| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Third Department of Internal Medicine Yamanashi Medical University Tamaho, Yamanashi 409–38, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and ß1, and acts as a potent positive regulator of various genes.
The effect of GABP on transcription of the TSH receptor (TSHR) gene in
rat FRTL-5 thyroid cells has now been investigated. Both
deoxyribonuclease I footprint analysis and gel mobility-shift assays
indicated that bacterially expressed glutathione
S-transferase fusion proteins of GABP subunits bind to a
region spanning nucleotides (nt) -116 to -80 of the TSHR gene. In gel
mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells
yielded several specific bands with a probe comprising nt -116 to
-80. Supershift assays with antibodies to GABP and to GABPß1
showed that GABP was a component of the probe complexes formed by the
nuclear extracts. Immunoblot analysis confirmed the presence of both
GABP subunits in the nuclear extracts. A reporter gene construct
containing the TSHR gene promoter was activated, in a dose-dependent
manner, in FRTL-5 cells by cotransfection with constructs encoding both
GABP and GABPß1. Both GABP binding to and activation of the TSHR
gene promoter were prevented by methylation of CpG sites at nt -93 and
-85. These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and ß1. GABP is a low-affinity DNA-binding
protein with an Ets domain, whereas GABPß1 contains a Notch-related
structural motif (1, 2). Both subunits show a widespread tissue
distribution (2). GABP is thought to act as a transcription factor for
various genes, including the aldose reductase gene, the adenovirus E4
gene, the ß2-integrin gene, the folate-binding protein gene, and the
cytochrome c oxidase subunit IV gene (3, 4, 5, 6, 7). We also recently showed
that GABP acts as a methylation-sensitive transcriptional activator
of the male-specific cytochrome P-450 gene Cyp 2d-9 (8). Many genes are regulated by specific combinations of widely expressed factors and tissue-specific factors. The TSH receptor (TSHR) gene is regulated by thyroid transcription factor-1 (TTF-1), a tissue-specific factor that binds to the region spanning nucleotides (nt) -189 to -175 of the 5'-flanking region of the TSHR gene and activates transcription (9, 10). The TSHR gene promoter also contains an octameric cAMP response element (CRE)-like sequence between nt -139 and -132. A 10-nt tandem repeat sequence between nt -162 and -141, immediately 5' to the CRE, acts as a repressive element with regard to constitutive CRE enhancer activity. This decanucleotide tandem repeat sequence, which interacts with single-stranded DNA-binding proteins, modulates the interaction of the CRE with CRE-binding proteins (11). The sequence GGAA, which is the core binding site for several members of the Ets family of transcription factors, is present within the TSHR gene minimum promoter (nt -220 to -1) (9, 10, 11). Ikuyama (10) previously showed that CpG sites at nt -93 and -85 are methylated in nonfunctioning FRT and proposed the importance of the methylation to TSHR gene expression by comparing methylation at these nucleotides in FRT and FRTL-5 thyroid cells. FRT cells are nonfunctioning thyroid cells that do not express the TSHR, while FRTL-5 cells are functioning and do express the TSHR. We have now therefore examined the effect of GABP and CpG site methylation on expression of the TSHR gene in FRTL-5 rat thyroid cells.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and GABPß1 bound to nt -116 to
-80 of the TSHR gene sense strand and nt -116 to -86 of the TSHR
gene antisense strand, respectively, but only in the presence of
both proteins (Fig. 1A
). Subsequent gel
mobility-shift assays showed that GST-GABP bound to the
sequence nt -116 to -80 only in the presence of GST-GABPß1 (Fig. 1B).
|
and GABPß1 (Fig. 2A). The same complex of GABP was also
observed with crude nuclear extracts prepared from FRTL-5 cells (Fig. 2B). To confirm the presence of GABP and GABPß1 in FRT and FRTL-5
cells, we subjected crude nuclear extracts from these cells to
immunoblot analysis. Antibodies to GABP and to GABPß1 detected 60-
and 52-kDa proteins, respectively (data not shown), sizes consistent
with those of the corresponding antigens.
|
, and Fig. 4 summarizes the methylation levels at
each site in FRT and FRTL-5 cells. The TSHR gene is expressed in FRTL-5
cells but not in FRT cells (10), and, consistent with this
cell-specific expression, all of these CpG sites of the TSHR gene were
completely demethylated in FRTL-5 cells. In contrast, the CpG/-154,
CpG/-142, CpG/-93, and CpG/-85 sites were highly methylated
(>71%), and the CpG/-190, CpG/-72, CpG/-38, CpG/-36, and CpG/-31
sites were methylated to a lesser extent (<32%) in FRT cells.
|
|
, GABP did not bind to either
methylated probe. Furthermore, whereas unlabeled wild-type probe
competed with the labeled wild-type probe for the binding of GABP,
unlabeled methylated probe [prepared with 5-methyl deoxycytidine CED
(ß-cyanoethyl-N,N-diisopropylamino)
phosphoramidite] had no effect on GABP binding to the labeled
wild-type probe (Fig. 5B). We further examined the effect of
methylation on GABP binding to the TSHR gene promoter by DNase I
footprint analysis (Fig. 5C). Nucleotides -116 to -86 of the TSHR
gene were protected by GST-GABP and GST-GABPß1 with a probe
subjected to mock methylation but not with a probe that had been
methylated by HpaII methylase.
|
). FRTL-5 cells were transfected with
mock-methylated or HpaII-methylated plasmids in the absence
or presence of GABP and GABPß1 expression vectors. The basal
activity of the HpaII-methylated pTRCAT5'-146 plasmid
was reduced by 60% compared with that of the mock-methylated
plasmid. Furthermore, whereas GABP increased transcription of the
mock-methylated pTRCAT5'-146 plasmid, it had no effect on the activity
of the HpaII-methylated plasmid. This activation of the
mock-methylated plasmid by GABP was dose dependent (data not shown).
GABP had no effect on the activity of either mock-methylated or
HpaII-methylated plasmid pTRCAT5'-90, which does not contain
the binding site for GABP. The basal activity of pTRCAT5'-90 or of the
promoterless plasmid p8CAT was not affected by methylation.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
DNA methylation is an important mechanism by which gene expression is
regulated during growth and development (21, 22, 23, 24). In general, DNA
methylation is associated with inhibition of gene expression. A high
degree of DNA methylation can result in cell transformation (25),
whereas demethylation of MyoD or another regulatory gene
results in the conversion of fibroblasts to myoblasts (26). The reduced
thyroglobulin gene expression in Ras-transformed FRTL-5
thyroid cells is also associated with methylation of the gene promoter
(27); treatment of the transformed cells with the DNA demethylating
agent 5-azacytidine reactivates the thyroglobulin gene promoter (28).
Various methylation-sensitive transcription factors, including
activator protein-2, CRE-binding protein/activating transcription
factor, and nuclear factor-
B, have been described (23). Thus,
methylation of the CRE of the human proenkephalin gene prevents
activator protein-2 binding and stimulation of transcription (29, 30).
Myeloid-specific transcription of the mouse M lysozyme gene is also
regulated by a single CpG methylation site within the enhancer
(31).
We recently added GABP to the list of factors sensitive to methyl-CpG (8). The CpG sites in the promoters of the sex-specific P-450 genes exhibit sex-specific patterns of methylation related to expression of the genes in the liver of mice. The CpG site at nt -97 in the promoter of the male-specific Cyp 2d-9 gene is preferentially demethylated in male mice. GABP transactivates the male-specific Cyp 2d-9 promoter through direct binding to the regulatory element 5'-TTC-97CGGGC; GABP does not bind to the promoter when the CpG/-97 site is methylated. Thus, we proposed that DNA demethylation and the methylation-sensitive transcription factor GABP underlie the sex-specific transcription of the Cyp 2d-9 gene. We have now shown that the methylation of CpG/-93 and CpG/-85 abolishes the binding of GABP to the TSHR gene promoter and reduces basal TSHR gene transcription. Thus, CpG sites located outside of the consensus-binding site of GABP affect the binding of GABP to the promoter of the TSHR gene.
The TSHR gene is expressed in FRTL5 cells but not in FRT cells, which are derived from rat thyroid (32) and have the characteristics of epithelial cells. FRT cells do not secrete thyroglobulin and do not express thyroperoxidase. Although they express Pax-8, they do not express TTF-1 (33, 34), which may largely explain the failure of FRT cells to express the endogenous TSHR gene. We have now also shown that CpG/-93 and CpG/-85 in the TSHR gene are highly methylated (>82%) in FRT cells, whereas these sites are completely demethylated in FRTL-5 cells consistent with the pattern of TSHR gene expression. It appears, therefore, that GABP does not transactivate the TSHR gene promoter when these CpG positions are methylated in FRT cells, and that GABP stimulates transcription of the TSHR gene when these CpG positions are demethylated in FRTL-5 cells. Thus, methylation of the promoter and the methylation-sensitive transcription factor GABP may also contribute to the failure of FRT cells to express the endogenous TSHR gene. All of the the CpG islands in the TSHR gene between -190 and -31 were completely demethylated in FRTL-5 cells, although only two of them appear relevant for GABP. Therefore, it remains possible that the other CpG sites, especially CpG/-154 and CpG/-142, which were also highly methylated in FRT cells in the TSHR gene promoter, and other methylation-sensitive transcription factors may also contribute to the regulation of this gene. In conclusion, the heteromeric transcription factor GABP can bind to, and thereby transactivate, the TSHR gene promoter. Moreover, the binding of GABP is sensitive to methylation of CpG sites at nt -93 and -85, and transcription of the TSHR gene is decreased by methylation of these CpG sites. Therefore, GABP may regulate the transcription of the TSHR gene in a manner dependent on the methylation status of the CpG sites at nt -93 and -85.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Plasmids
The plasmids pTRCAT5'-146, and pTRCAT5'-90, containing 146 and
90 bp, respectively, of the 5'-flanking region of the rat TSHR gene,
upstream of a CAT reporter gene, were kindly provided by Dr. L. D.
Kohn (10, 11). The promoterless CAT plasmid, p8CAT, was also provided
by Dr. L. D. Kohn. The in vitro methylation of plasmid
DNA was performed using HpaII methylase according to the
instructions of the supplier (New England Biolabs, Beverly, MA).
Unmethylated control plasmids (mock-methylated plasmids) were prepared
identically without addition of methylase. Before transfection,
the methylated and mock-methylated plasmids were
phenol-extracted and ethanol-precipitated. Complete methylation was
verified by digesting the DNA with an excess of HpaII
restriction enzyme.
Sequencing of the Sodium Bisulfite-Treated Promoter
Genomic DNAs were prepared from FRT and FRTL-5 cells using the
SDS/proteinase K method, digested with PstI, and then
subjected to a sequential reaction to determine CpG methylation pattern
according to Frommer et al. (12). The oligonucleotide
primers were synthesized based on the reported sequences of the TSHR
genes (10). The top strand of promoter sequence (-220/-1) of the TSHR
gene was amplified using 10 µl of the bisulfite-reacted DNA as a
template, and the oligonucleotides
5'-GGGGAAGCTTTTTGTTTGGATGGAGAGTTG and
5'-GGGGTCTAGATTTCCAAAAAACCTCCAATA as the 5'- and
3'-primers, respectively. The underlined regions indicate
that a HindIII site and a XbaI site were added at
each end of the amplified DNAs. Amplified DNAs were digested with
HindIII and XbaI and then cloned into M13 mp19
vectors for DNA sequencing.
Nuclear Extracts
Nuclear extracts were prepared from FRT and FRTL-5 cells. Cells
were harvested, washed with Dulbecco’s modified PBS without
Mg2+ and Ca2+ (pH 7.4), and, after
centrifugation at 500 x g, suspended in five pellet
volumes of buffer A [10 mM HEPES-KOH (pH 7.9), 10
mM KCl, 1.5 mM MgCl2, 0.1
mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5
mM phenylmethylsulfonyl fluoride, leupeptin (2 µg/ml),
and pepstatin A (2 µg/ml) ] containing 0.3 M sucrose and
2% (vol/vol) Tween-40. The cells were then frozen, thawed, and gently
homogenized, and nuclei were isolated by centrifugation of the
homogenate at 25,000 x g through a 1.5 M
sucrose cushion prepared in the same buffer. Nuclei were lysed in
buffer B [10 mM HEPES-KOH (pH 7.9), 420 mM
NaCl, 1.5 mM MgCl2, 0.1 mM EGTA,
10% (vol/vol) glycerol, 0.5 mM phenylmethylsulfonyl
fluoride, leupeptin (2 µg/ml), and pepstatin A (2 µg/ml)], and the
lysate was centrifuged at 100,000 x g for 1 h.
The resulting supernatant was dialyzed for use in DNase I footprint
analysis or gel mobility-shift assays.
Bacterial Expression of GABP
The pCRII vector containing the mouse GABP or GABPß1 coding
sequence was digested with EcoRI and ligated with
EcoRI-digested pGEX-2T (Pharmacia Biotech, Piscataway, NJ).
Ten milliliters of an overnight culture of transformed bacteria were
inoculated in 1 liter of LB medium supplemented with ampicillin (100
µg/ml) and glucose (0.2%). After incubation for 3 h at 37 C,
isopropyl-ß-D-thiogalactopyranoside was added to a final
concentration of 0.1 mM, and the cells were incubated for
an additional 2 h. The bacterial cells were then harvested and the
glutathione S-transferase (GST) fusion protein was purified
on a glutathione-Sepharose 4B column (Pharmacia Biotech).
DNase I Footprint Analysis
We performed DNase I footprint analysis with a Sure Track
footprinting kit (Pharmacia Biotech). The
AvrII-BssHII (nt -199 to -36) fragments of the
rat TSHR gene were end-labeled with [
-32P]ATP (>5000
Ci/mmol; Amersham, Arlington Heights, IL) with T4 polynucleotide
kinase, and then purified by agarose gel electrophoresis. Methylated
DNA was prepared using HpaII methylase according to the
supplier’s protocol and then end-labeled. Labeled DNA fragments
(30,000 cpm) were incubated with recombinant mouse GABP and/or
GABPß1 in 50 µl of 10 mM Tris-HCl buffer (pH 7.5)
containing 2.5 µg of poly(deoxyinosinic-deoxycytidylic)acid, 50
mM NaCl, 2.5 mM MgCl2, 1
mM DTT, 0.5 mM EDTA, and 5% glycerol for 30
min at room temperature. Then the DNAs were digested by 1 U of DNase I
for 30 sec, extracted with phenol-chloroform, and precipitated with
ethanol. As the sequence markers, the corresponding DNA fragment was
chemically cleaved at nucleotides G and A by the method of Maxam and
Gilbert (35). Finally, the digested DNA samples were electrophoresed on
an 8% polyacrylamide-7 M urea gel, and the gel was then
dried, exposed to an imaging plate, and analyzed with a Bas 2000 image
analyzer (Fuji, Tokyo, Japan).
Gel Mobility-Shift Assay
Each oligonucleotide was annealed to its complement and labeled
by using [-32P]dATP (>6000 Ci/mmol; Amersham) and DNA
polymerase Klenow fragment. Methylated oligonucleotides were prepared
by including 5-methyl deoxycytidine CED phosphoramidite (Pharmacia
Biotech) during the appropriate cycle of synthesis or with the use of
HpaII methylase. Each radioactive probe was incubated with 5
µg of nuclear proteins or 0.1 µg of GST fusion proteins of GABP
and GABPß1 in 10 µl of 20 mM Tris-HCl (pH 7.5)
containing 1 µg of poly(deoxyinosinic-deoxycytidylic)acid, 50
mM NaCl, 0.1 mM DTT, and 10% glycerol at room
temperature. In experiments using antiserum to GABP, nuclear extracts
were incubated with the antiserum (1 µl) in the same buffer for 30
min at room temperature before adding the labeled probes and processing
above. The following oligonucleotides were used in the studies as the
probes:-116CTCCTCCTTCCTCCCTTTCCCTCCGGCACCCCGGTCT-80,
and
-116CTCCTCCTTC-CTCCCTTTCCCTCm5CGGCACCCm5CGGTCT-80.
An Oct1 consensus oligonucleotide,
5'-AATTGCATGCCTGCAGGTGGACTCTAGAGGATCCATGCAAATGGATCCCCGGGTACCC-AGCTC,
was also used as a nonspecific competitor.
Transient Expression Analysis
FRTL-5 cells were grown to 80% confluency in 6H medium, shifted
to 5H medium for 1 day, and then returned to 6H medium for 1 day before
transfection by electroporation (300 V; capacitance, 960 µfarad)
(Gene Pulser; Bio-Rad, Richmond, CA). Cells were harvested, washed, and
suspended at 1.5 x 107 cells/ml in 0.8 ml PBS and
cotransfected with 20 µg of the pTRCAT plasmids or p8CAT plasmids, 10
µg of GABP (pCR3-), and GABPß1 (pCR3-ß1) expression
plasmids, and 5 µg of the ß-galactosidase expression plasmid
pCH110. The total amount of transfected DNA was adjusted to 45 µg by
adding carrier DNA. The cells were pulsed, then plated, and cultured
for 72 h. To measure CAT activity, the cells were lysed by
freezing and thawing and the lysate (30 µg of protein) was incubated
with [14C]chloramphenicol according to the method of
Gorman et al. (36).
|
ACKNOWLEDGMENTS |
|---|
and GABPß1 cDNAs and
anti-GABP antiserum. |
FOOTNOTES |
|---|
Received for publication December 31, 1997. Revision received March 30, 1998. Accepted for publication April 10, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. Xing BRAF Mutation in Papillary Thyroid Cancer: Pathogenic Role, Molecular Bases, and Clinical Implications Endocr. Rev., December 1, 2007; 28(7): 742 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Lin, P. J. Collins, N. D. Trinklein, Y. Fu, H. Xi, R. M. Myers, and Z. Weng Transcription factor binding and modified histones in human bidirectional promoters Genome Res., June 1, 2007; 17(6): 818 - 827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Xing Gene Methylation in Thyroid Tumorigenesis Endocrinology, March 1, 2007; 148(3): 948 - 953. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. T. Smith, R. D. Nicholls, and D. Reines The gene encoding the fragile X RNA-binding protein is controlled by nuclear respiratory factor 2 and the CREB family of transcription factors Nucleic Acids Res., February 25, 2006; 34(4): 1205 - 1215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Xing, H. Usadel, Y. Cohen, Y. Tokumaru, Z. Guo, W. B. Westra, B. C. Tong, G. Tallini, R. Udelsman, J. A. Califano, et al. Methylation of the Thyroid-stimulating Hormone Receptor Gene in Epithelial Thyroid Tumors: A Marker of Malignancy and a Cause of Gene Silencing Cancer Res., May 1, 2003; 63(9): 2316 - 2321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Shimura, H. Suzuki, A. Miyazaki, F. Furuya, K. Ohta, K. Haraguchi, T. Endo, and T. Onaya Transcriptional Activation of the Thyroglobulin Promoter Directing Suicide Gene Expression by Thyroid Transcription Factor-1 in Thyroid Cancer Cells Cancer Res., May 1, 2001; 61(9): 3640 - 3646. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
