Testis-specific TTF-D Binds to Single-stranded DNA in the
c-mos and Odf1 Promoters and Activates
Odf1*
Jessica H.
Oosterhuis
and
Frans A.
van der Hoorn§
From the Department of Biochemistry & Molecular Biology, University
of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1,
Canada
 |
ABSTRACT |
We recently identified testis-specific nuclear
factor binding sites in the testis-specific promoters of the
c-mos gene and the Odf1 gene, which are 80%
identical. Here we characterize a testis-specific nuclear factor,
TTF-D, which is able to complex with both binding sites and stimulates
Odf1 promoter activity. TTF-D is detectable in mouse testis
as early as day 11 postpartum and contains three peptides of 22, 25, and 35 kDa in size. Surprisingly, TTF-D binds specifically to its
cognate double-stranded DNA binding site as well as to its
single-stranded DNA binding site. Both double-stranded and
single-stranded binding site oligonucleotide DNA can specifically
repress Odf1 promoter activity. Our results suggest that
TTF-D is involved in positive transcription regulation of a pre-meiotic
and a post-meiotic gene in the testis.
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INTRODUCTION |
Spermatogenesis represents a model system for mammalian
development. Within a tightly controlled environment, spermatogonia undergo rapid successive divisions to form spermatocytes, which, upon
passage through a meiotic phase, enter a differentiation or
spermiogenic phase in which spermatids undergo morphological changes to
form mature sperm. The processes resulting in the formation of
spermatozoa are well defined at the morphological level. However, the
molecular signals responsible for tissue- and stage-specific gene
expression throughout germ cell development are less well defined.
Several genes have been identified that are only expressed in the
testis or express a testis-specific variant of a somatic gene (1-3).
Using transgenic mice, promoter regions in mouse protamine
mP11 (4), mP2 (5), human
phosphoglycerate kinase 2 (PGK-2) (6), testis angiotensin-converting
enzyme (7), and rat Odf1 (8, 9) genes that confer male germ
cell-specific transcription onto reporter genes have been delineated.
These studies identified transcription factor binding sites crucial for
their expression. Although the transcription factors that bind to such
sites have not been positively identified for most cases, a small
number have been characterized (reviewed in Refs. 10 and 11). The best
characterized testis-specific transcription factor is perhaps CREM
.
Full-length CREM
acts as a positive regulator of transcription in
spermatids (12, 13). Under the control of follicle-stimulating hormone,
a switch in the regulation of expression of CREM occurs during
spermatogenesis. Negative regulators CREM
, CREM
, and CREM
are
shut off, and the positive regulator CREM
is switched on (12, 14).
An unstable form of CREM
RNA is first expressed in spermatocytes. In
spermatids, CREM
RNA stability is greatly increased due to the use
of an alternative polyadenylation site, and the CREM
protein is only
produced in these cells (15). Phosphorylation sites on CREM
regulate
its activity (16). Finally, the appearance of CREM activator protein in
spermatids correlates with the transcription activation of
Odf1, which contains a CREM
binding site (17). Strong
positive regulation of Odf1 by CREM
was further shown by
in vitro transcription assays and transfection assays,
implicating an important regulatory role for CREM
in Odf1
transcription during spermatogenesis (17). The generation of CREM
knockout mice (18, 19) further showed that in addition to
Odf1, mP1, testis angiotensin-converting enzyme, and TP1 are also regulated by CREM
in spermatids. In addition to CREM
,
several genes have been cloned from testis cDNA libraries that
contain motifs found in transcriptional regulatory proteins. Two zinc finger-containing genes have been identified: Zfp-35, which is pachytene spermatocyte-specific (20), and Zfp-29, which is expressed in
round spermatids (21). Testis-specific genes encoding high mobility
group box motifs have also been cloned. Boissonneault and Lau (22)
isolated the testis-specific high mobility group that is predominantly
located in elongating spermatids and may be involved in the regulation
of gene expression of the haploid male genome. An SRY-related gene that
also encodes a high mobility group box designated Sox-5 was isolated
(23). Sox-5 is most highly expressed in round spermatids (reviewed in
Ref. 24). The function of these putative transcription factors in
spermatogenesis and the promoters that they interact with remain to be
determined. Putative transcription factors were also identified based
on specific protein-DNA interactions. Factors present in testis
extracts have been shown to interact specifically with
cis-acting elements in the promoters for several genes
including mP1 (25), testis-specific histone H1t (26), and PGK-2 (27,
28). The appearance of a 13-kDa protein and a 30-kDa protein, which
bind the H1t promoter, coincides with the onset of transcription of the
H1t gene (Ref. 29; reviewed in Ref. 30). Testis-specific and
promoter-specific regulation of PGK-2 transcription involves the
binding of TAP-I, resulting in stimulation of PGK-2 transcription (28).
We previously demonstrated that an uncharacterized testis-specific
transcription factor, which we called TTF-D, binds to the
Odf1 promoter (31). A related binding site was identified in
the c-mos promoter (32). Using testis-specific nuclear
extracts, we analyzed TTF-D and its pattern of expression in the
developing testis, and we investigated the role of TTF-D in
Odf1 promoter activity.
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EXPERIMENTAL PROCEDURES |
Nuclear Extract Isolation
Nuclei from rat liver and seminiferous tubules (STs) were
prepared as described by van der Hoorn and Tarnasky (31). The protein
concentrations in the dialyzed nuclear extracts ranged from 9 to 16 mg/ml. Nuclear extracts from mouse testes were prepared as described by
Lilienbaum and Paulin (33). Protein concentrations were determined by
colorimetric assay (Bio-Rad).
DNA-Protein Interactions
Gel Retardation Assay--
Gel retardation assays were performed
as described previously (34) using double-stranded (ds)
oligonucleotides Odf1D1 (5'-AATTGGCCTTAGGG-3') and c-mosD1
(5'-ATGGACTTAGGA-3') labeled with Klenow polymerase. In the indicated
experiments, ss oligonucleotides were labeled with polynucleotide
kinase. Reaction mixtures contained 1 ng of labeled oligonucleotide
DNA, 1.0 µg of poly(dI-dC)(dI-dC), and extract. The reaction products
were separated on 8% nondenaturing polyacrylamide gels and visualized
by autoradiography. In the indicated experiments, an excess of
unlabeled ds or ss oligonucleotide DNA was used as competitor DNA.
Oligonucleotides used in such competition experiments were D2
(5'-AATTACCTTAACTG-3'), B (5'-AATTGTGGCTCCTGCCC-3'), MUT1
(5'-AATTCTCCTTAGGG-3'), MUT2 (5'-AATTGGCACGAGGG-3'), and MUT3
(5'-AATTGGCCTTCTAG-3').
UV Cross-linking Assays--
To determine the molecular weight
of proteins capable of forming complexes in the gel retardation
assays, the radiolabeled oligonucleotide DNA-protein complexes were
cross-linked by UV irradiation using a UV Stratalinker 1800 (Stratagene, La Jolla, CA) before separation on native polyacrylamide
gels or on 8% or 12% SDS-polyacrylamide gels. The complexes
were visualized by autoradiography.
Southwestern Blotting Assays--
Liver and ST nuclear proteins
(50 µg) were separated by SDS-PAGE, transferred to NitroPlus
nitrocellulose filters (Micron Separations Inc., Fisher Scientific,
Nepean, Ontario, Canada), and incubated in blocking buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% skim milk
powder) (Becton Dickinson, Cockeysville, MD) for 1 h at 4 °C.
Filters were incubated overnight at 4 °C in binding buffer (25 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EDTA, and 100 µg/ml ss herring sperm DNA). The filters were then probed using
105 cpm/ml Odf1D1 DNA in binding buffer for 6 h at
4 °C. After four 8-min washes in binding buffer at 4 °C, filters
were exposed to Kodak XAR film.
In Vitro Transcriptions
In vitro transcriptions were performed as described
previously by van der Hoorn and Tarnasky (31, 32), using
pRT7-0.2C2AT construct as the test promoter and pAdMLP
construct as the internal control. Transcription of the constructs
generated the expected RNAs of 385 and 190 nucleotides for
Odf1 and Ad, respectively. Competitor oligonucleotides (ds
or ss) were added to reactions as indicated in the legends.
Autoradiograms were scanned by laser densitometry, and transcription
levels were normalized using the internal adenovirus major late
promoter control and expressed as a percentage of full Odf1
promoter activity as described previously (31).
 |
RESULTS |
Characterization of TTF-D--
Delineation of the testis-specific
c-mos (32) and testis-specific Odf1 (31)
promoters had revealed testis-specific nuclear factor binding sites
that are 80% identical and are located at the same distance from the
TATA box (at
120) and may therefore bind a common testis-specific
nuclear protein that we named TTF-D. To characterize TTF-D and to
demonstrate that both Odf1D1 and c-mosD1 oligonucleotides
bind the same proteins as suggested by the previous gel retardation
assays, UV cross-linking assays were performed. ST nuclear proteins
(and, as a control, liver nuclear proteins) were UV cross-linked to
radiolabeled oligonucleotides and separated according to size. We
observed that testis-specific peptides with approximate molecular
masses of 33, 23, and 20 kDa (after subtracting the molecular mass of
the oligonucleotides) bind to both oligonucleotide DNAs (Fig.
1, lanes b and e,
arrows). We had previously shown that the two oligonucleotides
could bind a nuclear protein present in somatic cells (24, 25). Our
experiments using liver nuclear extracts show that both
oligonucleotides bind a 40-kDa somatic protein (Fig. 1, lanes
a and d). As expected, we observe less of this somatic
protein when using ST extract (lanes b and e)
because seminiferous tubules contain few somatic cells. Formation of
all ST-generated complexes can be competed by self-oligonucleotide
(data not shown) as well as by cross-competition (Fig. 1, lanes
c and f). Odf1D1 oligonucleotide, but not
c-mosD1 oligonucleotide, also interacts with the 30- and
32-kDa peptides that are present in somatic cells but not in germ
cells. Their origin is unknown. These two proteins do not bind the
c-mosD1 oligonucleotide (lane d), and, as
expected, they are not efficiently competed by this oligonucleotide
(lane b).

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Fig. 1.
Odf1D1 and c-mosD1
oligonucleotides bind the same TTF-D components. Liver nuclear
proteins (lanes a and d) and ST nuclear proteins
(lanes b, c, e, and f) were UV cross-linked to
the indicated ds oligonucleotides (Odf1D1, lanes a-c;
c-mosD1, lanes d-f), and complexes were
separated by size using SDS-PAGE. Reactions in lanes c and
f contained a 50-fold molar excess of unlabeled
c-mosD1 and Odf1D1 oligonucleotide DNA, respectively, in
addition to the probe and extract. Molecular mass markers and
testis-specific complexes (arrows) are indicated.
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To independently obtain a more accurate estimation of the molecular
masses of the testis-specific peptides that are TTF-D components, we
performed a Southwestern blot analysis of two ST extracts and two liver
nuclear protein isolates using radiolabeled binding site
oligonucleotides as probes. The results obtained with the
c-mosD1 and Odf1D1 oligonucleotides are identical (the Odf1D1 results are shown in Fig. 2).
Somatic cells contain 38-, 30-, and 32-kDa nuclear factors capable of
binding the TTF-D oligonucleotides (Fig. 2, lanes a and
b). Male germ cells specifically express 35-, 25-, and
22-kDa-binding peptides (lanes c and d, arrows), in close agreement with our UV cross-linking results.

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Fig. 2.
Size determination of TTF-D DNA binding
components. Different isolates of liver nuclear proteins
(lanes a and b) and ST proteins (lanes
c and d) were separated by SDS-PAGE, transferred to
nitrocellulose filters, and probed with radiolabeled Odf1D1
oligonucleotide DNA. Arrows indicate the peptides present in
ST nuclear extracts but not in liver nuclear extracts.
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Developmental Expression of TTF-D--
We predicted that if TTF-D
binding to the c-mos and Odf1 promoters has
physiological significance, then TTF-D must be present in cells that
express c-mos and Odf1. The c-mos testis-specific promoter is active in pachytene spermatocytes and round spermatids, and
the Odf1 promoter is active in spermatids. In the mouse,
pachytene spermatocytes are produced for the first time at days 12-14
after birth, whereas round spermatids are not produced until days
21-22 after birth. To determine whether TTF-D is present in mouse
testis at the time of expression of the c-mos and
Odf1 genes, nuclear extracts were prepared from total testes
of 11-, 16-, 21-, and 25-day-old mice and an adult mouse. These
extracts were analyzed for TTF-D activity in gel retardation assays.
The results shown in Fig. 3A
demonstrate that TTF-D activity (indicated by the asterisks) can be detected as early as 11 days after birth (lane a).
TTF-D activity is detectable into adulthood (lane e) when a
full complement of germ cells is established. The 38-kDa somatic
protein that binds the oligonucleotide is present in all total testis
extracts (s, lanes a-e), as expected. These experiments
show that TTF-D is present at the time of first expression of both the
c-mos and Odf1 genes. The increased TTF-D signal
likely results from the increased percentage of male germ cells during
postnatal development from day 11 onward: control gel retardation
assays using SP1 oligonucleotides demonstrated no change in the amount
of complex formed (data not shown). To further document the expression
of TTF-D in male germ cells before and after meiosis, we performed gel
retardation assays using c-mosD1 oligonucleotide as a probe
and nuclear proteins isolated from elutriated pachytene spermatocytes
and round spermatids. The results (Fig. 3B) show that TTF-D
binding activity is detectable in these germ cell types (arrow,
lanes g and i, respectively) and that the pattern is
similar to that seen using ST extract (lane j).

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Fig. 3.
TTF-D activity during testis development.
A, gel retardation assays of total mouse testis nuclear
extracts isolated 11, 16, 21, and 25 days after birth (lanes
a-d) and adult mouse testis nuclear extract (lane e)
using radiolabeled Odf1D1 oligonucleotide DNA. TTF-D DNA complexes are
indicated by asterisks. s, the retarded complex
generated by the somatic nuclear protein. B, nuclear
proteins were isolated from pachytene spermatocytes (lanes f
and g) and round spermatids (lanes h and
i) purified by centrifugal elutriation and from total male
germ cells (lane j) and analyzed by gel retardation using
the c-mosD1 oligonucleotide probe. The reactions shown in
lanes f and h contained 0.5 µg of protein, the
reactions shown in lanes g and i contained 5 µg
of protein, and 25 µg of protein were used in lane j. The
TTF-D complex is indicated by an arrow.
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TTF-D Acts as a Weak Positive Transcription Factor--
Our
previous in vitro transcription studies have shown that the
Odf1 promoter region harboring the TTF-D and CREM
sites
acts as a testis-specific cis-acting element (31).
Transfection experiments and analysis of CREM
/
knockout mice have established CREM
as the major regulator of the
Odf1 promoter (13, 19). The testis-specific c-mos
promoter lacks a CREM
binding site but shares a TTF-D site with the
Odf1 promoter. It is weak compared with the Odf1
promoter. To demonstrate a role for TTF-D in promoter activation, we
analyzed promoter activity in in vitro transcription assays,
using ST nuclear extracts with and without an excess of Odf1D1,
c-mosD1, and other indicated oligonucleotides as competitors
for TTF-D binding. pAdMLP was included in all assays as an internal
control. The addition of excess ds Odf1D1 oligonucleotide and ds
c-mosD1 oligonucleotide resulted in a reduction of the
activity of the Odf1 promoter by approximately 30% (Fig.
4, lanes b and d,
respectively) in comparison with a competitor oligonucleotide (B
oligonucleotide) that had no effect on Odf1 promoter
activity (lane e). Interestingly, a related oligonucleotide
(D2) also repressed the activity of the Odf1 promoter
in vitro (lane c). None of the competitor
oligonucleotide DNAs used affected the activity of the adenovirus
promoter (compare lane a and lanes b-e). We
conclude from these assays that TTF-D acts as a testis-specific,
positive transcription factor.

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Fig. 4.
TTF-D is a positive-acting transcription
factor. pRT7-0.2C2AT, which contained the
ODF1 promoter region linked to a 390-bp G-free cassette, and
pAdMLP, which contained the adenovirus major late promoter region
linked to a 190-bp G-free cassette, were incubated with ST nuclear
extract in the absence (lane a) or presence of a 50-fold
molar excess of Odf1D1 oligonucleotide, D2 oligonucleotide,
c-mosD1 oligonucleotide, or B oligonucleotide (lanes
b-e, respectively). Transcribed RNAs were analyzed by denaturing
PAGE.
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TTD-D Specifically Interacts with ss Binding Site
Oligonucleotides--
In the course of the above-described gel
retardation experiments, we observed in preliminary experiments that
TTF-D might bind to ss DNA representing its binding site. Therefore, we
performed the following studies to determine the specificity of such an interaction, and we asked whether the same TTF-D peptides could bind to
ss DNA binding sites. We used ST nuclear proteins and sense and
antisense Odf1D1 binding site oligonucleotides in gel retardation
assays. In indicated experiments, we included an excess of ss
competitor oligonucleotide. The results for the sense and antisense
oligonucleotide were essentially identical: those for sense Odf1D1 ss
DNA are shown in Fig. 5A. The
results demonstrate that TTF-D binds efficiently to ss oligonucleotide
(lane a, asterisks) and that this binding cannot be competed
by a large excess of unrelated ss oligonucleotide (B-sense
oligonucleotide and B-antisense oligonucleotide, lanes g and
h, respectively). Binding to sense oligonucleotide is
efficiently competed by ds Odf1D1 oligonucleotide and by sense and
antisense self-oligonucleotide (lanes b-d, respectively). Competition was slightly less efficient with ss c-mosD1
oligonucleotides (lanes e and f).

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Fig. 5.
TTF-D specifically binds ss DNA binding
sites. A, radiolabeled ss Odf1D1 sense DNA was incubated
with ST nuclear extract in the absence (lane a) or presence
of a 50-fold molar excess of ds Odf1D1 oligonucleotide DNA (lane
b) or in the presence of a 50-fold molar excess of the following
ss oligonucleotide DNAs: Odf1D1 sense, Odf1D1 antisense,
c-mosD1 sense, c-mosD1 antisense, B sense, and B
antisense (lanes c-h, respectively). Complexes (indicated
by asterisks) were analyzed by nondenaturing PAGE.
B, UV cross-linking followed by SDS-PAGE was used to analyze
peptides that are components of TTF-D that bind to ds Odf1D1 and
c-c-mosD1 DNA (ds, lanes i and l), to
ss sense Odf1D1 and c-mosD1 DNA (s, lanes j and
m), or ss antisense Odf1D1 and c-mosD1 DNA
(a, lanes k and n). Molecular mass markers are
indicated, and TTF-D complexes are indicated by
arrows.
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UV cross-linking was used to ascertain whether the same or different
TTF-D peptides are involved in the binding to ds and ss DNA. The
results are shown in Fig. 5B and indicate that Odf1D1 (lanes i-k) and c-mosD1 (lanes l-n)
sense and antisense oligonucleotides bind the same peptides compared
with each other and with ds oligonucleotide. In this experiment, the
25-kDa TTF-D component appears resolved in two bands, a result that was
not always evident. The increased intensities of the ssDNA-TTF-D
complexes (lanes j, k, and n) could result from a
greater affinity of TTF-D for those particular ss DNA cognate sites
compared with the corresponding ds DNA binding sites. To further define
the specificity of binding, we designed mutant Odf1D1 oligonucleotides
called MUT1, MUT2, and MUT3 in which the first two, the middle three,
or the last three nucleotides of the binding site were changed,
respectively (see "Experimental Procedures"). Gel retardation
analysis using these mutant oligonucleotides as competitor DNA (Fig.
6A) shows that MUT1 and MUT3
oligonucleotides compete for binding of TTF-D to ss Odf1D1
oligonucleotide (lanes d and f, asterisks),
although with reduced efficiency compared with self-oligonucleotide
(lane c). MUT2 oligonucleotide did not compete (lane
e), nor did the unrelated B oligonucleotide (lane a).
Preliminary UV cross-linking experiments showed that the same peptides
bind to Odf1D1, MUT1, and MUT3 DNA, but not to MUT2 DNA (data not
shown). Thus, mutation of the central three nucleotides abolished TTF-D
binding, and mutation of the flanking sequences affected TTF-D binding
but did not abolish it.

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Fig. 6.
ss DNA binding by TTF-D is involved in
Odf1 promoter activation. A, the specificity of
TTF-D interaction with ss Odf1D1 DNA was analyzed. Radiolabeled ss
Odf1D1 DNA was incubated with ST extract and a 50-fold molar excess of
ss B oligonucleotide, ds Odf1D1, ss sense Odf1D1, or MUT1, MUT2, and
MUT3 ss DNA (lanes a-f, respectively), and complexes
(indicated by asterisks) were analyzed by gel retardation.
B, Odf1 promoter activity was analyzed as described in the
legend to Fig. 4 in the absence (lane g) or presence of a
50-fold molar excess of the following ss oligonucleotide DNAs: Odf1D1
sense, MUT1, MUT2, MUT3, D2 sense, and B sense (lanes h-m).
The internal control was pAdMLP, as described above.
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Single-strand TTF-D Binding Site Oligonucleotides Repress Odf1
Promoter Activity in Vitro--
Here we show that ds
c-mosD1 and Odf1D1 TTF-D binding site DNA reduced
Odf1 promoter activity in vitro. Because we
discovered that TTF-D also specifically binds to ss binding site DNA,
we set out to determine whether these ss DNAs can repress
Odf1 promoter activity. In vitro transcription
experiments were carried out using ST nuclear proteins,
Odf1, and AdMLP promoter constructs and the indicated sense
ss oligonucleotides as competitors for TTF-D binding. The results are
shown in Fig. 6B. This analysis shows that ss Odf1D1, MUT1,
MUT3, and D2 oligonucleotides reduce promoter activity by approximately
50% (lanes h, i, k, and l). ss MUT2 and the
unrelated ss B oligonucleotides did not significantly affect
Odf1 promoter activity in vitro (lanes
j and m). Thus, it appears that activation of the
Odf1 promoter by TTF-D can be competed specifically by both
ds and ss DNA representing the TTF-D binding site.
The 3' Half of the TTF-D Binding Site Interacts with the Somatic
38-kDa Protein--
The assays described above demonstrate that a
38-kDa somatic, nuclear protein can interact with the TTF-D binding
site. We used ds wild type and mutant DNAs to gather further
information on the nucleotides involved. Oligonucleotide DNA was
incubated with liver nuclear extract, and the complexes were analyzed
in the gel retardation assays shown in Fig.
7. The complex between the 38-kDa somatic
protein and Odf1D1 ds DNA (s, lane a) can be competed by
self-oligonucleotide (lane b) and c-mosD1
oligonucleotide (lane c) but cannot be significantly
competed by D2 oligonucleotide (lane d). Because D2 and
Odf1D1 only share sequences in the middle region, this shows that
either the 5' or 3' end of the Odf1D1 oligonucleotide binds the somatic
protein. We therefore analyzed whether the mutant oligonucleotides can
complex with the somatic protein. The results (Fig. 7) indicate that
the 3' nucleotides in the Odf1D1 sequence are essential for binding,
which is abolished by their mutation (lane g). Reduced
binding was observed for MUT2 (lane f), suggesting that
nucleotides in the middle of the Odf1D1 sequence contribute to the
affinity. These results suggest that the binding site for TTF-D and the
somatic 38-kDa protein overlap.

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Fig. 7.
A somatic protein binds specifically to 3'
nucleotides in Odf1D1. To analyze the specificity of the
interaction between the 38-kDa somatic nuclear protein and the TTF-D
binding site represented in the Odf1D1 oligonucleotide, the following
experiments were done. Radiolabeled ds Odf1D1 DNA was incubated with
liver nuclear proteins in the absence (lane a) or presence
of a 50-fold molar excess of self-oligonucleotide, c-mosD1,
or D2 (lanes b-d, respectively) and analyzed by gel
retardation. To analyze sequence requirements for the observed binding,
radiolabeled MUT1, MUT2, and MUT3 DNAs were incubated with liver
nuclear proteins. The complex (indicated by s) was analyzed
by gel retardation. An overexposure is shown for reactions in
lanes e-g to visualize the complex formed between MUT2 and
the somatic protein.
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DISCUSSION |
Transcriptional regulation of genes plays a key role in
their tissue- and cell-specific expression. In the testis, with the exception of CREM
, little is known regarding transcription factors that play a role in the positive or negative regulation of
testis-specific genes (10, 11). We have recently shown that CREM
is
a regulator of transcription of the post-meiotic testis-specific gene
Odf1 (17). We had previously identified a second putative
regulator of Odf1 transcription that we called TTF-D (31).
The sequence of the TTF-D binding site was deduced from DNase I
footprinting assays and gel retardation experiments and is called
Odf1D1 (31). In the c-mos promoter, an element that is 80%
identical to the Odf1D1 sequence was identified that binds a
testis-specific factor that we postulated could be related to or
identical to TTF-D (32). Here we show that TTF-D is a testis-specific
transcription factor, which binds to both the Odf1D1 oligonucleotide
and the c-mosD1 oligonucleotide. Mutational analysis
indicates that the central CTT nucleotides in the 5'-GGCCTTAGGG-3'
TTF-D binding site are essential for binding. The flanking GG and AGG
nucleotides, however, increase the affinity of TTF-D for its cognate
site. TTF-D contains three testis-specific peptides of 22, 25, and 35 kDa that surprisingly bind to both ds and ss Odf1 and
c-mos TTF-D binding site oligonucleotides. We do not know
the relationship between any of these peptides: it is possible that one
is a post-translationally modified version of another one, for
instance, by phosphorylation. Importantly, the same peptides bind to
both ds and ss versions of the Odf1 and the c-mos
TTF-D binding sites. TTF-D activation of Odf1 promoter activity in vitro is reduced by the addition of TTF-D
binding site oligonucleotides (both as ds and ss DNA), but not by
unrelated or mutant oligonucleotides.
Our functional data and binding results with TTF-D strongly suggest
that TTF-D plays a role in the transcription of the c-mos and Odf1 testis-specific promoters. The fact that TTF-D can
be detected as early as day 11 in germ cell development strengthens such a role for TTF-D in this transcriptional regulation, because c-mos is first detectable in spermatocytes that develop
12-14 days after birth in the mouse. The in vitro
transcription assays that we performed previously (31, 32) suggest that
the c-mos promoter is weak in comparison to the
Odf1 promoter. This finding can be explained by the facts
that (a) compared with CREM
, TTF-D is a weak transacting
factor, and (b) it is the only testis-specific factor that
binds to the c-mos promoter detectable by DNase I footprint
assays (32). It is more difficult to interpret a role for TTF-D in the
regulation of the post-meiotic Odf1 promoter. Clearly, TTF-D
is present in pre-meiotic cells that do not express Odf1. One
possibility is that the binding of TTF-D to the Odf1 promoter does happen in spermatocytes and that this event changes the
Odf1 chromatin structure to prepare the Odf1
promoter for the binding of CREM
. In this respect, it is interesting
that TTF-D can specifically bind to its ss cognate site. One can
envision two models: first, such interactions stabilize ss regions in
the Odf1 promoter that are important for the subsequent
regulation of activity by CREM
, and second, TTF-D binding to ss
promoter sequences transmits structural signals. The role and mode of
action of ss binding transacting factors is not clear at present.
Interestingly, Yiu et al. (35) recently isolated the mouse
testis-specific p54/p56 protein, a homolog of the Xenopus
FRG Y2 protein (36). Testis-specific p54/p56 can also bind mP2 (37);
binding, is preferentially to ss DNA (37). Consensus p54/p56 binding
sites are present in the testis-specific promoters of the mP1, TP2, PGK-2, and H2B genes (37). Other ss DNA binding, positive transacting factors include Pur
(38), Brn-3a (39), PYBP (40), and FBP (41). The
mechanism of action of any of these factors remains to be determined,
but these and our data suggest that gene regulation by ss binding
transacting factors may be a common theme in spermatogenesis. In an
alternative model, TTF-D does not have access to the
Odf1 promoter until after meiosis. Future experiments will
distinguish these possibilities.
TTF-D acts as a testis-specific positive-acting transcription factor
for the post-meiotic gene Odf1. We also observed that in
addition to TTF-D, a 38-kDa somatic nuclear protein absent from male
germ cells binds to the c-mosD1 and Odf1D1 oligonucleotides. The absence of this nuclear protein from male germ cells suggests that
it might play a role in repression of the c-mos and
Odf1 promoters in somatic cells. This possibility is
strengthened by our observation that the binding sites for the somatic
protein and TTF-D overlap. This suggests a model in which the binding of the 38-kDa protein to its site blocks the expression of Odf1 and/or
c-mos in cells that accidentally ectopically express TTF-D. Such a model, although unproven in this case, is attractive because it
has been shown to occur in other systems: one of the mechanisms of
action of the Cut homeodomain repressor protein is its ability to block
access of the transcriptional activator CTF/NF1 protein to its binding
site (42). For this reason, Cut protein had also been named CDP
(CCAAT-box displacement factor). The identity of the 38-kDa somatic
protein and its putative role in gene regulation remain to be determined.
 |
ACKNOWLEDGEMENTS |
We thank Heide Tarnasky and Rajesh Gupta, who
were involved in the initial characterizations of TTF-D.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Medical
Research Council of Canada (to F. A. v. d. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by an Alberta Cancer Board Studentship.
§
To whom correspondence should be addressed. Tel.: 403-220-3323;
Fax: 403-283-8727; E-mail: fvdhoorn{at}ucalgary.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
mP, mouse protamine;
ds, double-stranded;
ss, single-stranded;
PGK-2, phosphoglycerate
kinase 2;
ST, seminiferous tubule;
PAGE, polyacrylamide gel
electrophoresis;
bp, base pair.
 |
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