A Role for Basic Transcription Element-binding Protein 1
(BTEB1) in the Autoinduction of Thyroid Hormone
Pia Bagamasbad‡1, Kembra L. Howdeshell‡1,2, Laurent M. Sachs§, Barbara A. Demeneix§, and Robert J. Denver‡3
and§UMR5166CNRS,USM-501Muse ´umNationald’HistoireNaturelle,De ´partementRe ´gulationDe ´veloppementetDiversite ´
Mole ´culaire,Case32,7RueCuvier,75231 Paris Cedex 05, France
essary for tadpole metamorphosis. Among the earliest
responses to T3are the up-regulation of T3receptor ? (TR?;
autoinduction) and BTEB1 (basic transcription element-bind-
ing protein 1). BTEB1 is a member of the Kru ¨ppel family of
transcription factors that bind to GC-rich regions in gene pro-
moters. The proximal promoter of the Xenopus laevis Tr?A
gene has seven GC-rich sequences, which led us to hypothesize
that BTEB1 binds to and regulates Tr?A. In tadpoles and the
frog fibroblast-derived cell line XTC-2, T3up-regulated Bteb1
mRNA with faster kinetics than Tr?A, and Bteb1 mRNA corre-
lated with increased BTEB1 protein expression. BTEB1 bound
to GC-rich sequences in the proximal Tr?A promoter in vitro.
By using chromatin immunoprecipitation assay, we show that
BTEB1 associates with the Tr?A promoter in vivo in a T3and
duction of the Tr?A gene. This enhancement was lost in N-ter-
minal truncated mutants of BTEB1. However, point mutations
ing that BTEB1 can function in this regard through protein-
protein interactions. Our findings support the hypothesis that
ing activity. Cooperation among the protein products of imme-
diate early genes may be a common mechanism for driving
developmental signaling pathways.
Autoinduction of nuclear hormone receptors is a common
but poorly understood phenomenon in animal development
(1). The autoinduction of thyroid hormone (T3)4receptor
genes (Tr) during amphibian metamorphosis is a dramatic
sess two Tr genes designated Tr? and Tr? (also known as
NR1A1 and NR1A2, respectively); Xenopus laevis has two Tr?
and two Tr? genes each designated A or B because of its
pseudotetraploidy (2). Thyroid hormone is the primary mor-
dependent transcription factors. One of the earliest gene regu-
lation events during amphibian metamorphosis is the
up-regulation of Tr? genes by T3(3). This regulation depends
on TRs binding to thyroid hormone-response elements (TREs)
present in the Tr? promoters (receptor autoinduction; see
Refs. 2, 4). It is hypothesized that autoinduction of Tr? genes is
essential for metamorphosis (1). The gene regulation programs
induced by the T3?TR complex that lead to tissue morphogenesis
Basic transcription element-binding protein 1 (Bteb1) is an
immediate early gene induced by T3in most tadpole tissues
during metamorphosis (there are two Bteb1 genes in X. laevis
of the X. laevis Bteb1 genes by T3is explained by one or more
TREs located upstream of the transcription initiation sites (6,
14). BTEB1 is a member of the Kru ¨ppel family of transcription
factors (KLF; also known as KLF-9 (15) and first isolated in a
the basic transcription element or BTE (16, 17)). BTEB1 pos-
sesses a DNA binding domain (DBD) consisting of three Cys2-
tantly related to the specificity protein (Sp) family members,
including Sp1 (18, 19). The BTEB1 DBD shares 72% sequence
similarity with rat Sp1 (17), and the two proteins bind with
similar affinity to the BTE sequence (20). Although Sp1 and
BTEB1 have very similar DNA binding domains, and they bind
to similar or identical consensus DNA sequences, the two pro-
* This work was supported in part by National Science Foundation Grants
IBN9974672 and IBN0235401 (to R.J.D.), NINDS Grant 1 R01 NS046690 from
the National Institutes of Health (to R.J.D.), and funding from the CNRS and
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in
supplemental Table 1.
1Both authors contributed equally to this work.
2Supported by an NIEHS, National Institutes of Health, postdoctoral trainee-
ship through the Environmental Toxicology Research Training Grant, Uni-
ogy Division, MD-72, NHEERL, ORD, Environmental Protection Agency,
Research Triangle Park, NC 27711.
3To whom correspondence should be addressed: Dept. of Molecular, Cel-
of Michigan, Ann Arbor, MI 48109-1048. Fax: 734-647-0884; E-mail:
TRE, thyroid hormone-response element; UTR, untranslated region; RT,
reverse transcription; ChIP, chromatin immunoprecipitation; EMSA, elec-
variance; NF, Nieuwkoop and Faber.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 4, pp. 2275–2285, January 25, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
JANUARY 25, 2008•VOLUME 283•NUMBER 4JOURNAL OF BIOLOGICAL CHEMISTRY 2275
at University of Michigan on January 20, 2008
Supplemental Material can be found at:
other KLF family members, three proteins designated BTEB2,
-3, and -4 have been identified in mammals, although the
BTEB2 appears to be more distantly related to BTEB1 than
the other two proteins (19, 21–23). As with BTEB1 and Sp1,
the BTEB proteins share almost identical DNA binding
domains but are divergent in their N-terminal regions that
harbor domains necessary for their transactivation and in
some cases transrepression functions (15).
Basic transcription element-binding protein 1 mRNA and
protein is strongly up-regulated by T3in tadpole tissues (13),
but the genes that BTEB1 regulates, and thus its functions in
tadpole development, are unknown. It is noteworthy that
BTEB1 is the only KLF/Sp1-like family member known to be
up-regulated by T3in tadpole tissues (5–12). Earlier, we and
rodent brain where it promotes neurite outgrowth (24–26).
BTEB1 is expressed in uterine endometrial cells where it trans-
activates the uteroferrin gene, and it may influence cell prolif-
eration by regulating cell cycle and growth-associated genes
(27–29). The actions of BTEB1 in endometrial cells appear to
involve direct protein-protein interactions with the progester-
one receptor (30). We found that X. laevis BTEB1 is capable of
activating synthetic promoter constructs containing multiple
or single GC boxes (13). Mammalian BTEB1 also has transacti-
vation function on several synthetic and native promoters (17,
27, 31–34). BTEB proteins have been reported to activate or
repress transcription depending on the number of GC boxes
present in the promoter construct tested, the target gene ana-
teins function as transcriptional activators or repressors may
depend on the architecture of the specific promoter and the
chromatin environment (15).
Based on the early response kinetics of the Bteb1 and Tr?A
genes, the observation that the protein products of these genes
are expressed in the same cells (13), and the identification of
seven GC-rich regions in the proximal X. laevis Tr?A pro-
moter, we hypothesized that Tr?A may be a target gene for
BTEB1. We further hypothesized that the up-regulation of
tioning as an accessory transcriptional activator. Here we show
that the kinetics of Bteb1 mRNA up-regulation in response to
T3are faster than Tr?A and that BTEB1 binds to regions of the
proximal Tr?A promoter that contain GC boxes. Using chro-
associates with the Tr?A promoter in vivo in a T3- and devel-
opmental stage-dependent manner. Forced expression of
BTEB1 in the X. laevis fibroblast cell line XTC-2 (37) acceler-
ates the activation of the Tr?A promoter and expression of
endogenous Tr?A mRNA in response to T3. This action
depends on the first 30 amino acids of BTEB1, but not on its
DNA binding capacity, because point mutations in the zinc
fingers did not alter the activity. Taken together, our findings
support the hypothesis that the up-regulation of BTEB1 by T3
plays a role in the transcriptional regulation of the Tr?A gene
during tadpole development.
Animals and Hormone Treatments—Tadpoles of X. laevis
were reared in dechlorinated tap water (water temperature,
20–22 °C) and fed pulverized frog brittle (Nasco, Fort Atkin-
son, WI). Developmental stages were assigned according to
Nieuwkoop and Faber (NF) (38). Tadpoles were treated with
3,5,3?-L-triiodothyronine (T3; sodium salt; Sigma) by adding it
to the aquarium water to a final concentration of 10 nM for
various times; water was changed and hormone replenished
by immersion in 0.01% benzocaine (Sigma), and whole brains
and tails were collected for RNA or ChIP analyses (see below).
Animal care was in accordance with institutional guidelines.
RNA Extraction and Reverse Transcription (RT)-PCR
Analysis—Total RNA was isolated from tadpole brains or
manufacturer’s instructions. The RNA was treated with DNase
I (Roche Applied Science) prior to reverse transcription to
Manzon and Denver (39). The DNase-treated RNA was
reverse-transcribed using SuperScript II (0.5 ?l, 200 units/?l;
Invitrogen), and 0.2 to 2 ?l of the resulting cDNA was used for
Semi-quantitative RT-PCR—Standard PCRs were initiated
in 25 ?l containing 10? PCR buffer, 1.5 mM MgCl2, dNTP mix
(1.25 mM each), forward and reverse primers for each gene of
interest (10 ?M), and TaqDNA polymerase (1.25 units; Pro-
mega, Madison, WI). Each thermal cycle consisted of 94 °C for
1 min, 55 °C for 1 min, and 72 °C for 2 min. The number of
cycles for each gene was determined empirically by construct-
ing linear amplification curves. We used 32 cycles for Bteb1, 36
for Tr?A, and 28 for ribosomal protein L8 (rpL8; a housekeep-
sis). Oligonucleotide primer sequences for Bteb1 are given in
Table 1. Primer sequences used for rpL8 and Tr?A were as
described by Manzon and Denver (39). PCR products were
Oligonucleotides used for semi-quantitative and quantitative real
Oligonucleotide primers for semi-quantitative RT-PCR and TaqMan assays were
designed to span exon/intron boundaries. The primers for semi-quantitative RT-
PCR amplified mRNAs from both Bteb1A and Bteb1B genes.
FAM-AGG CAC AGG TGT CC-MGBNFQ
5?-CAC GGC CTG GAT CAT GGA-3?
VIC-AGG GTA TTG TGA AAG ACA-MGBNFQ
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Tr?A amplicons for each sample were normalized to the den-
sities of the rpL8 bands.
Quantitative Real Time PCR (qPCR)—For quantitative RT-
PCR (RTqPCR), we developed TaqMan assays and analyzed
ter City, CA). The primer/probe sets used are given in Table 1
and were designed to span exon/intron boundaries. Standard
curves were generated using cDNAs from the time point that
exhibited the highest expression level for each gene to provide
for a relative quantitation. Tr?A and Bteb1 mRNAs were nor-
malized to the level of rpL8 mRNA.
Plasmid Constructs—The pCMV-xBTEB1 expression plas-
mid was described by Hoopfer et al. (13). The X. laevis Tr?A
promoter-luciferase plasmid (40) was a generous gift of Dr.
Yun-Bo Shi. Full-length and N-terminal truncated mutants of
fragments were directionally cloned into the pCS2 vector. The
choice of deletions was based on the location of two putative
transactivation domains (A and B) located in rat BTEB1 (41)
that are highly conserved in Xenopus BTEB1 (13). The plasmid
transactivation domains A and B removed; and pCS2-
xBTEB1?120 represents only the DNA binding domain.
The plasmid construct pCS2-xBTEB1 C2AH harbors histi-
dine to alanine substitutions (H211A, H241A, and H296A) in
the first histidine residues of each of the three zinc fingers of
BTEB1. This construct was generated using the QuikChange
multisite-directed mutagenesis kit (Stratagene) with pCS2-
xBTEB1 as template and three primers shown in Table 2.
al. (13) with minor modifications. The BTE and mutated BTE
probes used were as described by Yanagida et al. (16). Bacterial
cell lysate containing the fusion protein GST-xBTEB1[DBD]
was prepared as described by Hoopfer et al. (13). Recombinant
wild type BTEB1 or BTEB1 C2AH mutant were produced in
vitro using the TNT SP6 Quick Coupled Translation System
(Promega). For EMSA, 1 ?l of a 1:512 dilution of GST-
xBTEB1[DBD] lysates or varying volumes of the in vitro trans-
lated proteins were incubated in a volume of 35 ?l with 20,000
cpm of32P-BTE and 1.4 ?g of double-stranded poly(dI-dC) in
buffer containing 20 mM HEPES (pH 7.8), 1 mM dithiothreitol,
0.1% Nonidet P-40, 50 mM KCl, and 20% glycerol. For antibody
supershifts, proteins were preincubated for 20 min prior to the
addition of32P-BTE with 1 ?g of normal rabbit serum IgG or
affinity-purified anti-xBTEB1 IgG that recognizes only the
tinued at room temperature for 40 min before fractionation by
nondenaturing 6% PAGE in 0.25? Tris borate/EDTA (TBE).
Gels were fixed in 30% methanol, 10% acetic acid, dried, and
processed for autoradiography.
The ability of regions of the proximal X. laevis Tr?A pro-
moter (GenBankTMaccession number U04675) to displace
petitive EMSA (1.89 ?M for each competitor DNA). The X.
laevis tr?A promoter fragments were generated by PCR and
gel-purified using QIAEX II (Qiagen, Valencia, CA). The
regions of the promoter that we analyzed are shown in Fig. 2
and supplemental Table 1, and the oligonucleotides used to
amplify the sequences by PCR are given in Table 3.
containing predicted GC boxes in the Tr?A promoter frag-
ments that demonstrated competitive binding to GST-
xBTEB1[DBD] (see above). Oligonucleotides (24 bp) were syn-
Oligonucleotide primers used to generate truncated mutants and point mutations in the three zinc fingers of X. laevis BTEB1
indicates forwards, and Rev indicates reverse.
Primers used to generate N-terminal truncated xBTEB1 mutants
?30 xBTEB1 For
?99 xBTEB1 For
?120 xBTEB1 For
Primers used to generate point mutations in the three zinc finger domains of xBTEB1
Oligonucleotide primers used for the analysis of the X. laevis Tr?A
For each pair of oligonucleotides the top sequence is the forward primer, and the
bottom sequence is the reverse primer. Capital letters in parentheses correspond to
promoter regions given in Fig. 2 and supplemental Table 1. qPCR indicates primer
sets that were used for quantitative real time PCR using SYBR Green. All other
primer sets were used for standard PCR with radiolabeled precursor.
Tr?A promoter region
5?-CAG TGG AGT AAC TAC CAG-3?
5?-GTA CAC ATG CCT GCA CTA-3?
5?-TAG TGC AGG CAT GTG TAC-3?
5?-GAG CAG GTG CAG CAT CTA-3?
5?-TAG ATG CTG CAC CTG CTC-3?
5?-ACT ATG GCA TGT TAC AGC-3?
5?-GCT GTA ACA TGC CAT AGT-3?
5?-GCC TGA GTG AAG ACC CAT-3?
5?-ATG GGT CTT CAC TCA GGC-3?
5?-GTC ATG AAA CTC CTC GGT-3?
5?-ACC GAG GAG TTT CAT GAC-3?
5?-TAT AGA CAC AGG CAG CTT A-3?
5?-TAA GCT GCC TGT GTC TAT A-3?
5?-TGA CAG TCA GAG GAA CTG A-3?
Exon 3/exon 4
5?-CAG AAA CCT GAA CCC ACA CAA-3?
5 ?-CAC TTT TCC ACC CTC GGG CGC ATT-3?
5?-TTG TGC CTG CTT GCT TGC TA-3?
5?-ACT ATA ATA GGC GGG CCA AGC TGA-3?
5?-AGC TGC CTG TGT CTA TAC TGA TGG-3?
5?-ACA GGG AGA TCT ACA GCT GAT CGT-3?
qPCR exon 5
5?-CCC CGA AAG TGA AAC TCT AAC GT-3?
5?-AAA CCA CTC CAA GTC CTC CAT TTT-3?
5?-TGC ACA GTT GGC GCA GTG-3?
5?-TGA GGA AGA GAG CGA ACC-3?
5?-ATA GCA GCA GGT GGT TGC G-3?
5?-GGC CAC AAG ATC TAC TCG-3?
JANUARY 25, 2008•VOLUME 283•NUMBER 4JOURNAL OF BIOLOGICAL CHEMISTRY 2277
at University of Michigan on January 20, 2008
thesized, each of which encompassed one or two GC boxes
within each Tr?A promoter region (see Fig. 2 and Table 4).
Each GC-box containing oligonucleotide was labeled with
[32P]dCTP and used as a probe in EMSA.
Cell Culture and Transfection Assays—We plated XTC-2
expression and transfection assays. For ChIP assays, we plated
cells at a density of 1 ? 106cells per 100-mm plate. Before
transfections or hormone treatments, cells were cultured over-
cultured in Leibovitz-15 medium (L-15; diluted 1:1.5 for
amphibian cells; Invitrogen) supplemented with sodium bicar-
bonate (2.47 g/liter), penicillin G sodium (100 units/ml), strep-
tomycin sulfate (100 ?g/ml), and 10% fetal bovine serum that
had been stripped of thyroid hormone following the method of
assays, cells were treated for different times with or without 5
nM T3before harvest.
cells using the polyethyleneimine (Sigma) method (43). The
vector (pCMVneo). All cells were cotransfected with the
pRenilla-luciferase plasmid for normalization of cell transfec-
er’s instructions (Promega, Madison, WI). Just prior to trans-
fection, the cells were washed twice with serum-free L-15, and
the polyethyleneimine/DNA solution was added directly to the
wells. After 1 h the transfection medium was replaced with
growth medium, and the cells were incubated overnight. Cells
were then treated with or without T3for different times before
harvest and analysis of luciferase activity. Luciferase activity
was quantified (measured as relative light units) using a lumi-
transfection experiment was conducted three times with 4–5
wells per treatment.
mutant BTEB1 on endogenous Tr?A mRNA, we used the
pCS2-based expression vectors described above and trans-
fected XTC-2 cells using FuGENE 6 transfection reagent
(Roche Applied Science). Each well of a 6-well plate received 1
?g of plasmid DNA, and the total amount of DNA per well was
normalized by adding empty vector (pCS2). Forty eight hours
after transfection, cells were treated with or without T3for dif-
Western Blotting and Immunocytochemistry—We prepared
Western blots following the methods of Ranjan et al. (40) with
protein extracts of XTC-2 cells transfected with pCS2-xBTEB1
or pCS2 and extracts of XTC-2 cells treated ?T3.Forty micro-
grams of total protein for each sample were separated by elec-
trophoresis on 10% denaturing SDS-polyacrylamide gels. Pro-
antiserum was generated in a rabbit against the full-length X.
laevis BTEB1 protein and affinity-purified such that the IgGs
(13) (0.2 ?g of purified IgG/ml). These antibodies do not rec-
ognize the DBD of xBTEB1 (13), which is critical to the speci-
We conducted immunocytochemistry for BTEB1 protein
following the methods that we described previously (13).
Briefly, NF stage 52 tadpoles were treated with or without T3
(10 nM) for 24 h before sacrifice. Brains were fixed for 24 h at
for 24 h. Tissues were embedded in M-1 embedding matrix
(Shandon Lipshaw Inc., Pittsburgh, PA), frozen, and cryosec-
a goat anti-rabbit horseradish peroxidase secondary antibody
Inc., Burlingame, CA) or with a goat anti-rabbit Cy3-conju-
gated fluorescence secondary antibody (Jackson Immuno-
Research Laboratories, West Grove, PA). To test for the speci-
ficity of the immunohistochemical reaction, we preabsorbed
the antibody with Escherichia coli-expressed GST-xBTEB (10
?g/ml) (13). Tissue sections were analyzed using an Olympus
IX81 inverted fluorescence microscope.
Chromatin Immunoprecipitation Assay—We conducted
used the ChIP assay kit from Upstate Biotechnology, Inc. (Lake
Placid, NY), following the manufacturer’s instructions. The
negative controls included no primary antibody, replacement
of the primary antibody with normal rabbit serum, and the
analysis of regions outside of the proximal Tr?A promoter
(Ef1? promoter, Tr?A exon 3/4, Tr?A exon 5, intestinal fatty
ity-purified IgGs against X. laevis BTEB1 (4 ?g of purified IgG/
reaction). The PCRs for ChIP on tadpole brain or tail included
[32P]dCTP (1 ?Ci/reaction), and the PCR products were ana-
lyzed on 6% polyacrylamide gels followed by autoradiography,
or using an Agilent Technologies 2100 Bioanalyzer (Agilent
Technologies, Inc., Santa Clara, CA). ChIP assays on XTC-2
cells were analyzed using quantitative, real time PCR using the
iCycler iQ real time PCR detection system from Bio-Rad. We
used iQ Syber Green Supermix (Bio-Rad) following the manu-
Short oligonucleotides corresponding to GC-rich regions (bold,
underlined) of the X. laevis Tr?A promoter used as probes and
competitors in EMSA
sequence is the reverse primer.
aNumbering is based on position within the Tr?A gene as depicted in Fig. 2.
2278 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 283•NUMBER 4•JANUARY 25, 2008
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facturer’s protocol with annealing temperatures adjusted for
each primer set. Oligonucleotide PCR primers used for ChIP
analyses are shown in Table 3.
Bteb1 mRNA Is Up-regulated by T3in Premetamorphic Tad-
premetamorphic (NF stage 52) tadpoles to T3(10 nM in the
aquarium water) resulted in signifi-
cant time-dependent increases in
brain Bteb1b (F ? 222.35, p ?
0.0001; ANOVA) and Tr?A (F ?
74.03, p ? 0.001) mRNA levels (Fig.
mRNA was detected was 8 h (p ?
0.0001; Scheffe’s test), and the
mRNA level continued to increase
up to 16 h. By contrast, a significant
increase in Tr?A mRNA expression
was not detected until 16 h.
Thyroid Hormone Up-regulates
BTEB1 Protein in Premetamorphic
Tadpole Brain—Similar to results
that we reported earlier (13), we
protein expression in premetamor-
phic tadpole brain (NF stage 52) fol-
lowing treatment with T3(10 nM for
24 h; representative brain sections
strong nuclear staining for BTEB1
was completely abolished by preab-
sorption with GST-xBTEB (Fig. 1B,
Western blot analysis with affini-
ty-purified anti-xBTEB1 IgG on
XTC-2 cells showed that the anti-
serum detected the overexpressed
act with endogenous cellular pro-
teins (Fig. 1C, upper panel). Native
BTEB1 protein was increased in
untransfected XTC-2 cells by 24 h
of treatment with T3(Fig. 1C, lower
panel). We routinely detected two
bands by Western blot that corre-
sponded to the BTEB1 protein. The
basis for BTEB1 protein heteroge-
neity is currently unknown, but
modifications (X. laevis BTEB1 is
predicted to have up to four phos-
phorylation and two N-linked gly-
cosylation sites (13)).
BTEB1 Binds to the Proximal
Tr?? Promoter in Vitro—Com-
puter analysis of the proximal X. laevis Tr?A promoter
sequence showed the presence of seven GC-rich regions com-
monly characterized as Sp1-binding sites (based on 40). The
approximate locations of these GC-rich regions are shown in
Tr?A promoter in vitro. We generated ?200–300-bp frag-
ments of the Tr?A promoter by PCR (Fig. 2, supplemental
FIGURE 1. Thyroid hormone up-regulates Bteb1 mRNA in tadpole brain with faster kinetics than Tr?A
(bottom) mRNAs in premetamorphic X. laevis tadpole brain (NF stage 52) following exposure to T3(10 nM)
significant differences from the zero time point (p ? 0.0001; Scheffe’s test). B, treatment with T3increases
stage 52 tadpole brain (optic tectum shown) but is increased dramatically by T3treatment (panel 2; 10 nM in
52 tadpole treated with T3in the aquarium water (10 nM; 24 h). Strong BTEB1 staining was restricted to cell
nuclei. Panel 4, immunostaining for BTEB1 was eliminated by preabsorption with GST-xBTEB. BTEB1 immuno-
reactivity was detected by Cy3 immunofluorescence (panels 1 and 2) or by horseradish peroxidase staining
(panels 3 and 4). C, Western blot analysis of xBTEB1 in protein extracts from pCS2 or pCS2-xBTEB1-transfected
(30 nM) for 24 h (lower panel). This dose of T3causes a maximal response in Tr?A and Bteb1 mRNA (data not
shown). Immunoblotting was conducted using affinity-purified IgG that recognizes the N-terminal region of
xBTEB1 (see “Experimental Procedures”). Arrows point to the two BTEB1 bands.
JANUARY 25, 2008•VOLUME 283•NUMBER 4JOURNAL OF BIOLOGICAL CHEMISTRY 2279
at University of Michigan on January 20, 2008
Table 1, and Table 3), and we used them as competitors in
(13) to a32P-labeled probe consisting of the BTE sequence of
ments with GC-rich sequences competed for binding in the
ber of GC boxes contained within the fragment. By contrast,
competition in the EMSA (Fig. 3A).
We next synthesized short oligonucleotide probes (24 bp)
encompassing one or two GC boxes within the proximal Tr?A
and Table 4) and tested for BTEB1 binding to these DNA ele-
ments by EMSA. Radioinert oligonucleotides were used as
competitors to verify the specificity of binding. This experi-
ment showed that BTEB1 bound to all but one of these GC box
sequences and that the binding could be competed with unla-
beled probe (Fig. 3B). We observed no binding with probe 5,
elements found in other regions. As a positive control for the
quality of the oligonucleotide probes, we conducted EMSAs
with nuclear extracts from X. laevis tadpole brain, which has
abundant GC box binding activity (13). This showed that
nuclear proteins formed complexes to an equal extent with
each of the radiolabeled DNAs (including probe 5; data not
BTEB1 Associates with the Proximal Tr?? Promoter in Vivo
in a T3and Developmental Stage-dependent Manner—To
determine whether BTEB1 associates with the proximal Tr?A
promoter in vivo, we conducted ChIP assays on the brain and
tail of premetamorphic X. laevis tadpoles that had been treated
with or without T3for 48 h before sacrifice. We found BTEB1
associated with the proximal Tr?A promoter in vivo, and the
signal was increased in a T3-dependent manner in both brain
and tail in most regions (not region G in brain or tail, nor
region B in tail; Fig. 4). As controls for the ChIP assays we
included the elimination of the primary antibody or the
replacement of the primary antibody with normal rabbit
serum. In each case the ChIP signal was below or at the limit
control was the analysis of regions outside of the proximal
Tr?A promoter (Ef1? promoter, Tr?A exon 3/4, Tr?A exon 5,
and Ifabp promoter), which showed little or no association of
BTEB1. It should be noted that although we analyzed the pro-
bp), the nature of the ChIP assay, in which genomic fragments
ranging from 500 to 1000 bp are produced by sonication, does
not allow us to determine with precision where within the pro-
moter BTEB1 is associating. Nevertheless, our data show that
that the signal is increased following T3treatment.
in supplemental Table 1. The numbering of the seven GC boxes corresponds to that given in Table 4. Thedark gray filled box represents the upstream region,
the S indicates the transcription start site, the light gray filled box represents the 5?-UTR, and the asterisk indicates a TRE that has been characterized and
proposed to mediate T3-dependent transactivation (40, 58).
FIGURE 3. A, binding of GST-xBTEB1[DBD] to regions of the proximal X. laevis
Tr?A promoter in vitro. We used EMSA to test the ability of radioinert DNA
imal Tr?A promoter (generated by PCR; see Fig. 2 and supplemental Table 1)
to displace GST-xBTEB1[DBD] binding to the32P-BTE probe. mBTE, mutated
BTE. B, binding of GST-xBTEB1[DBD] to GC-rich regions of the proximal Tr?A
promoter. We used EMSA to test whether GST-xBTEB1[DBD] could bind to
Tr?A promoter. The numbering of the GC boxes included in each oligonu-
cleotide probe is based on that given in Fig. 2 and Table 4. In each case
2280 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 283•NUMBER 4•JANUARY 25, 2008
at University of Michigan on January 20, 2008
is down-regulated by T3(45).
matic increases in tadpole brain during spontaneous or T3-in-
duced metamorphosis (13). We therefore tested whether the
increased BTEB1 protein expression in brain during spontane-
ous metamorphosis resulted in increased association of BTEB1
the amount of BTEB1 associated with the Tr?A promoter
(regions A/B were analyzed) was increased in animals at meta-
morphic climax (NF stage 62) when T3production and BTEB1
protein are the highest (13) compared with premetamorphic
tadpoles (NF stage 54) when T3and BTEB1 are low (Fig. 4B).
BTEB1 Associates with the Proximal Tr?? Promoter in XTC-2
Cells—We found statistically significant, time-dependent
effects of T3on Bteb1 (F ? 11.255, p ? 0.0001; ANOVA) and
Tr?A (F ? 36.936, p ? 0.0001) mRNA expression in XTC-2
cells (Fig. 5A). Significant up-regulation of Bteb1 mRNA
occurred by 3 h (p ? 0.009; Scheffe’s test), which was the max-
48 h of treatment. By contrast, Tr?A mRNA was not signifi-
cantly increased until 6 h (p ? 0.001) and then reached a max-
imum by 12 h that was maintained through 48 h.
for 24 h, we observed association of BTEB1 with two regions of
the proximal Tr?A promoter. Real time PCR analysis of the
ChIP assay showed significantly greater association of BTEB1
Fig. 2 and 3), compared with the region located in the 5?-UTR
(region G; Fig. 5B; F ? 12.957, p ? 0.0001; ANOVA). The
BTEB1 signal at exon 5 of the Tr?A gene (which is far down-
stream from the transcription start site) was not significantly
T3and developmental stage-dependent manner. ChIP assay was con-
promoter in tadpole brain and tail. Premetamorphic (NF stage 52) X. laevis
tadpoles were treated with 10 nM T3added to the aquarium water for 48 h
prior to tissue collection for ChIP assay (see “Experimental Procedures”). The
lettered Tr?A promoter regions analyzed correspond to those given in Fig. 2
and supplemental Table 1. The Tr?A exon 3/exon 4, and the Ef?1 and Ifabp
promoters were used as negative controls. B, developmental stage-depend-
ent association of BTEB1 with the proximal Tr?A promoter in early prometa-
morphic (NF stage 54) and climax stage (NF stage 62) X. laevis tadpole brain.
Only region A of Tr?A promoter, which showed robust T3-dependent associ-
ation of BTEB1 was targeted for ChIP analysis in this experiment. Each of the
ChIP experiments was repeated three times with similar results.
FIGURE 5. Bteb1 and Tr?A mRNAs are up-regulated by T3, and BTEB1
treated with T3(5 nM) for various times before harvest for RNA isolation and
expression was normalized to the level of rpL8 expression (a housekeeping
gene). Bteb1 mRNA was maximally induced at 3 h (p ? 0.009; Scheffe’s test)
and maintained through 48 h of treatment. Tr?A mRNA was significantly
through 48 h. Bars represent the mean ? S.E. (n ? 6 wells/time point), and
letters above the means indicate significant differences among time points
XTC-2 cells were treated with T3(5 nM) for 24 h, and we used ChIP assay
Tr?A gene. We found significantly greater association of BTEB1 at an
Fig. 2; ?885 to ?752), which contains multiple GC boxes compared with a
region in the 5?-UTR (region G; ?166 to ?322) that has only one GC box, or
the exon 5 of the Tr?A gene which has no GC boxes. Letters indicate signifi-
(region A/B) as analyzed by ChIP assay (*, p ? 0.043; t test).
JANUARY 25, 2008•VOLUME 283•NUMBER 4JOURNAL OF BIOLOGICAL CHEMISTRY 2281
at University of Michigan on January 20, 2008
data not shown; see Fig. 5B). We observed a small but statisti-
cally significant (p ? 0.043; t test) T3-dependent increase in
BTEB1 association with the upstream region of the Tr?A pro-
moter (region A/B) in XTC-2 cells (Fig. 5C). Note that the level
compared with the brain in vivo (?10.5-fold).
Induced Expression of BTEB1 in XTC-2 Cells Accelerates
Autoinduction of the Tr?? Gene—We used XTC-2 cell trans-
BTEB1 enhances autoinduction of the Tr?A gene. Treatment
with T3caused significant time-dependent increases in lucifer-
55.564, p ? 0.0001) and pCMV-xBTEB1 (F ? 601.043, p ?
0.0001; luciferase activity was significantly elevated by 2 h in
both treatments; p ? 0.05; Scheffe’s test; Fig. 6A). Forced
expression of BTEB1 had no effect on basal promoter activity
but resulted in a significant acceleration of Tr?A promoter
autoinduction. Luciferase activity in pCMV-xBTEB1-trans-
We also used XTC-2 cells to determine whether forced
expression of BTEB1 could alter the autoinduction of the
endogenous Tr?A gene. Treatment with T3caused a time-de-
pendent increase in endogenous Tr?A mRNA in cells trans-
fected with empty vector (pCS2; ANOVA; F ? 86.02, p ?
0.0001) and pCS2-xBTEB1 (ANOVA; F ? 215.2, p ? 0.0001;
Fig. 6B). At all time points measured, Tr?A mRNA was signif-
icantly greater in pCS2-xBTEB1-transfected cells compared
with empty vector controls (p ? 0.05 for 0 h; p ? 0.01 for 2, 4,
and 6 h; unpaired t test). Furthermore, the increase in Tr?A
increasing Tr?A mRNA 1.2- and 1.4-fold, respectively, over
empty vector controls (data not shown).
BTEB1 Transactivation Domain Is Required for Tr??
Autoinduction—Two N-terminal transactivation domains in
rodent BTEB1 that were identified by mutagenesis are highly
cated xBTEB1 mutants in which one or both of these transac-
tivation domains were removed to determine whether they are
necessary for the action of BTEB1 on Tr?A autoinduction in
XTC-2 cells. Removal of transactivation domain A (pCS2-
xBTEB1?30) or both domains A and B (pCS2-xBTEB1?99 or
pCS2-xBTEB1?120) abolished the activity of BTEB1 on Tr?A
autoinduction (compare with cells transfected with pCS2-
xBTEB1; Fig. 7A; p ? 0.001). Deletion of only transactivation
domain A (pCS2-xBTEB1?30) resulted in apparent dominant
negative activity, for it also reduced the T3-induced Tr?A
mRNA as compared with the empty vector control (p ? 0.001;
DNA Binding Capacity of BTEB1 Is Not Required for Tr??
Autoinduction—We introduced point mutations into the zinc
to alanine substitutions of the first histidine residue in each of
mutagenesis. The histidine to alanine substitution was shown
previously to eliminate the DNA binding capacity of KLF1 (46)
and another zinc finger protein JAZ (47). The mutant BTEB1
EMSA (Fig. 7C). Similar amounts of wild type BTEB1 and
BTEB1 C2AH mutant were used in the EMSA as verified by
Western blotting (data not shown).
Thyroid hormone initiates programs of gene expression in
diverse tadpole tissues that underlie the dramatic transforma-
expression screens code for transcription factors (6–10, 49).
FIGURE 6. Expression of BTEB1 enhances Tr?A autoinduction in XTC-2
of the Tr?A promoter. XTC-2 cells were cotransfected with the X. laevis Tr?A
promoter-luciferase plasmid, pCMV-xBTEB1, and pRenilla (to normalize for
transfection experiment (n ? 4/treatment group), and the experiment was
data not shown.) Asterisks denote significant differences from empty vector
sion of BTEB1 increases the expression of endogenous Tr?A mRNA. XTC-2
cells were cotransfected with 1 ?g of pCS2-xBTEB1 or pCS2 empty vector.
6 h before harvest. Gene expression analysis was done by RTqPCR. Data
shown are the means ? S.E. from one transfection experiment (n ? 6/treat-
**, p ? 0.001; unpaired t test).
2282 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 4•JANUARY 25, 2008
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These proteins are hypothesized to regulate a secondary
response program of genes necessary for adult phenotypic
expression (50). The transcription factor BTEB1, whose
located upstream of the transcription start site, is the earliest
The Tr?A gene is strongly up-regulated by T3, which requires
direct binding of the T3?TR complex to the Tr?A promoter (a
phenomenon referred to as autoinduction; see Ref. 4). Here we
show that BTEB1 associates with the promoter region of the
Tr?A gene and can enhance T3-dependent transcription. Our
findings support the hypothesis that the early up-regulation of
BTEB1 during tadpole metamorphosis plays a role in the auto-
induction of Tr? genes, which is
hypothesized to be essential for
metamorphosis (50). Therefore, the
protein products of two primary
promoter. Cross-regulation among
primary response transcription fac-
tors is likely to be an important
means for developmental gene reg-
ulation causing robust gene expres-
sion responses necessary for driving
The autoinduction of Tr? genes
liest molecular response to T3in
tadpole tissues (1, 3). Previous stud-
ies that relied on Northern blotting
suggested that the up-regulation of
Tr?A and Bteb1 mRNAs by T3was,
by and large, coordinate (14, 52).
However, several lines of evidence
support the view that the Bteb1
genes are the most rapidly respond-
ing genes yet identified in tadpole
tissues (14, 51). By using RT-PCR
we clearly show that BTEB1 is
induced by T3with faster kinetics
than TR?, both in the tadpole in
vivo and in the X. laevis fibroblast-
derived cell line XTC-2. We found
detectable accumulation, and maxi-
mal induction of BTEB1 transcripts
several hours earlier than TR? (the
precise timing depends on whether
tissues or cultured cells are ana-
lyzed; see also Refs. 13, 53). Also,
BTEB1 protein is up-regulated dur-
ing spontaneous metamorphosis or
by exogenous T3in tadpoles (see
expressed in the same cells (13).
These findings are consistent with
the hypothesis that BTEB1 is pres-
ent within the cell, either commen-
surate with or prior to the up-regu-
lation of TR? and could thus influence transcription of the
The presence of GC-rich sequences in the proximal Tr?A
promoter (commonly referred to as Sp1 sites; see Ref. 40), and
the early and robust T3response kinetics of BTEB1 led us to
hypothesize that this protein binds to and regulates the Tr?A
gene. We used EMSA to test whether regions of the proximal
an EMSA is suggestive of the presence of a transcription factor
binding site, it does not determine whether the DNA-binding
protein actually associates with the gene of interest in vivo. To
BTEB1 are required for Tr?A autoinduction. A, N-terminal truncated forms of xBTEB1 fail to enhance Tr?A
cells were treated with 5 nM T3for 6 h. Gene expression analysis was done by RTqPCR. Data shown are the
means ? S.E. for the T3--treated cells only; n ? 6/treatment. Letters indicate significant differences among
treatments (i.e. means with the same letter are not significantly different; p ? 0.05; Bonferroni’s multiple
comparison test). B, mutations in the three zinc fingers of BTEB1 do not affect activity on Tr?A autoinduction.
The first histidine residue in each of the Cys2-His2zinc finger DNA binding domain of BTEB1 was mutated to
48 h later cells were treated with 5 nM T3for 6 h. Data shown are the means ? S.E. from one transfection
experiment (n ? 6/treatment) and the experiment was repeated twice with similar results. Letters indicate
significant differences among treatments (i.e. means with the same letter are not significantly different; p ?
0.05; Bonferroni’s multiple comparison test). C, electrophoretic mobility shift assay showed that the BTEB1
C2AH mutant does not bind to DNA. Recombinant wild type BTEB1 and BTEB1 C2AH mutant proteins were
generated by coupled in vitro transcription/translation, and varying amounts were tested for their ability to
and antibody supershift was used to verify the presence of BTEB1 protein in the protein-DNA complexes
formed. Western blot analysis confirmed that equal amounts of wild type and mutant BTEB1 proteins were
used in the EMSA (data not shown).
JANUARY 25, 2008•VOLUME 283•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2283
at University of Michigan on January 20, 2008
test this for BTEB1 and Tr?A, we used ChIP assay that
depended on a specific affinity-purified antiserum directed
against the unique N-terminal region of the frog BTEB1 pro-
with the proximal Tr?A promoter in vivo in a hormone- and
developmental stage-dependent manner. Earlier, we showed
vivo and is highly expressed during metamorphic climax (com-
pared with premetamorphosis (13)). The enhanced association
of BTEB1 with the Tr?A promoter with T3treatment and at
metamorphic climax could be due to the increased expression
of BTEB1 and/or an active T3-dependent recruitment of
BTEB1 to the promoter.
Similar to our findings in the tadpole in vivo we found that
Bteb1 and Tr?A mRNAs are up-regulated in XTC-2 cells, and
that Bteb1 exhibits faster kinetics than Tr?A. We also found
that BTEB1 associates with the proximal Tr?A promoter in
XTC-2 cells by ChIP assay and that the degree of association
and versus the Tr?A exon 5 where there are no identifiable GC
boxes; see Fig. 5B). Furthermore, association of BTEB1 with
region A/B in XTC-2 cells was T3-dependent.
Given that BTEB1 and TRs are expressed in the same cells
vitro and in vivo, and frog BTEB1 possesses transactivation
function (13), we hypothesized that BTEB1 positively regulates
the Tr?A gene. In support of this hypothesis we found that
induced expression of BTEB1 in XTC-2 cells resulted in faster
kinetics and greater absolute magnitude of induction by T3of
the Tr?A gene, as determined by promoter-reporter transfec-
By contrast to the full-length BTEB1, forced expression of
N-terminal truncated mutants of BTEB1 in which one or both
transactivation domains were removed eliminated activity on
Tr?A autoinduction. Kobayashi et al. (41) identified two trans-
activation domains in rat BTEB1 by mutagenesis. Earlier we
showed that frog BTEB1 has transactivation activity, and the
identified transactivation domains are very similar among the
frog and rodent proteins, suggesting conserved functions (13).
of BTEB1 for activity on Tr?A autoinduction.
Up to this point our results were consistent with BTEB1
binding to GC-rich regions of the frog Tr?A gene leading to
enhanced autoinduction. We were therefore surprised to dis-
cover that this DNA binding capacity was dispensable for
BTEB1 action. Substitution of alanines for each of the zinc-
chelating histidine residues in the three zinc fingers of BTEB1
destroyed DNA binding but did not alter activity of the protein
on Tr?A. Thus, although BTEB1 associates with chromatin at
the Tr?A promoter in vivo, binding to DNA is not required for
tions in this regard through protein-protein interaction. The
GC boxes present in the Tr?A promoter could facilitate the
targeting of the protein to this genomic region.
Members of the KLF and Sp factor families have been found
to synergize with nuclear hormone receptors through protein-
protein interactions. For example, Sp1 interacts with the estro-
shown to interact with progesterone receptor in the regulation
of progesterone receptor target genes in endometrial epithelial
cells (30). However, in a preliminary study we found no direct
interaction between BTEB1 and TRs using coimmunoprecipi-
tation assays.5To our knowledge, other than the PR, BTEB1
interactions with nuclear proteins have not been studied.
BTEB1 is a member of a family of proteins (KLF/Sp1-like) that
bind to GC- or GT-rich regions in gene promoters (15). It is
possible that other KLFs or Sp-like factors regulate the Tr?A
promoter, and this deserves further study. However, it is note-
worthy that BTEB1 is the only KLF identified in several gene
expression screens of tadpole tissues that is strongly up-regu-
lated by T3during metamorphosis (5–12, 49). Also, to our
knowledge, BTEB1 is the only KLF/Sp1-like family member
found to be regulated by T3in mammalian cells (24). Thus, if
do so as basal or constitutive factors. We propose here that the
strong up-regulation of BTEB1 by T3is critical to the role that
BTEB1 plays in regulating the Tr?A promoter in vivo.
In conclusion, our results support the hypothesis that the
protein product of the immediate early gene Bteb1 associates
with the Tr?A genomic region in vivo and can enhance autoin-
duction, i.e. it forms a positive regulatory loop. The surge in
plasma T3that occurs during metamorphic climax in the tad-
pole is accompanied by a dramatic autoinduction of Tr? genes
(57). The autoinduction of Tr? genes is thought to be essential
for metamorphosis, especially for later developmental events
such as cell differentiation and programmed cell death (e.g. tail
to initiate tissue transformation may require that TRs bind
to and activate the Tr? promoters (4) and induce the expres-
sion of BTEB1, which cooperates with TRs in the autoinduc-
tion of their genes. Such cooperativity among the protein
products of immediate early genes may be a common phe-
nomenon in animal development.
Acknowledgments—During this work, we used the Molecular Core of
the Michigan Diabetes Research Training Center, which is funded by
NIDDKD Grant 5P60 DK20572 from the National Institutes of
Health. We thank Eric Hoopfer for providing valuable input during
the initial stages of this work. We are very grateful to Dr. Yun-Bo Shi
for supplying the X. laevis Tr?A promoter-luciferase plasmid and
Dr. David Turner for the pCS2 plasmid. We thank Dr. Dan Buchholz
for assistance with ChIP assay and Keith Williamson, Cyrus
Kholdani, and Jessica Kim for technical assistance.
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Supplemental Table 1. The X. laevis TrβΑ promoter divided into seven segments for
analysis, and location of GC-rich sequences.
A) -1138/-823 Three GC boxes (-1029/-1024; -1020/-1015; -973/-968)
B) -841/-604 Two GC boxes (-767/-762; -720/-715)
C) -622/-414 One GC box (-538/-533)
D) -432/-266 No GC box
E) -283/-92 No GC box
F) -109/+182 No GC box
G) +164/+366 One GC box (+231/+236)
GC boxes are indicated in boldtype capitals. TRE half sites are boldtype lowercase
underlined. The transcription start site is the capital, boldtype, shaded ‘G’ in fragment F
(based on 58). Genbank Accession # U04675.