyrotrope-derived TtT-97 thyrotropic
tumors to investigate tissue-specific expression of the TSH
promoter. TSH
promoter activity in thyrotropes is conferred by
sequences between -270 and -80 of the 5`-flanking region.
We have recently reported that the proximal region from -133 to
-100 (P1) is required for promoter expression in thyrotropes.
This region interacts with the pituitary-specific transcription factor
Pit-1. While Pit-1 appears necessary for TSH
promoter activity in
thyrotropes, this transcription factor is not alone sufficient for
promoter activity in pituitary-derived cells. In this report, we have
generated a series of promoter mutations in the P1 region to identify
additional protein-DNA interactions and determine their functional
significance. We have found that Pit-1 interacts with the distal
portion of the P1 region, and a second protein interacts with the
proximal segment of this region. Each protein is able to independently
interact with the TSH
promoter, but neither alone can maintain
promoter activity. Both proteins appear to be necessary for full
promoter activity in thyrotropes. Southwestern analysis with the
proximal segment of the P1 region (-117 to -88) reveals
interaction with a 50-kDa protein. Interestingly, this protein is not
found in the pituitary-derived GH3 cells and may represent a
thyrotrope-specific transcription factor. Further characterization of
this newly identified DNA-binding protein will further our
understanding of the tissue-specific expression of the TSH
gene.
Thyrotropin (TSH) ()is a glycoprotein hormone that is
produced only by thyrotrope cells of the anterior pituitary
gland(1) . TSH contains two dissimilar, noncovalently
associated subunits: the
-subunit, which is shared among other
glycoprotein hormones, and the
-subunit, which is functionally and
immunologically unique and has expression limited to thyrotropes. Gene
expression is affected by trans-acting factors that bind
directly to cis-acting elements within promoter regions of
specific genes(2) . Transfection experiments in thyrotrope
cells have shown that the cell-specific activity of the mouse TSH
promoter is localized between -270 and -80 of the
5`-flanking region(3, 4) . DNase I protection studies
using nuclear protein extracts from a TSH
-expressing mouse
thyrotropic tumor, TtT-97, have identified four cis-acting
elements in the -270 to -80 region: D1 (-253 to
-222), D2 (-196 to -176), P1 (-133 to
-100) and P2 (-86 to -64)(4, 5) . We
have been studying thyrotrope-specific expression of the TSH
promoter and factors involved in this expression.
Three proteins
(Pit-1/GHF-1, thyrotrope embryonic factor (TEF), and mLIM-3) have been
shown to interact with the TSH promoter in the region from
-270 to
-80(6, 7, 8, 9, 10, 11) .
Pit-1 is a well characterized POU-homeodomain, pituitary-specific
transcription factor(12, 13, 14) . Pit-1
protein expression is limited to thyrotropes, lactotropes, and
somatotropes in the anterior pituitary gland(14) . Pit-1 has
been shown to be required for efficient transcription of the growth
hormone and prolactin genes (15, 16) as well as
autoregulation of its own expression(17, 18) . Pit-1
has also been shown to mediate thyrotropin-releasing hormone (TRH) and
cAMP response of TSH
promoter
activity(7, 8, 19) . Pit-1 interacts with
three of the four cis-acting elements in the -270 to
-80 region of the TSH
promoter, D1, P1, and P2(5) .
Studies with the rat and human promoters suggested that the cognate D1
and P1 regions confer Pit-1-mediated stimulation with TRH and
cAMP(8, 19) . We have recently shown that the P1
region is necessary for basal activity of the TSH
promoter in
thyrotropes(20) . A mutation in this region, which disrupted
Pit-1 binding, decreased basal promoter activity to a level seen with a
promoter deletion to -80. While Pit-1 is necessary for basal
activity of the TSH
promoter in thyrotropes, it does not appear to
be sufficient to stimulate promoter activity when transfected into
cells that lack
Pit-1(5, 8, 9, 19, 21) . A
thyrotrope-specific splice variant of Pit-1,
Pit-1T(10, 20) , stimulates the TSH
promoter
through the P1 region, but its role in cell-specific promoter activity
is unclear.
TEF is a member of the leucine zipper (bZIP) gene family
of transcription factors(11) . TEF mRNA appears in the rat
anterior pituitary gland on embryonic day 14, before TSH mRNA. TEF
interacts with a region of the mouse TSH
promoter which is similar
to the D2 thyrotrope extract protected region of the mouse promoter (4) . TEF stimulates TSH
promoter activity in CV-1 cells,
but TEF mRNA expression in adult tissues is not restricted to
thyrotropes, raising questions as to its role as a thyrotrope-specific
transcription factor. Recently, a study of mouse pituitary development
has shown that TEF may be responsible for the initiation of the
thyrotrope phenotype, but Pit-1 and perhaps other factors are necessary
for the persistence of mature thyrotropes(22) .
mLIM-3 is a
member of the LIM homeodomain family of transcription factors and
appears to be pituitary-specific(23, 24) . Bach and
colleagues (23) have shown that mLIM-3 mRNA is present in
developing and adult mouse pituitaries as well as a number of
pituitary-derived cell lines, including the thyrotrope-derived aTSH
cells. They also showed that in combination with Pit-1, mLIM-3
stimulated mTSH promoter activity in CV-1 cells and interacted
with the -120 to -60 region of the promoter in gel
retardation assays.
The P1 region of the mouse TSH promoter is
necessary for cell-specific basal promoter activity and Pit-1, which
interacts with this region, does not appear to be sufficient for full
promoter activity observed in thyrotrope cells. We recently observed
that the DNase I protected footprints in this region differed at the 3`
end between recombinant Pit-1 and the TtT-97 thyrotrope
extract(20) , which suggests that other thyrotrope proteins may
be interacting with the mTSH
promoter. Steinfelder et al.(7) demonstrated that a human TSH
promoter fragment
(-128 to -61) yeilded five distinct protein-DNA complexes
with a thyrotrope extract by gel mobility shift analysis. None of these
complexes were seen using non-pituitary HeLa cell extract. Furthermore,
they proved that four of these complexes contained Pit-1 and one
complex appeared to be due to an unrelated thyrotrope protein. These
data suggest that more than one protein may be interacting with the
TSH
promoter in this functionally critical region. In this report,
we have undertaken a detailed mutagenesis analysis of the P1 region and
identified a second protein that interacts with the proximal portion of
this region and appears to be necessary for basal activity of the
mTSH
promoter in thyrotropes.
Figure 1:
Schematic of mutations of the TSH
promoter P1 region. The -140 to -80 region of the wild-type
TSH
promoter is shown. Putative Pit-1 sites are noted by the boxed sequence. Specific mutation nucleotides are highlighted
by boldface and italic and identified as P1M1, P1M2,
etc.
Figure 2:
DNase I
protection footprinting of the TSH promoter and P1 mutations. The
-392 to +40 mTSH
promoter wild-type and mutation
fragments were excised from pGEM7zf+, gel-purified, and
selectively labeled with [
-
P]dATP and dTTP
by reverse transcription. DNase I protection assays were performed with
30,000 cpm radiolabeled promoter fragment. A, protection assay
with 20 µg of bovine serum albumin, 120 µg of recombinant
rPit-1 extract, 75 µg of TtT-97 nuclear protein extract, or 60
µg of GH3 nuclear protein extract. Undigested probe is shown at the far left. Regions protected by TtT-97 extract are designated
D0, D1, D2, P1, and P2. Regions that interact with Pit-1 are identified
at the far right. B, protection assay with the
individual TSH
promoter mutations. WT, wild type. Lanes
are identified as: 0, bovine serum albumin; P,
recombinant rPit-1 protein; T, TtT-97 nuclear extract. Pit-1
binding and TtT-97 extract extended protections are noted at the right. PP-1 is the proximal P1-protected area. Sequences of
the wild-type promoter and specific mutations are noted at the bottom. Putative Pit-1 binding sites are noted by the boxed sequence.
Fig. 2B shows DNase I protection with recombinant Pit-1 and TtT-97 extract
protein on the eight different P1 region mutations. Mutations P1M1,
P1M2, P1M6, and P1M8 appeared to have no effect on either Pit-1 or
TtT-97 protein binding. In contrast, mutations P1M3 and P1M4 disrupted
recombinant Pit-1 binding as well as the distal portion of the
footprint generated with TtT-97 extract, but the proximal P1-protected
region (PP-1) was preserved with the TtT-97 extract only, suggesting
that a protein other than Pit-1 in the thyrotrope nuclear extract
interacts with the proximal portion of the P1 region. The P1M5 mutation
abrogated binding of Pit-1 and TtT-97 extract. This mutation
corresponds to a previously reported random mutation (20) which
also disrupted Pit-1 and TtT-97 extract binding. The P1M7 mutation
disrupted binding of TtT-97 extract to the proximal P1 region (PP-1),
but this mutation had no effect on Pit-1 binding or the binding of
Pit-1 in the TtT-97 extract. These data suggest that two separate
thyrotrope proteins interact with the P1 region of the mTSH
promoter and that each protein interacts independently of the other.
Figure 3:
Transfection of TSH promoter plasmids
in TtT-97 cells. 7-10 million TtT-97 cells were co-transfected
with 20 µg of each TSH
promoter-luciferase plasmid and 1
µg of pCMVbgal by electroporation. After 16 h of incubation at 37
°C, cell extracts were prepared and luciferase, and
-galactosidase activities were measured. Activity is shown as
percent of a wild-type promoter ± S.E. The number of individual
transfections are designated by n. Asterisks note a
significant reduction (p < 0.05) in activity compared with
the M6 and M8 promoter activities.
Figure 4:
Southwestern blot of nuclear extract
proteins. 50 µg of each protein extract was size-separated on a 10%
SDS-acrylamide gel and transferred to nitrocellulose. After
denaturation-renaturation with guanidine hydrochloride, binding
reaction was carried out with a [-
P]dATP
end-labeled duplex oligonucleotide corresponding to the -177 to
-88 wild-type (lanes 1-3) and M7 mutated (lanes 4-6) region of the mTSH
promoter. TtT-97 (lanes 1 and 4),
TSH (lanes 2 and 5), and GH3 (lanes 3 and 6) are noted.
Molecular size standards are noted at the left.
In this report we have used mutagenesis, DNA footprinting,
and gene transfer studies to dissect the functionally important P1
region of the mTSH promoter. These results strongly suggest that
more than one protein interacts within this region, a predictable
conclusion when one considers the size of the native thyrotrope-derived
footprint (47 base pairs). Many lines of evidence indicate that the
pituitary transcription factor Pit-1 or one of its isoforms is one of
the factors that interacts with this region and appears necessary for
activation of the TSH
promoter. Pit-1 interacts with three regions
in the -390 to +40 segment of the mTSH
promoter which
confers cell-specific activity, D1 (-295 to -222), P1
(-133 to -86), and P2 (-80 to -62) ((5) , Fig. 2A). Several reports have shown
that Pit-1 is necessary for TRH- and cAMP-mediated stimulation of the
TSH
promoter, occurring through the D1- and P1-related regions in
the rat and human promoters
respectively(6, 7, 8, 9, 19, 28) .
Kim et al.(9) propose that Pit-1 acts together with
an AP-1-like factor, which interacts with a TGGGTCA motif at -1
to +6 of the hTSH
promoter to mediate TRH stimulation.
Steinfelder et al.(19) observed that mutations of
either Pit-1 site in the P1 equivalent region of the hTSH
promoter
reduced activity by approximately 50% in GH3 cells, while forskolin and
TRH stimulation was reduced by more than 60% in the upstream Pit-1 site
(P1M3 equivalent) and only 20-30% in the downstream site (P1M6
equivalent). Lin and colleagues (22) mutated two putative Pit-1
binding sites, the P1M3 equivalent at -122 to -116 and a
proximal site at -76 to -69. This double mutant promoter
was no longer stimulated by Pit-1 in CV-1 cells. Basal activity in
pituitary-derived cells was not studied.
We have recently shown that
a mutation in the P1 region of the mTSH promoter, which disrupted
Pit-1 binding, abrogated basal activity of the promoter in TtT-97
thyrotropes, but had no effect in GH3 somatotropes(20) ,
suggesting that both Pit-1 and the P1 region are necessary for
cell-specific basal activity of the mTSH
promoter only in
thyrotropes. However, introduction by gene transfer of Pit-1 into
various Pit-1-deficient cell types has little or no effect on TSH
promoter
activity(5, 8, 9, 19, 21) ,
indicating that while Pit-1 may be necessary for TSH
promoter
activity, it is not sufficient for promoter activity.
In this study,
we have identified a segment of the P1 region, the proximal P1 region
(PP-1), which interacts with a non-Pit-1 protein and is separately and
equally critical to the region that interacts with Pit-1 for activity
of the mTSH promoter in thyrotropes. Systematic scanning
mutagenesis of this region has revealed separate contact areas for
these two distinct proteins. While all of the mutations affected
promoter activity in thyrotropes, they appeared to segregate into
mutations which did not affect protein-DNA interactions (less than 45%
reduction in activity) and mutations which disrupted binding (greater
than 60% reduction). The reduction of promoter activity seen with
mutations that did not affect protein-DNA interactions may reflect
sensitivity differences between functional and structural assays. P1M1
and P1M2 mutations (Fig. 1) appear to have little effect on
protein binding and modest decreased function of the mTSH
promoter. Interestingly, the P1M2 mutation changes the final T to a G
in the Pit-1 binding site AATNCAT. P1M3 and P1M4 mutations both affect
Pit-1 interaction with the P1 region, but do not alter protein binding
at the PP-1 region. These mutations cause a greater reduction in basal
activity of the mTSH
promoter than the mutations which do not
affect protein-DNA interaction. The P1M3 mutation significantly alters
the Pit-1 binding sequence, while the P1M4 mutation does not directly
alter this sequence but creates changes seen with a previously
described mutation (20) that also affected Pit-1 interaction
with the promoter. The P1M4 mutation does change the AA preceeding the
Pit-1 binding site to CC. This A/T-rich region preceeding the core
binding site is necessary for high-affinity Pit-1 interaction as well
as promoter function(20, 29, 30) . The P1M5
mutation appears to alter interaction of both proteins with the
promoter. Promoter activity is reduced greater than 60%, but not to the
level seen with the M3, M4, and M7 mutations which disrupt individual
protein binding. It appears that this mutation affects binding of both
proteins, but Dnase I protection (Fig. 2B) shows that
Pit-1 and TtT-97 footprints are slightly different than the bovine
serum albumin control and may reflect partial protein-DNA interaction.
Surprisingly, the P1M6 mutation, while altering a putative Pit-1
binding sequence (AAANCAT), has no effect on protein-DNA interaction
and modestly affects promoter function. This data implies that the
upstream Pit-1 binding element is the critical element for protein-DNA
interaction. The P1M7 mutation does not affect Pit-1 interaction with
the promoter, but the novel protein no longer binds to the PP-1 region.
This is the only mutation that affects binding in the PP-1 region and
yet does not alter Pit-1 interaction with the promoter. Basal activity
of the promoter is greatly reduced by this mutation, suggesting that
the two factors can interact with the promoter independently of binding
by the other, but both are necessary for full basal activity of the
promoter. These systematic mutations clearly illustrate that two
separate proteins are interacting at overlapping regions on the
mTSH
promoter and that neither factor alone is capable of
conferring full promoter activity in thyrotropes. Identification of the
factor that interacts with the PP-1 region, and the nature of its
functional interaction with Pit-1 will greatly increase our
understanding of thyrotrope-specific activity of the TSH
promoter.
Using Southwestern blot analysis with a duplex oligonucleotide
corresponding to the PP-1 segment of the P1 region (-117 to
-88), we have shown that a 50-kDa protein in thyrotrope-derived
cells interacts with this region. Examination of the sequence in this
PP-1 region reveals an AGATAA motif at -98 to -93. The M7
mutation (Fig. 1), which disrupts this AGATAA sequence,
abrogates both functional and structural interaction of the TSH
promoter and the 50-kDa protein. This sequence resembles a consensus
binding site for the GATA family of transcription factors designated as
GATA-1 through
GATA-6(31, 32, 33, 34) . Steger et al.(34) have shown that the pituitary-derived
T3-1 gonadotrope cell line contains mRNA for mGATA-2 as well as an
mGATA-4 related factor, which has a different message size than the
previously reported mGATA-4(35) . They also demonstrated by gel
mobility shift analysis that two proteins interacted with a human
-subunit gene GATA-containing element (-224 to -97),
and a mutation of this GATA element decreased transcriptional activity
of the
-subunit promoter 2.5-fold in
T3-1 cells. In this
report, we show that a mutation of this GATA element (M7) reduces
transcriptional activity of the TSH
promoter 5-fold in
thyrotropes. Since the proximal portion of the P1 element in the
mTSH
promoter contains this consensus AGATAA sequence, and GATA
proteins are present in pituitary-derived cell lines, a GATA-related
protein may be present in thyrotropes and involved in expression of the
TSH
gene in thyrotropes. Furthermore, the 50-kDa protein
identified by Southwestern blot analysis is consistent with sizes of
GATA factors reported previously(34) .
Two groups have
recently described another homeodomain transcription factor, mLIM-3 (24) or P-Lim(23) . mLIM-3 is a LIM (Lin-11, Isl-1,
Mec-3) homeodomain protein which has a cysteine-rich domain which
contains two adjacent zinc-coordinated structures, referred to as the
LIM domain. This transcription factor mRNA has been identified in adult
mouse pituitary as well as pituitary-derived GH3, GH4C1, and T3
cell lines(24) . In the developing mouse pituitary, detection
of mRNA for mLIM-3 was coincident with the appearance of Rathke's
pouch at embryonic day 9 (e9)(23) . In the adult mouse, mRNA
was confined to the anterior pituitary gland and neurointermediate lobe
and not in other areas of the brain or the
liver(23, 24) . Bach et al.(23) performed gene transfer studies in CV-1 cells by
co-transfection of the mTSH
promoter (-1.2 kb),
cytomegalovirus promoter-directed mLIM-3, and CMV-directed rPit-1.
While mLIM-3 alone did not stimulate the TSH
promoter, the
combination of Pit-1 and mLIM-3 cooperatively stimulated the promoter
(50-fold) greater than Pit-1 alone (10-fold). However, mLIM-3 alone
stimulated the
-subunit promoter. They extended their studies to
protein-DNA interaction which showed that a truncated version of mLIM-3
interacted with the -120 to -60 region of the mTSH
promoter, but the full-length protein did not. The -120 to
-60 region of the mTSH
promoter contains the proximal
portion of the P1 footprint (-133 to -98) as well as the P2
footprint (-86 to -64). Both of these regions are AT-rich
and could conceivably interact with a homeodomain protein such as
mLIM-3. mLIM-3 is reported to contain 400 amino acids and has a
predicted protein size of 50 kDa. Interestingly, both groups reported
that mLIM-3 mRNA was present in GH3 cells, and our data indicate that
the 50-kDa protein is not present in GH3 cells by footprint or
Southwestern blot analysis. This suggests that the 50-kDa protein we
have identified is unlikely to be mLIM-3. Therefore, mLIM-3 may be
interacting with the mTSH
promoter in the P2 region.
Alternatively, mLIM-3 protein may not be sufficiently expressed in GH3
cells to be detectable by footprint or Southwestern blot analysis.
In summary, we have identified a 50-kDa protein in thyrotropes that
appears to be necessary for basal activity of the mTSH promoter.
Further characterization of this factor will lead to a better
understanding of TSH
gene regulation and cell-specific gene
expression.