Full text of DOI:10.1089/105072503322511328

THYROID
Volume 13, Number 10, 2003
© Mary Ann Liebert, Inc.
Production and Application of Polyclonal Antibody to
Human Thyroid Transcription Factor 2 Reveals Thyroid
Transcription Factor 2 Protein Expression in Adult Thyroid
and Hair Follicles and Prepubertal Testis
Melwyn Sequeira,1,2 Farakid Al-Khafaji,1 SooMi Park,3 Mark D. Lewis,1 Malcolm H. Wheeler,2
V. Krishna K. Chatterjee,3 Bharat Jasani, and Marian Ludgate1
Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate,
indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying
an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector,
production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits
were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of
thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was ob-
tained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid fol-
licular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No
staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues
tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactiv-
ity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In con-
clusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this
transcription factor in adult human thyroid and hair follicles and prepubertal testis.
Introduction
with other thyroid transcription factors (11,12), TTF2 may be
expressed in tissues other than the thyroid. Furthermore,
northern analysis of a range of human tissues using a probe
HYROID TRANSCRIPTION FACTOR 2 (TTF2) is a member of
the forkhead/winged helix family of transcription fac- specific for the 3 untranslated region (UTR) of TTF2 revealed
tors, present in the developing thyroid. TTF2 is a helix- a major transcript of 5.3 kb in the thyroid and a second tran-
turn-helix DNA binding protein, which functions as a tran- script of 3.2 kb that was also present in the testis (9).
9
T
In a previous study (13), we investigated TTF2 gene ex-
pression in a range of benign and malignant thyroid lesions.
In normal thyroid tissues TTF2 transcript levels are low; 18
scriptional activator in adult thyroid (1,2). Tissue-specific ex-
pression of thyroglobulin (Tg) and thyroid peroxidase (TPO)
is regulated at the transcriptional level and both genes have
binding sites for TTF2, which binds the DNA as a dimer and of 36 were weakly positive and 18 of 36 negative. TTF2 tran-
is regulated by the redox state (3,4). There is some contro- scripts were detected in 8 of 8 Graves’ disease (GD); 3 of 7
versy as to the precise role of TTF2 in the adult thyroid be- Hashimoto’s disease; 2 of 2 follicular hyperplasia; 15 of 21
follicular adenoma; 11 of 13 multinodular goiters, and 0 of 1
hyalinizing trabecular adenoma. In the malignant thyroid le-
sions, TTF2 transcripts were detected in 8 of 18 follicular can-
cers; 0 of 2 anaplastic carcinomas, and 11 of 17 papillary can-
cause, despite indirect evidence that it is a transcriptional ac-
tivator (2,5), more recently it has also been found to function
as a repressor (6). TTF2 is the main mediator of thyrotropin
(TSH) and insulin regulation of TPO gene expression (7).
TTF2-null mice display features of either thyroid agenesis cers. Confirmation that the transcripts are translated into the
or maldescent (8). In humans, germline mutations of the TTF2 protein in these thyroids and lesions requires evalua-
tion of the tissue sections by immunohistochemistry (IHC),
but antibodies to human TTF2 are lacking. Antibodies we
TTF2 gene result in thyroid agenesis, cleft palate, choanal
atresia, and spiky hair (9,10). This suggests that, in common
Departments of 1Medicine (EMD Section), 2Surgery and Pathology, University of Wales College of Medicine, Cardiff, U.K.
3Department of Medicine, Addenbrooke’s Hospital, Cambridge, U.K.
927

928
SEQUEIRA ET AL.
raised to synthetic TTF2 peptides yielded disappointing re- vested by centrifugation, 5000 rpm for 5 minutes, 4°C, and
sults in IHC. In a recent study a polyclonal antibody to rat resuspended in 10 mL of native binding buffer (20 mM phos-
TTF2 was described (14) and applied to mouse embryo sec- phate, 500 mM NaCl, pH 7.8). Lysozyme was added to the
tions. It was not reported whether the antibody cross-reacts lysate to a final concentration of 100 g/mL and incubated
m
with the human protein.
on ice for 15 minutes. The suspension was kept on ice, and
The aim of this work was to produce a TTF2 fusion pro- sonicated at medium intensity with three 10-second bursts
tein, use it to raise an antibody to human TTF2, suitable for and then submitted to three rounds of freeze/thawing. In-
immunohistochemical analysis of archival formalin-fixed, soluble debris was removed by centrifugation at 3000 for
g
paraffin-embedded tissue sections, and apply it to investi- 15 minutes, at 4°C. The lysate was filtered through a 0.45-
gate the extrathyroidal expression of TTF2 protein.
m filter and then applied to the equilibrated nickel
m
(ProBond, Invitrogen, Carlsbad, CA) column in two 5-mL
aliquots. The polyhistidine tagged proteins were allowed to
bind to the column by gently rocking the column with the
cell lysate for 15 minutes at 4°C. The column was washed
three times with 4 mL of native binding buffer (pH 7.8) fol-
Materials and Methods
Subcloning human TTF2 into a procaryotic
expression vector
The entire coding region of human TTF2 (accession num- lowed by three washes with 4 mL of native wash buffer (pH
ber 51294, National Center for Biotechnology Information 6.0). The protein was eluted by applying four 2.5-mL aliquots
[NCBI]), was excised from pGEM III and subcloned into of increasing concentrations of imidazole (50–500 mM). The
H1 and
III restriction sites, in the poly linker of eluate was collected in 1-mL aliquots and quantified using
Bam
pTrcHis (InVitrogen, Carlsbad, CA), using standard ligation 60% trichloroacetic acid precipitation with bovine serum al-
protocols (15). The vector uses the promoter (regulated bumin (BSA) as the control standard. The aliquots with the
Hind
trc
by the lac operator) for high-level, inducible expression of highest protein concentrations were pooled together.
recombinant proteins, which are tagged with six histidine
residues for nickel column purification. The resulting
pTrcHis-TTF2 construct was confirmed by complete se-
quencing of the TTF2 coding region and 200 base pairs of
flanking vector sequence. Plasmid DNA was amplified us-
ing a Qiagen (Valencia, CA) maxiprep kit according to the
Western blotting
Equal quantities (volume) of the cell lysate, the flow
through and the purified protein were separated by 10%
PAGE and analyzed both by Coomassie blue staining and
Western blotting. The blot was incubated in a 1:1000 dilu-
tion of a rabbit antibody raised against a synthetic peptide
of TTF2 (GVPGEATGRGAGGRRRKRPLQ–C). The blots
were then incubated with a second antibody (anti-rabbit IgG-
HRP conjugate, 1:5000 at room temperature for 1 hour) and
detected using a chemiluminescent system (ECL Plus, Amer-
sham Pharmacia Biotech, Uppsala, Sweden), films were ex-
posed for 2 minutes. The membrane was stripped and
reprobed with a 1:1000 dilution of the preimmune serum of
the same rabbit used to generate the peptide antibody.
manufacturer’s instructions and used to transform the
Esch-
Topp 10 strain.
erichia coli
Optimization of soluble TTF2 protein production
In pilot studies, 50 ml of LB with ampicillin was inocu-
lated with 0.5 mL of an overnight culture of Topp 10 trans-
formed with the pTrcHis-TTF2 construct and incubated in
an orbital shaker at 230 rpm at 30°C or 37°C, until the opti-
cal density (OD)600 reached 0.3. Expression of recombinant
protein was induced by the addition of isopropylthiogalac-
topyranoside (IPTG) to a final concentration of 0.1 or 1 mM.
One-milliliter aliquots of the culture were taken at hourly in-
tervals for up to 4 hours after induction with IPTG, cen-
trifuged to separate the pellet from the supernatant and the
Immunization protocol
Prior to immunization the protein was dialyzed exten-
sively against PBS to remove the imidazole. Two rabbits
were immunized subcutaneously in several sites, with 300
pellet resuspended in 100 L of phosphate-buffered saline
m
g purified and dialyzed TTF2 fusion protein, initially in
m
(PBS) and either treated immediately or kept on ice
overnight. The samples were put through three cycles of
freeze/thawing and the pellet (insoluble protein) and su-
pernatant (soluble protein) analyzed separately. The samples
complete Freund’s adjuvant and subsequently boosted at ap-
proximately 3-week intervals with 300 g TTF2 in incom-
plete Freund’s adjuvant.
m
were resuspended in 100 L of sodium dodecyl sulfate-poly-
m
acrylamide gel electrophoreses (SDS-PAGE) loading buffer
ELISA screening
(16), boiled for 5 minutes, and 20 L of each of the insolu-
m
Anti-TTF2 antibody activity in rabbit serum was deter-
mined by enzyme-linked immunosorbent assay (ELISA).
ble and soluble protein fractions were analyzed by 10%
PAGE. A number of variables were evaluated for optimal
protein production including incubation time after induc-
tion, immediate or delayed processing, IPTG concentration,
and incubation temperature. The gels were stained in
Coomassie blue solution, destained, and dried.
Polypropylene 96-well microplates were coated with 100
L
m
of a 10 g/mL dilution of the purified TTF2 fusion protein
m
in coating buffer (15 mM carbonate, 35 mM bicarbonate, pH
9.6) overnight at 4°C. The plates were washed with ELISA
wash buffer (10 mM phosphate, 150 mM NaCl, 0.1% Tween,
5% BSA, pH 7.4) and incubated with the immune sera from
various bleeds at concentrations of 1:500, 1:1000, and 1:5000
for 2 hours. The plates were washed three times and incu-
bated for 30 minutes in a 1:2000 dilution of anti-rabbit horse-
radish peroxidase conjugate solution containing a 1:50 con-
Large-scale production of TTF2 and
nickel column purification
The cells from a 1 L optimized culture of Topp 10-TTF2 in
LB medium containing 100 g/mL ampicillin, were har-
m

POLYCLONAL ANTIBODY TO HUMAN TTF2
929
for 25 minutes in 1 mM ethylenediaminetetraacetic acid
(EDTA) solution. Endogenous biotin activity was blocked us-
ing a commercially available avidin/biotin blocking kit
(Vector Labs, Burlingame, CA) by incubating the slides for
15 minutes each in 2–3 drops per slide of avidin followed by
biotin. The slides were thoroughly washed in PBS between
each step and prior to application of the primary antibody,
overnight at 4°C. Multi-Link biotinylated anti-IgG (Bio-
Genex, San Ramon, CA) and streptavidin peroxidase label
(BioGenex) were utilized at a 1:40 dilution, each for 30 min-
utes at room temperature. Sections were washed with PBS
between each step of the procedure. The chromogenic sub-
strate utilized was diaminobenzidine tetrahydrochloride
(DAB) made up in PBS with two drops of hydrogen perox-
ide. The sections were counterstained with Harris’ hema-
toxylin.
A
B
Optimization of TTF2 antibody staining was performed
using doubling dilutions of the antibody from 1:500 to 1:8000.
Optimum dilution was set at the point where specific stain-
ing of thyroid follicular cell nuclei/cytoplasm was visible
and there was little or no staining of the interstitium, the con-
nective tissue, smooth muscle, or endothelium.
In later experiments, the sections were also incubated in
5% milk in PBS for 30 minutes prior to incubation in the pri-
mary antibody (the rest of the procedure being as detailed
above) in order to further reduce any background staining.
Results
Coomassie blue stain of bacterial lysate sepa-
FIG. 1. A:
Generation of TTF2 fusion protein and polyclonal antibody
rated on 10% polyacrylamide gel. MW, molecular weight
markers (Biolabs); Lanes 1 and 2, insoluble pellet and solu-
ble fraction prior to induction with 0.1 mM IPTG (isopropyl-
thiogalactopyranoside); Lanes 3 and 4, insoluble pellet and
soluble fraction 3 hours postinduction with 0.1 mM IPTG.
Optimal expression of the polyhistidine tagged TTF2 fu-
sion protein in Topp 10 was achieved after 4 hours induc-
tion with IPTG. The majority of the protein was in the in-
soluble (pellet) form. Incubation at 30°C, produced a
marginal improvement compared to 37°C but there was no
difference between induction with 0.1 or 1 mM of IPTG.
However, in subsequent experiments we found that the sol-
ubility of the TTF2 protein was improved by maintaining the
crude cell lysate on ice overnight prior to the freeze/thaw
cycles as shown in Figure 1A in which the TTF2 protein at
48 kD is clearly visible.
(Note the increased intensity of the band at 48 kd).
West-
B:
ern blot of nickel column purified protein lysate using preim-
mune serum (left) and TTF2 peptide antibody (right) con-
firming the identity of the 48-kd band. Lanes 1 and 3, protein
produced at 30°C; lanes 2 and 4, protein produced at 37°C.
centration of sheep serum. After washing, the antibody was
detected by incubating in substrate solution (i.e., ABTS (Azi-
nobisethylbenzthiazoline sulfonic acid, Sigma Chemical Co,
St. Louis, MO) in citrate phosphate buffer for 30 minutes).
The OD at A410 was recorded at 5-minute intervals for half
an hour. Negative controls included preimmune serum at
1:250 concentration in addition to uncoated wells.
After nickel purification of the cell lysate, the 48-kD pro-
TABLE 1. OPTICAL DENSITY READINGS AT 450 nm OF
PREIMMUNE AND IMMUNE SERA FROM RABBITS IMMUNIZED
WITH THE PURIFIED TTF2 FUSION PROTEIN
Rabbit A
Rabbit B
Immunohistochemistry
Preimmune 1 250
0.165
2.68
2.61
1.9
2.85
2.64
1.85
0.140
2.65
2.49
0.96
2.72
2.39
1.00
;
Paraffin-embedded human thyroid sections and sections
of the testis, skin, appendix, lymph node, uterus, fallopian
tube, liver, prostate, and gallbladder were retrieved from the
archives of the Pathology Department at UWCM. The poly-
clonal TTF2 antibody was initially applied to sections of thy-
roid from a patient with GD using the streptavidin-biotin
complex immunoperoxidase procedure, with and without
antigen retrieval. In brief, the sections were deparaffinized,
then incubated in methanol (98.4%) and hydrogen peroxide
(1.6%) to block endogenous peroxidase activity and if anti-
gen retrieval was to be performed, microwaved at full power
Immune-I 1 500
;
Immune-I 1 1000
;
Immune-I 1 5000
;
Immune-II 1 500
;
Immune-II 1 1000
;
Immune-II 1 5000
;
Immune-I, serum obtained after three immunizations; immune-II,
serum obtained after four immunizations.
Optical density readings are the mean of at least duplicates which
agree to within 10%.
TTF2, thyroid transcription factor 2.

930
SEQUEIRA ET AL.
tein was the major component. Its identity as TTF2 was con- lium as shown in Figure 2B. No staining was obtained with
firmed in a Western blot using an antibody to a TTF2 pep- the preimmune serum in the same conditions in the Graves’
tide (Fig. 1B), which revealed bands at 48 kd and 25 kd. Im- thyroid (Fig. 2A). In the normal thyroid, approximately one
munoreactivity to the lower molecular weight protein was third of the follicles displayed TTF2 staining, predominantly
also present in the preimmune serum from the rabbit used in the cytoplasm, although there were a few ( 5%) posi-
,
to raise the TTF2 peptide antibody.
tively staining nuclei (data not shown). In contrast, in the
We were able to produce approximately 9 mg of purified GD thyroid, the majority ( 90%) of the thyrocytes displayed
.
TTF2 per liter of culture medium. The purified TTF2 fusion nuclear and cytoplasmic staining with only a minority being
protein was used for the immunization of two rabbits and negative.
generated two high-titer antibodies, as summarized in
Table 1.
No staining was obtained with appendix, lymph node,
uterus, fallopian tube, liver, prostate, or gallbladder tested
with the TTF2 polyclonal (data not shown). The specificity
of the TTF2 antibody was further confirmed using Chinese
hamster ovary (CHO) cells stably transfected with full-length
human TTF2 that displayed TTF2 immnoreactivity, in con-
trast to nontransfected cells that did not (data not shown).
TTF2 staining was detected in the epidermis of the skin
but was most prominent in the hair follicles, as shown in Fig-
ure 2D. The immunoreactivity was detected in 50%–60% of
nuclei in the epithelial cells of the outer root sheath. In con-
Immunohistochemistry with the TTF2 polyclonal
The optimum protocol to produce specific staining of
Graves’ thyroid follicular cell nuclei/cytoplasm was prein-
cubation of the sections with 5% milk in PBS for 30 minutes
before incubation in 1:500 diluted TTF2 antibody, without
antigen retrieval. This resulted in little or no staining of the
interstitium, connective tissue, smooth muscle or endothe-
A
C
E
B
D
F
Paraffin sections showing immunohistochemical staining.
Graves’ thyroid with preimmune serum. Graves’
B:
FIG. 2.
thyroid stained with the thyroid transcription factor 2 (TTF2) polyclonal, note the predominantly nuclear with some cyto-
plasmic staining in the majority of thyroid follicular cells. Section of skin with pre-immune serum. Section of skin
A:
C:
stained with TTF2 polyclonal showing cytoplasmic and nuclear staining of cells in the outer root sheath of the hair follicle.
Prepubertal testis with preimmune serum. Section of prepubertal testis showing TTF2 immunoreactivity predomi-
D:
E:
F:
nantly in the cytoplasm and with some nuclear staining in the seminiferous tubules. Magnification, 40
.
3

POLYCLONAL ANTIBODY TO HUMAN TTF2
931
trast, TTF2 staining was absent from the more differentiated
cells in the suprabasal layer.
We have demonstrated for the first time, the presence of
TTF2 protein in the prepubertal testis (inferred from our pre-
In sections of prepubertal testis, TTF2 immunoreactivity vious Northern blot analysis) and defined its location as be-
was present in the exocrine cells of the seminiferous tubules ing the exocrine seminiferous tubules. The possible function
but absent from the germinal cell layer, as shown in Figure of TTF2 in the testis is not clear but it may effect spermato-
2F. No staining was obtained with the preimmune serum in genesis. The reproductive potential of human subjects with
skin or testis, as shown in Figure 2C and 2E, respectively.
TTF2 mutations is as yet unknown. Another forkhead pro-
tein, HFH-4, has been linked to the regulation and mainte-
nance of the ciliated cell phenotype in epithelial cells and
hence to cell motility (26). TTF2-null mice have either thy-
roid agenesis or maldescent, suggesting that it may control
migration of the gland. The recent cloning of a TTF2 homo-
logue, AmphiFoxE4, identified in amphioxus supports this
hypothesis (27).
The spiky-hair phenotype of patients harboring germline
mutations in TTF2 indicate that the protein has a role and is
expressed in the hair follicle, at least during embryogenesis
and this has been confirmed in the mouse (14). Our studies
demonstrate for the first time that human adult hair follicles
also express TTF2 protein and that it is located in the same
region of the follicle as in the embryo, the outer root sheath,
whose precise role in hair development is not known. Fu-
ture studies will investigate whether TTF2 is present at all
stages of the hair follicle life cycle, anagen, telogen, and cata-
gen.
In conclusion, we have developed an antibody to TTF2,
which has enabled us to demonstrate the presence of this
transcription factor in adult human thyroid and in several
extrathyroidal tissues, confirming our results obtained at the
transcript level (9,13). Future studies will apply the antibody
to investigate TTF2 protein expression in a range of benign
and malignant thyroid lesions to determine whether it is af-
fected by and/or contributes to malignant transformation.
Discussion
TTF2 is a forkhead transcription factor encoded by a sin-
gle exon. It differs from other members of the family, many
of which are encoded by two exons, with the forkhead do-
main located in the first (17), but may have a more com-
plex genomic organization (e.g., FKHL16, which spans 10
exons on human chromosome 12; 18). The absence of in-
trons, makes transcript-based detection methods for TTF2
prone to false-positives because, for example, reverse tran-
scription-polymerase chain reactions (RT-PCR) reactions
could amplify contaminating genomic DNA and produce
an amplicon of the expected size.
hybridization pro-
In situ
vides one solution but to confirm the presence of TTF2 pro-
tein requires IHC, which in turn depends on a suitable an-
tibody.
Previous attempts to generate an antibody to a synthetic
peptide of TTF2 yielded disappointing results. We opted to
express the entire TTF2 coding region in bacteria, but expe-
rienced considerable difficulty in obtaining the protein in sol-
uble form. This was slightly surprising, because TTF2 lacks
membrane-spanning regions, which are known to hamper
production in bacteria. However, TTF2 has a polyalanine
tract, which is highly hydrophobic and probably contributed
to the difficulties we encountered. This region is interesting,
because it can be associated with transcriptional repression
(19). A recent study has found variations in the number of
alanine repeats in thyroid dysgenesis patients, although sub-
Acknowledgements
We are grateful to the Royal College of Surgeons and the
Wellcome Trust for grant support.
sequent
expression revealed that the transcriptional
in vitro
activity of the polymorphic TTF2 variants was equivalent to
the wild-type (20).
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Marian Ludgate
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