(Received for publication, May 14, 1997)
From the The major histocompatibility complex
(MHC) class I gene cAMP response element (CRE)-like site, Thyrotropin (TSH)1
suppresses major histocompatibility (MHC) class I gene expression in
association with its action to increase the growth and function of rat
FRTL-5 thyroid cells in continuous culture (1, 2). Since enhanced class
I expression has been demonstrated in thyrocytes from patients with
autoimmune thyroid disease (ATD) (3), we proposed (1, 2, 4-6) that TSH suppression of class I levels might be a normal mechanism to preserve self-tolerance in the face of increases in gene products associated with growth and function and that its loss or attenuation might cause
ATD. The importance of suppressing class I to preserve self-tolerance and prevent autoimmunity is becoming clear in multiple disease states.
For example, methimazole and iodide, agents used to treat patients with
Graves' disease, one form of ATD, act in part by suppressing class I
levels in thyrocytes (2, 7), also methimazole prevents the development
of a systemic lupus erythematosus syndrome or autoimmune blepharitis in
experimental models in mice (8, 9). Class I-deficient mice are
resistant to developing these experimental diseases (9, 10), and the
action of methimazole mimics the class I-deficient state in these
experimental diseases (8-10).
TSH/cAMP coordinately decrease expression of the TSH receptor (TSHR)
and class I genes (5, 6), while increasing thyroglobulin (TG) and
thyroid peroxidase (TPO) gene expression. We suggested that
TSH-decreased MHC class I and TSHR gene expression might involve common
transcription factors and that this allowed the cross-talk necessary
for preserving self-tolerance to gene products increased during
TSHR-directed function and growth. Similarly, we considered the
possibility that transcription factors involved in TSH/cAMP-increased
TG and TPO gene expression might suppress class I, since TG and TPO are
major thyroid autoantigens in ATD.
Transcription factors involved in TSH/cAMP regulation of TSHR gene
expression (11-20) include CREB, which binds to the CRE in the TSHR
minimal promoter and is necessary for efficient TSHR expression
(11-13). Thyroid transcription factor-1 (TTF-1), which requires a
double-strand element, and the single-strand binding protein, SSBP-1,
which binds to a noncoding strand element overlapping the 5 In this report, we show that the CRE-like site (TGACGCGA) at Highly purified bovine TSH was obtained from the
hormone distribution program, NIDDKD, National Institutes of Health
(NIDDK-bTSH; 30 units/mg), or was a previously described preparation,
26 ± 3 units/mg, homogeneous by ultracentrifugation, about 27,500 in molecular weight, with the amino acid and carbohydrate composition of TSH (25). [ FRTL-5 rat thyroid cells (Interthyr Research
Foundation, Baltimore, MD; ATCC No. CRL 8305) were a fresh subclone
(F1) that had all properties previously detailed (1, 2, 11-13,
15-20). They were grown in 6H medium consisting of Coon's modified
F12 supplemented with 5% heat-treated, mycoplasma-free calf serum (Life Technologies, Inc.), 1 mM nonessential amino acids
(Life Technologies, Inc.), and a mixture of the following six hormones: bovine TSH (1 × 10 The swine PD15 The sequences of all constructs were confirmed (35), and DNA was
prepared by CsCl gradient centrifugation (36). pSVGH, used to evaluate
transfection efficiency, was a BamHI-EcoRI
fragment encoding the human growth hormone (hGH) gene inserted into the BamHI-XbaI site of the pSG5 expression vector
(Stratagene, La Jolla, CA) (11).
Transient transfections in
FRTL-5 cells were performed as described (11-13, 15-20, 32). In one
procedure, cells were cultivated in 6H medium to approximately 80%
confluency, harvested, washed, and resuspended (1.5 × 107 cells/ml) in 0.85 ml of electroporation buffer (272 mM sucrose, 7 mM sodium phosphate buffer, pH
7.4, and 1 mM MgCl2). Plasmid DNA, 30 µg of
the CAT chimera together with 5 µg pSVGH, was added; 10 µg of
pRcCMV-TTF-1, pRcCMV-TSEP-1, pRcCMV-Pax-8, or control pRcCMV vector was
used when present. Cells were pulsed (330 V; capacitance 25 microfarads), plated (6 × 106 cells/10-cm dish), and
cultured in 6H medium. After 24 h, the medium was aspirated and
aliquots taken for radioimmunoassay of hGH (Nichols Institute). Cells
were rinsed with phosphate-buffered saline at pH 7.4 and then
maintained in 3H or 5H medium plus 5% calf serum supplemented or not
with 10 In the second procedure, FRTL-5 cells were grown to 80% confluency and
then maintained 6 days in 5H medium plus 5% calf serum. Cells were
returned to 6H medium for 12 h and transfected with the CAT
chimeras as described above. Twelve h later, fresh 5H medium with 5%
calf serum was added, supplemented or not with 10 CAT activity was measured as described (1, 11-13, 15-20, 32, 37),
using 10-30 µg of cell lysate and a 130-µl assay volume. Incubation was at 37 °C for 4 h with acetyl-CoA supplementation (20 µl of a 3.5 mg/ml solution) after 2 h. Acetylated
chloramphenicol was separated by thin layer chromatography and
autoradiographed; spots were quantitated in a scintillation
spectrometer. Protein concentration was determined by Bradford's
method (Bio-Rad); recrystallized bovine serum albumin was the
standard.
Cell extracts were made by a modification
of the method of Dignam et al. (38). Briefly, FRTL-5 cells
were harvested by scraping, after being washed twice in ice-cold
phosphate-buffered saline and pelleted. The pellet was resuspended in 2 volumes of Dignam buffer C (20 mM HEPES at pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
pepstatin). The final NaCl concentration was adjusted on the basis of
cell pellet volume to 0.42 M. Cells were lysed by repeated
cycles of freezing and thawing. Extracts were centrifuged at
100,000 × g and at 4 °C for 20 min. The supernatant was recovered, aliquoted, and stored at Oligonucleotides used for EMSA were synthesized or were
purified from 2% agarose gel using QIAEX (Qiagen, Chatsworth, CA) following restriction enzyme treatment of the chimeric CAT constructs described above. They were labeled with [ Electrophoretic mobility shift assays were performed as described
previously (12, 13, 14-20, 32, 40). Binding reactions, in a volume of
20 µl, were for 20 min at room temperature. Reaction mixtures
contained 1.5 fmol of [32P]DNA, 3 µg of cell extract,
and 0.5 or 3 µg of poly(dI-dC) in 10 mM Tris-Cl at pH
7.9, 1 mM MgCl2, 1 mM
dithiothreitol, 1 mM EDTA, and 5% glycerol. Where
indicated, unlabeled double- or single-stranded oligonucleotides were
added to the binding reaction as competitors and incubated with the
extract for 20 min prior the addition of labeled DNA. In experiments
using antisera, extracts were incubated with the serum in the same
buffer for 1 h at 20 °C before being processed as above.
Reaction mixes were electrophoresed on 4 or 5% native polyacrylamide
gels at 160 V in 1 × TBE at 4 °C. Gels were dried and
autoradiographed.
Footprinting, using 1,10-phenanthroline-copper ion, was
carried out essentially as described (41). After a scaled-up EMSA using
an end-labeled fragment, Fr168, comprising All experiment were repeated at
least three times with different batches of cells. Values are the
mean ± S.E. of these experiments where noted. Significance
between experimental values was determined by two-way analysis of
variance and are significant if p values were <0.05 when
data from all experiments were considered.
The CRE-like Sequence between The ability of TSH/cAMP to repress MHC class I transcription has
been mapped to within 127 bp of initiation of transcription (1).
Examination of this 128-bp DNA segment revealed the presence of an 8-bp
sequence,
The ability of the CRE-like element to silence a heterologous promoter
was also assessed by introducing a 38-bp DNA segment, spanning TSH or forskolin significantly decreased the activity of the
p( Table I.
Effect of TSH or forskolin (Fsk) on p( Cell Regulation Section,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
107 to
100 base pairs, is a critical component of a previously unrecognized
silencer,
127 to
90 bp, important for thyrotropin
(TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer
activity is associated with the formation of novel complexes with the
38-base pair silencer, whose appearance requires the CRE and involves
ubiquitous and thyroid-specific proteins as follows: the CRE-binding
protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and
Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and
TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with
the silencer, thereby decreasing maximal class I expression; TSH/cAMP
enhance TSEP-1 and Pax-8 complex formation in association with their
repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8,
prevent formation of the TSH/cAMP-induced complexes associated with
TSH-induced class I suppression, i.e. TSEP-1 appears to be
the dominant repressor factor associated with TSH/cAMP-decreased class
I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or
Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH
receptor gene in thyrocytes, suppression of MHC class II, and
up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of
common transcription factors may, therefore, be the basis for
self-tolerance and the absence of autoimmunity in the face of
TSHR-mediated increases in gene products that are important for thyroid
growth and function but are able to act as autoantigens.
-end of the
TTF-1 site, are enhancers that work together with CREB to maximize TSHR
gene expression. TSH/cAMP decreases the RNA levels of each, decreases
complex formation with the TSHR promoter, and decreases TSHR promoter
activity (15-17, 20). TSHR suppressor element protein-1 (TSEP-1), a
Y-box protein, is a suppressor of the enhancer activity of the TSHR CRE
(19); TSH/cAMP-induced phosphorylation of TSEP-1 is implicated in its
suppressor action (19). Pax-8 is a positive regulator of TG and TPO
gene expression (21, 22); it interacts with some TTF-1 sites on those
promoters but does not interact with TTF-1 sites on the TSHR promoter
(14-16). TSH/cAMP decrease TTF-1 but increase Pax-8 complex formation
and action, accounting for TSH/cAMP-positive regulation of the TG and
TPO genes, despite TSH/cAMP-induced negative regulation of TSHR (15,
16).
107 to
100 bp in the class I promoter, which is homologous to a consensus
CRE (TGACGTCA) (23, 24), is critical for the activity of a hitherto
unrecognized 38-bp (
127 to
90 bp) constitutive silencer of the
class I promoter. TSH/cAMP induce the formation of specific and novel
protein-DNA complexes with the silencer; the induced complexes reflect
the ability of TSH/cAMP to regulate the interaction of multiple
transcription factors with the silencer, the net result of which is
suppression of class I gene expression. We show that TSH/cAMP-induced
suppression of the class I, TG, and TSHR genes involves common
transcription factors, as hypothesized (1, 2, 4-6).
Materials
-32P]Deoxy-CTP (3000 Ci/mmol) and
[14C]chloramphenicol (50 mCi/mmol) were from NEN Life
Science Products and [
-32P]ATP from Amersham Corp.
Anti-CREB-327 or -activating transcription factor-2 (ATF-2), and their
preimmune counterparts, was the gift of Dr. James P. Hoeffler,
Invitrogen, San Diego, CA. Anti-CREB-2 was from Dr. J. M. Leiden
(University of Michigan Medical Center, Ann Arbor, MI) and anti-mXBP
from Dr. L. H. Glimcher (Harvard School of Public Health and
Department of Medicine, Harvard University Medical School, Boston). The
TTF-1 expression vector, pRcCMV-TTF-1, was that used previously (15,
16, 26) and was the kind gift of Dr. Roberto Di Lauro (Stazione
Zoologica A. Dohrn, Villa Comunale, Naples, Italy); the pRcCMV plasmid
used in its construction was from Invitrogen. The Y-box and Pax-8
expression vectors, pRcCMV-TSEP-1 and pRcCMV-Pax-8, were constructed by
ligating their full-length coding sequences with the pRc/CMV vector
(19, 21). All other materials were from the Sigma unless otherwise
noted.
10 M), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml),
glycyl-L-histidyl-L-lysine acetate (10 ng/ml),
and somatostatin (10 ng/ml) (27). Cells were diploid and between their
5th and 25th passage. Fresh medium was added every 2 or 3 days; cells were passaged every 7-10 days. In different experiments, as noted, cells were maintained in 5H medium that contains no TSH or 3H medium
which contains no TSH, insulin, or hydrocortisone.
-flanking sequence-CAT chimeras,
p(
1100)CAT, p(
203)CAT, and p(
127)CAT have been described
(28-32). Other PD1-CAT-chimeras were created by polymerase chain
reaction (PCR; Ref. 33) using 25 ng of p(
203) or p(
127) CAT as
template and 100 pmol each of a forward primer with a BamHI
site on the 5
-end and a reverse primer having the PD1 sequence from
13 to +1 bp of the transcription start site plus a HindIII
site on the 3
-end. The reaction was performed at 94 °C (1 min),
55 °C (2 min), and 72 °C (3 min) for 30 cycles; final extension
was for 7 min at 72 °C; and the amplified fragment was purified
using 1.5% agarose gel electrophoresis. Mutants of p(
127)CAT were
created by two-step, recombinant PCR (33, 34). In the first step, two
PCR products that overlap the sequence were created, both of which
contain the same mutation introduced as part of the PCR primers. The
second step PCR was performed using these overlapped PCR products as template and DNA sequence of the 5
- or 3
-end of the final products as
primer. The PCR products were inserted into the multicloning site of
pSV3CAT (28)2 or
pCAT-promoter or pCAT-Basic vectors purchased from Promega (Madison,
WI). In the case of the pCAT vector, the CRE-like sequence and its
mutants were created with a BamHI site on both ends of the
primers.
10 M TSH or 10 µM
forskolin. After 2 additional days, medium was collected for
radioimmunoassay of hGH and cells were harvested for CAT assay.
10
M TSH or 10 µM forskolin. CAT activity was
assayed 36 h later and conversion rates were normalized to hGH
levels and protein.
70 °C.
-32P]dCTP
using Klenow or with [
-32P]ATP using T4 polynucleotide
kinase and then purified on an 8% native polyacrylamide gel (11-13,
14-20, 32, 39).
168 through
1 bp of the
PD1 promoter, the gel was immersed in 200 ml of 50 mM
Tris-HCl at pH 8.0 and 20 ml of the following solutions were added: 2 mM 1,10-ortho-phenanthroline, 0.45 mM CuSO4, and 58 mM 3-mercaptopropionic acid. After 15 min at room temperature, 20 ml of 28 mM 2,9-dimethyl ortho-phenanthroline was used to
quench the reaction; 2 min later, the gel was rinsed in distilled
H2O and autoradiographed for 40 min at 4 °C, until the
retarded bands were visible. Bands were excised and eluted overnight at
37 °C in 0.5 M ammonium acetate containing 0.1% sodium
dodecyl sulfate and 10 mM magnesium acetate. The eluted DNA
was ethanol-precipitated and resuspended in distilled H2O.
Equal numbers of counts from each sample were dried, resuspended in
98% formamide containing 10 mM EDTA, 0.025% bromphenol
blue, and 0.025% xylene cyanol, and then separated on an 8%
sequencing gel along with G + A and C + T Maxam-Gilbert sequence
reactions (42) performed using the same probe. Autoradiography was at
80 °C overnight.
107 and
100 bp Functions as a
Constitutive Silencer and Is a Target for TSH/cAMP-mediated Repression
of the Class I Promoter
107 to
100 bp (Fig.
1A), with homology to
characterized CREs (23, 24). To determine whether this element
functioned to regulate class I promoter activity, a set of derivative
constructs was generated from a parental construct containing 127 bp of
5
-flanking sequence p(
127CAT). In one derivative, the 8-bp CRE-like
sequence was deleted; in the other, we substituted a nonpalindromic
mutation of the CRE-like octamer (Fig. 1B). Both constructs
displayed increased promoter activity, relative to the parental
construct, when transfected into FRTL-5 cells maintained in the absence
of TSH (Fig. 1B).
Fig. 1.
Effect of modifications of the CRE-like
element on the activity of the p(127)CAT promoter (B) or
on the CRE-like element linked to a heterologous promoter
(C). A, the sequence of the MHC class I gene
between
127 and
90 bp is presented to show the position of the
CRE-like site. The numbering system used is that in our previous report
(32). B, the 8-bp CRE-like element in p(
127)CAT was either
deleted (
CRE) or mutated to a nonpalindromic sequence (NP
CRE) as noted. The CAT activities of these derivative constructs
were compared with that of the parental p(
127)CAT following
transfection into FRTL-5 cells and their incubation in 5H medium plus
5% calf serum for 2 days. Conversion rates were normalized to hGH
levels and protein; then CAT activities were expressed relative to the
parental p(
127)CAT, which averaged 2-fold higher than the pSV0-CAT
control. Statistically significant differences (p < 0.05) from p(
127)CAT are noted by the asterisk. C, the sequence between
127 and
90 bp (designated
CRE) was introduced at the 3
-end of constructs containing
the SV40 promoter ligated to the CAT gene and then transfected into
FRTL-5 cells ("Experimental Procedures"). CAT activities were
measured after maintaining the cells for 2 days in 5H medium plus 5%
calf serum. Constructs are diagrammatically represented on the
left of the figure; arrows depict the orientation
and the number of CRE copies. CAT activities are presented relative to
the parental promoter construct, pCAT Promoter, which contains a
minimal SV40 promoter; statistically significant decreases are
indicated as p < 0.05 (*) or p < 0.01 (**). Data in B and C are the mean ± S.E.
for three separate experiments; results in both panels were duplicated
with cells maintained in 3H medium plus 5% calf serum. For example, in
B, when cells were incubated in 3H medium plus 5% calf
serum for 2 days after transfection, CAT activities were 100%,
196 ± 9%, and 208 ± 8% for p(
127)CAT, p(
127)
CRE
CAT, and p(
127) NP CRE CAT, respectively, in three separate
experiments (mean ± S.E.).
[View Larger Version of this Image (41K GIF file)]
127 to
90 bp, downstream of an SV40 minimal promoter (Fig. 1C).
The choice of a 38-bp segment was derived from the experiments
described below. When placed in a 5
to 3
orientation, a single copy
of this DNA segment was able to significantly reduce SV40 promoter
activity, and the magnitude of the effect increased with the number of
copies of the 38-bp segment inserted (Fig. 1C). When placed
in a 3
to 5
orientation, two copies of this DNA segment were also
able to significantly reduce SV40 promoter activity (Fig.
1C). Derivatives of the 38-bp segment, containing either a
deletion (
CRE 1 (+)) or nonpalindromic (NP CRE 1 (+)) mutation of
the CRE-like element, did not similarly decrease SV40 promoter activity
(Fig. 1C).
127)CAT construct in FRTL-5 cells relative to untreated controls (Table I, A, columns 3-5), i.e. TSH enhanced the silencer
activity. Deletion or mutation of the CRE, which increases the
constitutive level of promoter activity in the absence of TSH,
diminished the repressive response to TSH (Table I, A, columns 3-5).
The role of the CRE-like site in conferring TSH/cAMP responsiveness was confirmed in studies using the 38-bp silencer linked to the
heterologous promoter (Table I, B). Although the SV40 promoter alone
did not respond to TSH or forskolin, the promoter activities of a
construct containing a single copy of the CRE 1 in a sense orientation
(CRE 1 (+)) or of constructs containing one or two copies of CRE 1 in a
3
to 5
orientation (CRE 1 (
), CRE 2 (
)), but not their nonpalindromic mutations, were significantly reduced by TSH or forskolin (Table I, B, columns 3-5).
127)CAT or pCAT promoter
constructs with an intact, deleted, or mutated (nonpalindromic) CRE
127)CAT promoter construct was
either deleted (
CRE) or mutated to a nonpalindromic sequence (NP
CRE) as noted (Fig. 1B). The CAT activities of these derivative constructs were compared with that of the parental p(
127)CAT following transfection into FRTL-5 cells and their incubation in 5H medium plus 5% calf serum with or without 1 × 10
10 M TSH or 10 µM forskolin for
36 h. B, class I DNA sequence between
127 and
90 bp
(designated CRE) were introduced at the 3
end of constructs containing
the SV40 promoter ligated to the CAT gene (Fig. 1C) and
transfected into FRTL-5 cells as in Fig. 1. CAT activities were
measured after maintaining the cells for 36 h in 5H medium plus
5% calf serum with or without TSH or forskolin. A and B, conversion
rates were normalized to hGH levels and protein ("Experimental
Procedures"); CAT activities were then expressed relative to the
parental p(
127)CAT or pCAT promoter control. Data are the mean ± S.E. for three separate experiments.
CAT activity relative to p(
127)CAT or pCAT promoter
control (%)
Promoter Construct
Control
+TSH (1 × 10
10 M)
+Forskolin (10 µM)
Relative effect
(+/
TSH)
(+/
Fsk)
A.
p(
127)CAT
control
100
35
± 1.5
33 ± 2.5
0.35
0.35
p(
127)
CRE
CAT
160 ± 11a
120 ± 6.0b
134
± 5.0b
0.75b
0.84b
p(
127)
NPCRE
220 ± 15b
165 ± 15b
176
± 9.0b
0.75b
0.8b
B.
pCAT
promoter control
100
100 ± 5
102
± 4
1.0
1.02
pCAT promoter CRE 1 (+)
68
± 7c
27 ± 8d
24
± 6d
0.4d
0.35d
pCAT promoter CRE
1 (
)
103 ± 5
28 ± 7d
31
± 5d
0.27d
0.30d
pCAT promoter
CRE 2 (
)
53 ± 4c
20 ± 5d
22
± 7d
0.38d
0.42d
pCAT promoter
NPCRE 1 (+)
172 ± 8
169 ± 9
181
± 8
0.98
1.05
pCAT promoter
CRE 1 (+)
167
± 9
170 ± 8
170 ± 4
1.02
1.02
a
Increased activity relative to p( 127)CAT control,
p < 0.05 or better.
b
Decreased activity relative to control cells in the absence
of TSH, p < 0.05.
c
Decreased activity relative to pCAT promoter control in the
absence of TSH, p < 0.05.
d
Decreased activity induced by TSH or forskolin,
p < 0.05 or better.
From these data, it is concluded that the 8-bp CRE-like site is
important for the function of a constitutive silencer located in a
38-bp fragment of the class I 5-flanking region,
127 to
90 bp from
the start of transcription. The 38-bp silencer is responsive to TSH or
its cAMP signal; the CRE-like element within it is necessary for this
functional response. The residual suppressive effect of TSH in
p(
127)CAT chimeras containing a CRE deletion or mutation (Table I, A)
suggests, nevertheless, that this may not be the sole site of TSH/cAMP
action and that additional sites downstream of
90 bp might be
TSH/cAMP-responsive.
TSH/cAMP Induces Novel Complexes Whose Formation Depends on the CRE-like Element
When a DNA fragment encompassing the silencer, 168 to
1 bp
(termed Fr168 (Fig. 2A, bottom), was used in gel
mobility shift assays with extracts derived from FRTL-5 cells cultured
with or without TSH, a multiplicity of protein-DNA complexes was formed with either extract (Fig. 2A).
Whereas protein-DNA complexes A to E were common to both extracts, TSH
treatment of the FRTL-5 cells induced the appearance of two novel
complexes, F and G (Fig. 2A, lane 2 versus 1). Formation of
the TSH-induced complexes was specific, since their appearance could be
prevented by unlabeled Fr168 (Fig. 2A, lane 3). More
importantly, formation of only the F and G complexes could be prevented
by the 38-bp silencer fragment,
127 to
90 bp, containing the
CRE-like site and termed CRE-1 (Fig. 2A, lane 4).
Forskolin (10 µM) could substitute for TSH to induce the
formation of the F and G complexes with the Fr168 probe (Fig.
2B). Moreover, with either forskolin- (Fig. 2B)
or TSH-treated cells (data not shown), formation of the F and G
complexes could be prevented by a derivative 38-bp fragment, 127 to
90 bp, in which a consensus CRE sequence (CON CRE) was substituted
for the native CRE-like sequence (Fig. 2B, lanes 3 and
4 versus 2) but not by derivative oligonucleotides from
which the CRE-like element had been deleted (
CRE) or mutated to a
nonpalindromic (NP CRE) substitution (Fig. 2B, lanes 5 and
6, respectively, versus lane 2). The region of
the 38-bp silencer 5
to the CRE (termed 5
CRE) did not inhibit formation of the TSH/cAMP-induced complex (Fig. 2B, lane 7 versus 2) nor did a shortened form of CRE-1, termed CRE-2, with only 6 base pairs on either side of the CRE octamer (Fig. 2B, lane 8 versus 2).
Phenanthroline-copper ion footprint analysis of the TSH-induced F or G
complex identified a protected region, 131 to
95 bp, bounded by two
strong hypersensitive sites (Fig. 3) that
encompasses the CRE-like site,
107 to
100 bp. A less prominent
hypersensitive band at
110 bp suggests this region may bind more than
one factor as will be shown below and in a separate
report.3 Although not the
only protected region in the footprint, these data, together with the
data in Fig. 2, established that coincident with TSH/forskolin-induced
suppression of class I RNA levels (1, 2) and TSH/forskolin-activated
silencer activity (Fig. 1; Table I),
TSH/forskolin-induced the appearance of novel complexes with the class
I promoter, whose formation required the CRE-like sequence, as did
silencer activity. The data (Fig. 2) additionally suggested that
sequences flanking the CRE-like site are involved in complex formation,
consistent with the extended footprint (Fig. 3).
The 38-bp DNA Fragment with CRE-dependent Silencer Activity Forms CRE-dependent Complexes with Multiple Proteins: the Effect of TSH on These Complexes and the Functional Role of the Proteins Involved
Identification of a Multiplicity of Transcription Factors That Interact with the 38-bp DNA Fragment Exhibiting CRE-dependent Silencer ActivityTo characterize
proteins capable of interacting with the 38-bp silencer element, we
radiolabeled a double-stranded oligonucleotide spanning the segment
127 to
90 bp (CRE-1) and used it in gel shift assays with extracts
from FRTL-5 cells maintained in the absence of TSH (Fig.
4); subsequently (see below), we
evaluated the effect of TSH on the characterized complexes.
Four sets of complexes were observed (Fig. 4, A-D) with
extracts from cells without TSH, all of which appeared to be specific, since their formation was prevented by competition with unlabeled CRE-1
(Fig. 4A, lanes 3-6 versus 2), albeit with different
affinities. More importantly, formation of all the complexes was
dependent on the CRE-like element. Thus, whereas their formation was
inhibited by the native 38-bp CRE-1 silencer, no comparable competition was evident using the silencer fragment in which the CRE was deleted (Fig. 4A, CRE-1, lanes 7-9, versus 2). An
oligonucleotide derived from the somatostatin receptor (Promega CRE),
which contains a consensus CRE but otherwise unrelated flanking
nucleotides, was unable to duplicate the action of CRE-1 as a
competitor (Fig. 4A, lanes 10-12 versus 2-6). These data
are consistent with interpretation that multiple proteins interact with
the 38-bp silencer, that the CRE is a critical element required for
their binding, but that sequences flanking the CRE are involved in the
binding of most.
As is the case for the TSHR CRE (11-13), one protein interacting with the CRE-like site in the 38-bp silencer is CREB-327 or an immunologically related CRE binding protein (23, 24, 43-45). Thus, the CRE-containing oligonucleotide derived from the somatostatin receptor, which contains a consensus CRE but otherwise unrelated flanking nucleotides, appeared to inhibit the formation of one component within the A complex (Fig. 4A, lanes 10-12 versus 2). Moreover, one of components in the A complex could be super-shifted (Fig. 4B, lane 4) by an antibody to CREB-327 (43) but not by anti-CREB2, anti-mXBP, or anti-activating transcription factor-2 (ATF2-BR).
Three points should be noted with respect to these data. First, in the A complex region, proteins other than CREB are likely to interact with the CRE-like sequences, given the limited effect of anti-CREB antibody in supershifting the complexes. One will be identified as Pax-8 below; however, other CRE binding protein analogs of CREB (23, 24) may be components of the A complex and remain to be identified. Second, the CRE-like element is a functional CRE, since it interacts with CREB and is TSH/forskolin-responsive (11-13, 23, 24, 43-45). Third, the consensus CRE from Promega, while decreasing the formation of a component in the A complex region, appears to enhance the formation of one or more components in the C region (Fig. 4, lane 12). This observation may be relevant to data that reveal a similar action of anti-CREB (see below).
We pursued the identity of other factors forming complexes with the 38-bp silencer. As noted in the introduction, we had hypothesized that transcription factors that negatively regulate class I promoter activity might be the same as some involved in TSH/cAMP-induced negative regulation of the TSHR and/or positive regulation of the TG or TPO promoters (4-6). Two obvious candidates are the tissue-specific factors, TTF-1 and Pax-8. Maximal TSHR gene expression in the absence of TSH is associated with the binding of TTF-1; additionally, TTF-1 acts synergistically with proteins such as CREB, which bind the CRE-like sequence of the TSHR (15). In the TG and TPO promoters, some TTF-1 sites also interact with Pax-8, i.e. the oligo(C) site in the TG promoter (21, 22, 26). TSH/cAMP decreases TTF-1 complex formation with the TSHR and TG promoters but increases Pax-8 complex formation with the TG promoter, coincident with increased TG gene expression (15, 16). We evaluated the possibility that TTF-1 and/or Pax-8 might interact with the class I 38-bp silencer.
DNA binding sites for both TTF-1 and Pax-8 are contained within
oligonucleotide C derived from the TG promoter (Fig.
5C). Oligonucleotide C is able
to completely inhibit formation of complex B and reduce the amount of
complex A, whereas a mutant of oligonucleotide C, which loses the
ability to bind TTF-1 or Pax-8 (21, 22, 26), also loses its inhibitory
effect (Fig. 5A, lanes 4 and 5 versus 2; Fig.
5B, lanes 3 and 4 versus 2). This suggests that either TTF-1 or Pax-8 (or related factors) contribute to these complexes.
To distinguish between TTF-1 and Pax-8 binding, an oligonucleotide that binds only TTF-1, oligonucleotide TTF-1 from the TSHR promoter, or its mutated counterpart (Fig. 5C) which loses TTF-1 binding and activity (15, 16), was substituted in the competition assays (Fig. 5B). Whereas oligonucleotide C affected both complexes A and B, oligonucleotide TTF-1 completely inhibited complex B but did not affect complex A (Fig. 5B, lane 5 versus 2-4). In contrast, the mutant derivative of oligonucleotide TTF-1 does not compete for B complex formation (Fig. 5B, lane 6 versus 2-4). Taken together, these data suggest that complex B contains TTF-1 and that Pax 8 is a component of complex A, in addition to CREB.
Two additional points should be noted in these experiments. First, both
the Pax-8 and TTF-1 complexes require CRE-dependent interactions with the 38-bp silencer. Thus, in the same experiment (Fig. 5A) the 38-bp CRE-1 oligonucleotide, but not its
CRE-1 derivative, prevented the formation of both A and B complexes (Fig. 5A, lanes 1 and 3, respectively,
versus 2).
Second, the concentration of poly(dI-dC) in the assay could change the appearance of the complexes (Fig. 5, A versus B). Higher poly(dI-dC) concentrations (3 µg/assay) were observed to significantly enhance formation of the A and B complexes (Fig. 5, A versus B) but attenuate formation of the C complexes (Fig. 5, B versus Fig. 4). Since TTF-1, CREB, and Pax-8 are double strand-specific in their DNA interactions (12-16, 21, 22, 26), one possibility to explain this phenomenon was that higher concentrations of poly(dI-dC) were suppressing the binding of proteins with lesser affinity or specificity, such as single strand DNA-binding proteins, and that these comprised the protein/DNA adducts in complex C.
To assess this possibility, each of the component single strands of the
38-bp silencer was tested for its ability to inhibit complex formation
in low poly(dI-dC) conditions (Fig.
6A). Neither strand affected
the formation of complexes A or B; however, both single strand
oligonucleotides reduced the formation of complex C, the non-coding
strand sequence more efficiently than the coding strand (Fig.
6A). Thus, whereas the A and B complexes involved double
strand DNA interactions, formation of the components of complex C
appeared to result from the interaction of factors that can bind to
individual strands of the 38-bp CRE-1 silencer.
Two proteins involved in the regulation of the TSHR gene are known to
be single-strand DNA binding proteins. One is a single strand binding
protein (SSBP-1) which was identified by its ability to bind to the
noncoding strand of the TSHR promoter, immediately 5 to and contiguous
with the TTF-1 binding site (17, 20). The second is a Y-box protein
that we cloned and termed TSEP-1 (TSHR suppressor element protein-1),
because of its ability to suppress the constitutive enhancer activity
of the TSHR CRE (19). TSEP-1, like other Y-box family proteins, binds
to both single or double strand DNA (13, 19, 46-48). The ability of
the cognate DNA binding sites of these two factors on the TSHR (Fig.
6C) to inhibit formation of complex C bands was assessed
(Fig. 6B).
An oligonucleotide corresponding to the SSBP binding site, 194 to
169 bp on the non-coding strand of the TSHR minimal promoter, competed for complex C entirely, without affecting either complexes A
or B (Fig. 6B, lanes 3 and 4 versus 2). A second
oligonucleotide containing the TSEP-1 binding site derived from the
coding strand of the TSHR promoter also competed for complex C but not
A or B (Fig. 6B, lanes 6 and 7 versus 5). In
contrast to oligonucleotide SSBP, however, oligonucleotide TSEP-1
inhibited formation of only the major center band within complex C,
sparing adjacent weaker bands. Association of TSEP-1 was, therefore,
clearly specific, since only one of the complexes was competed; it was
unclear why oligonucleotide SSBP competed for all of the bands within
complex C, although we speculated that its binding to the noncoding
strand might exclude the binding of TSEP-1 to the coding strand. To
further assess the specificity of these complexes and characterize
their single strand binding ability, we performed the following
experiments using radiolabeled single strand oligonucleotides with the
sequence of the 38-bp silencer termed CRE-1.
Using a radiolabeled oligonucleotide with the sequence of the CRE-1
coding strand on the class I promoter, we showed that formation of the
complex with the slowest mobility in Fig. 6A was inhibited
by the presence of excess unlabeled wild type oligonucleotide TSEP-1
(Fig. 6C). TSEP-1 binds to a CCTC motif on the terminus of
the 5-tandem repeat (Fig. 6C, TR); mutation of the CCTC
motif, but not a mutation not involving the CCTC motif, resulted in a loss of this competition. These data were consistent with the interpretation that TSEP-1 binding to the 38-bp silencer is one component of the C complex.
SSBP sites on the TSHR are on the noncoding strand of the TSHR, 5 and
contiguous with an upstream and downstream TTF-1 double strand binding
site; each has a GXXXXG binding motif (17, 20), the G
nucleotides of which are underlined in Fig. 6C.
Mutation of the 5
and 3
terminal G nucleotides in the SSBP sites
results in a loss of SSBP binding and function (17, 20). Using the CRE-1 noncoding strand as radiolabeled probe, we showed that formation of one of the protein complexes with the noncoding strand of class I
38-bp silencer (Fig. 6A) was reduced by including an excess of wild type, single strand oligonucleotide able to bind SSBP from the
TSHR (Fig. 6C) but much less so by the SSBP oligonucleotide with its GXXXXG site mutated. These data were consistent
with the interpretation that SSBP-1 or a related protein binds to the 38-bp silencer and is another component of the C complex.
We believe it is reasonable to conclude from these data that complex C includes two major components formed by the interaction of single strand binding proteins, SSBP-1 and TSEP-1, which interact with the TSHR and that the binding of either requires the CRE-like site. We conclude from this series of experiments (Figs. 4, 5, 6) that the CRE is part of a constitutive silencer that associates with a multiplicity of transcription factors, of which CREB appears to be a minor component. The formation of the complexes depends on both the CRE-like element, as well as additional flanking sequences. Among the factors that interact with this silencer are factors that interact with the TSHR (TTF-1, SSBP-1, TSEP-1, and CREB) as well as TG or TPO promoters (TTF-1 and Pax-8). They include, therefore, factors that exhibit tissue specificity (TTF-1 and Pax-8), as well as factors that are ubiquitous (CREB, TSEP-1, and SSBP-1).
Effect of TSH on the Multiplicity of Protein-DNA Complexes Formed with the 38-bp DNA Fragment with CRE-dependent Silencer ActivityTSH treatment of FRTL-5 thyroid cells decreases class I
promoter activity, in part by acting through a 38-bp silencer whose activity is dependent on a CRE-like element, as evidenced above (Fig. 1
and Table I). This is consistent with the observation that TSH
treatment for as little as 12-18 h causes a significant decrease in
class I RNA levels (1, 2). We now show that extracts from cells treated
with TSH for this period alter the amount and composition of the
protein-DNA complexes formed with the 38-bp silencer region whose
activity and binding depends on the CRE (Fig.
7).
Thus, TSH treatment of the cells results in markedly diminished formation of the A and B complexes, which contain CREB, Pax-8, and TTF-1 (Fig. 7, lane 4 versus lane 2). The decreased CREB interaction is evidenced by a diminished ability of anti-CREB-327 to supershift the A complex (Fig. 7, lane 5 versus 3, dashed arrow). The residual component of the A complex involves Pax-8, as evidenced by inhibition of its formation by oligonucleotide C of the TG promoter (data not shown). The simultaneous decrease of the TTF-1 and CREB complexes may be related, since CREB and homeodomain-containing binding proteins such as TTF-1 are known to act synergistically on the minimal promoter of the TSHR (15) and somatostatin receptor (49-51).
In contrast to the A or B complex, the intensity of the C complex region is enhanced by TSH treatment, and the complexes become evident even in the presence of high poly(dI-dC) concentrations (Fig. 7, lane 4 versus 2). Since the C complex is now seen, despite the high levels of poly(dI-dC), this suggests that the specificity and/or affinity of binding by proteins within the C complex are enhanced, also that one or both single strand binding proteins, SSBP-1 or TSEP-1, is now better able to bind double strand DNA.
Interestingly, the addition of anti-CREB-327 in vitro mimics the in vivo effect of TSH treatment of FRTL-5 cells in its ability to increase C complex formation (Fig. 7, lane 3 versus 4 by comparison to lane 2). This action is specific since other antisera, i.e. anti-CREB2, anti-mXBP, or ATF2-BR, do not similarly increase the amount of complex C (data not shown). Furthermore, anti-CREB-327 does not change B complex formation (Fig. 7, lane 3 versus 2 or 4 versus 3). The TSH-induced increase in C complex may, therefore, reflect a TSH-induced decrease in CREB binding mimicked by anti-CREB-327 in vitro. Nevertheless, it seems clear that TSH increases the binding of TSEP-1 and/or SSBP-1 (complex C) while decreasing CREB and TTF-1, but not Pax-8 binding, to the 38-bp silencer (complexes A and B).
These data raised the possibility that the novel complexes, which are
evident when TSH/forskolin-treated extracts are incubated with Fr168 of
the 5-flanking region of the class I promoter, reflect the TSH-induced
change in complexes with the 38-bp silencer, particularly the binding
of TSEP-1 and/or Pax-8, since both are suppressors of class I gene
expression as will be shown below. To evaluate this possibility, we
tested the ability of oligonucleotides containing these binding sites
on the TSHR or TG promoters to compete for the TSH-induced complexes
formed with radiolabeled, double strand Fr168 or Fr127 (Fig.
8).
Oligonucleotide TIF from the TSHR insulin response element (18), like
oligonucleotide TSEP-1, contains a CCTC motif and TSEP-1 binding site
(Fig. 8C). In the presence of excess, unlabeled single
strand oligonucleotide TIF(), formation of the TSH-induced complexes
with either double strand Fr168 or Fr127 are inhibited (Fig. 8A,
lane 1 versus 2 and Fig. 8B, lane 3 versus
2, respectively), as is the case for the positive control, double
strand oligonucleotide CRE-1, the 38-bp silencer itself (Fig. 8B,
lane 7 versus 2). In contrast, a single strand form of
oligonucleotide TIF(
), with a mutation in the CCTC motif that is
important for TSEP-1 binding (mutant 2), did not compete (Fig.
8A, lane 3 versus 2; Fig. 8B, lane 5 versus 2),
whereas the oligonucleotide TIF(
)-derivative with a mutation that
does not involve the CCTC motif, mutant 1, remained a competitor (Fig.
8A, lane 4 versus 2; Fig. 8B, lane 6 versus 2).
These results were duplicated by wild type and mutant single strand
oligonucleotide TSEP-1(+) and oligonucleotide S-box(+) from the TSHR
(Fig. 8C), which also have TSEP-1 sites (data not shown).
Oligonucleotide C, an oligonucleotide that binds the double-stranded binding proteins TTF-1 and Pax-8 (Fig. 5), did not affect the TSH-induced complex (Fig. 8B, lane 4 versus 2). The oligonucleotide with the sequence of the SSBP site also did not inhibit formation of the TSH-induced complex (data not shown).
We conclude from these data (Fig. 8), that TSEP-1, a Y-box protein
involved in TSH/cAMP suppression of TSHR gene expression in FRTL-5
cells, is a dominant component in the formation of the TSH/cAMP-induced
novel complex with the CRE-like silencer of the MHC class I gene.
Formation of this novel complex is associated, therefore, not only with
TSH/cAMP-induced suppression of class I gene expression (Fig. 1; Table
I), which requires an intact CRE-like site, 107 to
100 bp, but also
with the ability of TSH to increase the binding of the Y-box protein to
the 38-bp silencer in a CRE-dependent manner (Fig. 7).
We evaluated the
functional effect of TSEP-1, TTF-1, and Pax-8 on the activity of the
construct containing 127 bp of 5-flanking sequence, p(
127)CAT, and
on a derivative, in which the 8-bp CRE-like sequence was deleted
p(
127
-CRE)CAT. Each was cotransfected into FRTL-5 cells,
maintained in the absence of TSH, along with plasmids containing the
TSEP-1, TTF-1, and/or Pax-8 cDNAs: pRcCMV-TSEP-1, pRcCMV-TTF-1,
pRcCMV-Pax-8, respectively (Fig. 9).
Transfections with pRcCMV, the vector's only construct, served as the
control in each case.
Cotransfection of pRcCMV-TSEP-1 decreased the promoter activity of
p(127)CAT but not the activity p(
127
-CRE) CAT (Fig. 9A); the same result was obtained comparing the effect of
pRcCMV-TSEP-1 on p(
127)CAT versus p(
127 NP CRE)CAT,
which has a nonpalindromic mutation of the CRE as described in Fig.
1B. In contrast to pRcCMV-TSEP-1, cotransfection of
pRcCMV-TTF-1 increased the promoter activity of p(
127)CAT but not the
activity p(
127
-CRE)CAT (Fig. 9B). pRcCMV-Pax-8
decreased the promoter activity of p(
127)CAT but not the activity
p(
127
-CRE)CAT (Fig. 9C), and its presence prevented
the enhancer activity of pRcCMV-TTF-1 (Fig. 9D). The effect
of Pax-8 to reduce p(
127)CAT activity in Fig. 9C is
ascribed to its action on endogenous TTF-1, whose levels are maximally expressed in FRTL-5 cells maintained in 5H medium with no TSH (15). In
each case, the activity was not duplicated by the vector control,
pRcCMV.
These data indicated that TSH, which increased the binding of TSEP-1 but decreased the binding of TTF-1 to the 38-bp silencer, was increasing the binding of a suppressor of class I activity (TSEP-1) while decreasing the binding of an enhancer (TTF-1). Moreover, Pax-8, whose binding to the 38-bp silencer is not decreased by TSH, also suppresses the enhancing activity of TTF-1. Since TSH-induced decreases in TTF-1 RNA levels and complex formation in the TSHR are only partial (15, 16), the Pax-8 action may explain the apparent complete loss of TTF-1 complex formation associated with TSH effects on the silencer complex of class I (Fig. 8).
MHC class I molecules are expressed on the cell surface and are vehicles for the presentation of antigenic peptides to immune cells; quantitative and qualitative variations in class I expression play important roles in determining the T-cell response (52, 53). MHC class I can be positively or negatively regulated in response to virus infections or lymphokines (54); recently we showed that class I levels could also be hormonally regulated and suggested this hormonal regulation might be important to suppress autoimmunity during hormonally induced changes in growth and function which resulted in altered levels of gene products known to be autoantigens (1, 2, 4-6).
The present experiments were aimed at characterizing the molecular
mechanisms governing class I transcription in thyrocytes and those
mediating the TSH/cAMP-induced decrease in transcription. With respect
to the former, we show, for the first time, that a CRE-like sequence,
107 to
100 bp from the start of transcription, functions as a
constitutive silencer of the class I gene. Thus, not only does its
deletion or mutation in the homologous class I promoter result in
increased activity, its insertion within a 38-bp surrounding region
into a heterologous promoter decreases promoter activity as a function
of copy number, so long as the CRE is intact. With respect to the
latter, we also show that TSH activates the silencer, that the TSH
action requires an intact CRE-like sequence, that this is one mechanism
by which the hormone decreases class I expression, and that the action
of TSH is cAMP-mediated, since it is duplicated by forskolin.
The CRE-like sequence appears to be a functional CRE and can bind CREB, although the binding of other CRE binding proteins as homo- or heterodimers is not excluded and is even likely. The ability of TSH to decrease CREB interactions with the silencer, in association with decreased class I transcription, suggests that CREB binding to the silencer normally functions as an enhancer of class I gene activity, in accord with its known mode of action in other genes (23, 24). One possibility for the action of TSH to decrease CREB binding is to alter its homo- or heterodimer structure. This is evidenced in our separately submitted report.3 The ability of anti-CREB to alter the CREB complex, and thereby duplicate the in vivo effect of TSH/cAMP-treatment of cells to increase the CRE-dependent formation of complex C and its component TSEP-1 suppressor, supports the important role of CREB binding. The decrease in CREB binding appears to be an important component of the TSH-increased binding of TSEP-1 and the TSH/cAMP-induced class I repressive effect. Since CREB and TSEP-1 are ubiquitous proteins, this result may be relevant to other tissues where hormones regulate the growth or function of a cell via the cAMP signal transduction system.
TSH/forskolin treatment of FRTL-5 cells induce the formation of a novel
complex with the class I promoter, whose existence is dependent on the
38-bp silencer and its CRE. Thus, its formation with class I promoter
fragments containing 168 or 127 bp of 5-flanking region is prevented
by an unlabeled oligonucleotide with the sequence of the 38-bp silencer
containing the CRE but not by the same unlabeled oligonucleotide
wherein the CRE is mutated or deleted. We show that in the absence of
TSH, the 38-bp silencer binds a multiplicity of proteins in a
CRE-dependent fashion in addition to CREB: TTF-1; a Y-box
protein termed TSEP-1 which is homologous to human YB-1; a single
strand binding protein, SSBP-1 (or a related protein), which binds to
the TSHR; and Pax-8 which binds to the TG and TPO promoters. In
addition to decreasing the interaction of CREB with the silencer, TSH
treatment of the cells decreases the formation of a complex which we
associate with the binding of TTF-1, but retains or enhances, at least
relatively, the binding of complexes associated with TSEP-1 and Pax-8
binding. At this time, we have not determined its effect on complexes
associated with SSBP-1 binding, although we anticipate these might
decrease, consistent with TSH/cAMP-induced decreases in SSBP-1 RNA
levels and SSBP-1 complex formation with the TSHR (20).
In these experiments, we show that TTF-1 is an enhancer of CRE-dependent class I promoter activity, that TSEP-1 is a suppressor, and that Pax-8 acts as a suppressor of TTF-1-dependent enhancer activity. Additionally, we show that oligonucleotides that interact with TSEP-1, but not those interacting with Pax-8, inhibit the formation of the TSH/forskolin-induced novel complexes with the class I promoter. TSEP-1 is, therefore, the dominant component of the TSH-induced novel complex; since TSEP-1 is a suppressor, it is not surprising class I expression is decreased.
Together with previous observations, the above results can be incorporated into a reasonable model of TSH/cAMP control of class I gene expression via the novel silencer described herein. In previous experiments in FRTL-5 cells, we have shown (15, 16) that TSH/cAMP decrease TTF-1 RNA levels and that this decrease can account for a decrease in complex formation with TTF-1 binding sites. Similarly, we have previously shown (17, 20) that TSH/cAMP decreases SSBP-1 RNA levels and that this decrease can account for a decrease in complex formation with SSBP binding sites. We have shown in FRTL-5 cells that TSH/cAMP, in contrast to decreasing TTF-1 complex formation with the TTF-1 sites, increases Pax-8 complex formation to sites that bind both TTF-1 and Pax-8 (15, 16). Finally, we have shown in FRTL-5 cells that TSEP-1 binding can be increased by protein kinase A pretreatment of extracts from cells incubated without TSH, i.e. phosphorylation can increase TSEP-1 binding (19). This is consistent with studies of Y-box proteins in oocyte development (55).
A reasonable model to interpret the data in this and our previous
reports is, therefore, as follows. The 38-bp silencer binds a
multiplicity of proteins. In the absence of TSH, TTF-1 and CREB form
prominent complexes and are enhancers that maximize class I expression
in the cells. In the presence of TSH and its activated cAMP signal,
CREB and TTF-1 binding are decreased, the former by an unknown
mechanism possibly related to altered heterodimer formation, the latter
reflecting a TSH-induced decrease in TTF-1 RNA and protein levels. This
results in decreased maximal class I expression. We suggest that
TSH/cAMP, simultaneously with decreased maximal class I expression,
cause a protein kinase A-mediated phosphorylation of TSEP-1. TSEP-1
acts as a suppressor of class I activity. Pax-8 binding is preserved or
relatively enhanced, thereby additionally minimizing TTF-1 enhancer
binding and activity. The net result of the changes in the interaction
of these proteins (CREB, TTF-1, Pax-8, and TSEP-1) with the 38-bp
silencer is the formation of new complexes with the class I promoter
and class I suppression. Formation of the complexes is dependent on the CRE-like element, 107 to
100 bp, and appears to reflect a dominant role of TSEP-1 and an ancillary one for Pax-8.
TSH up-regulates the function and growth of the thyroid cell; during the early stages of the cell cycle, it coordinately decreases TSHR and MHC class I gene expression (1, 2, 56). We have hypothesized that this is necessary to maintain self-tolerance in the face of the increase in proteins associated with TSH-induced growth and function (1, 2, 4-6). The present data are consistent with this hypothesis. First, Pax-8 suppresses TTF-1-induced class I promoter activity, while simultaneously acting as an enhancer of TG or TPO gene expression (21, 22, 26). Pax-8 is, therefore, a transcription factor whose binding activity is increased by TSH/cAMP (15, 16), whose activity is associated with increased thyroid function, TG synthesis, or TPO activity, and yet whose activity simultaneously helps suppress class I gene expression. Second, human Y-box homologs of TSEP-1 are able to bind to c-myc (46, 47, 57, 58) and to other genes associated with activation of growth: the epidermal growth factor receptor and c-Ki-ras (59-61). Sabath et al. (62) have suggested that TSEP-1 might stimulate the transcription of numerous growth-associated genes. TSEP-1, therefore, functions not only in the TSH/cAMP-induced suppression of TSHR and MHC class I, but also, simultaneously, is likely to be a positive regulator of the growth of FRTL-5 cells.
In sum, common transcription factors are involved in TSH/cAMP-induced negative regulation of class I and the TSHR as hypothesized (1, 2, 4-6) and in TSH/cAMP-induced positive regulation of TG and TPO regulation. The net result is cross-talk which suppresses class I and preserves self-tolerance, while allowing TSH-induced thyroid growth and function to proceed.
Y box proteins are known to suppress MHC class II gene expression and are important in cAMP-induced down-regulation of class II (63-65). Since aberrant class II, as well as increased class I expression, is associated with thyroid autoimmunity (3, 6), coordinate suppression of class I and class II by a common transcription factor would additionally preserve self-tolerance in the thyroid cell challenged with TSH (6).
There is a feature of the Y-box protein family members that may be relevant to understanding the break down of tolerance and the development of autoimmunity. Infections by hepatitis C, retro-, and foamy viruses have been associated with the appearance of thyroid autoantibodies and frank thyroid autoimmunity of various types (66-73). All are single strand RNA viruses; foamy is a retrovirus family member with a similar long terminal repeat (74). We have identified a binding site for TSEP-1 on the hepatitis C virus upstream region, which is important in strand replication.4 10 Studies by Kashanchi et al. (75) reveal the involvement of Y-box proteins in the transcription of human T-cell lymphotrophic virus type-1. Thus, the downstream regulatory element 1 in the long terminal repeat of human T-cell lymphotrophic virus type-1, as well as the site A region of human immunodeficiency virus, both contain Y-box elements. In addition, cotransfection of Jurkat T-cells with a YB-1 expression vector and wild type or mutant viral promoter-CAT constructs demonstrated that the Y-box sequence was essential for efficient transactivation. Kashanchi et al. (75) suggest that interleukin-2 induction of YB-1, as described earlier for T-cell proliferation, might lead to stimulation of viral gene expression and viral replication (75). We speculate that autoimmunity may be precipitated by the action of positive strand RNA viruses infecting cells, "capturing" TSEP-1, and using it, and/or other single strand binding proteins such as SSBP-1 which also regulates class I and the TSHR, to promote viral replication. This results in the loss of normal negative regulation of host genes important for self-tolerance.
Concern can be expressed that the present data and studies in
thyrocytes in culture are not relevant to the whole animal. In this
respect, previous work in transgenic animals has shown that downstream
sequences including the silencer region can influence the pattern of
tissue-specific class I expression and the ability of the gene to
respond to -interferon (76). The present report is consistent with
the former point; our preliminary
report,5 which shows the CRE
is a critical element in the
-interferon response, further supports
this.
We thank Dr. J. Hoeffler and J. Haebner for antisera.