(Received for publication, August 14, 1996, and in revised form, October 19, 1996)
From the A conserved hormone response element, CNTFR-DR1
(5 Members of the steroid/thyroid hormone receptor superfamily are
transcription factors that can bind to specific DNA sequences called
hormone response elements (HRE)1 and
thereby regulate the expression of their target genes (1). This
superfamily includes receptors for steroid, thyroid, vitamin D3, and retinoids, and a large number of orphan receptors
whose cognate ligands are still unknown (1). These members have been grouped into three categories according to their binding preference to
HREs, the palindromic half-site AGAACA is preferred for the binding by
the androgen, glucocorticoid, mineralocorticoid, and progesterone
receptors; the direct repeat with various spacing is preferentially
recognized by the estrogen, thyroid, retinoic acid, retinoid X, vitamin
D, and many orphan receptors; and the single half-site of AGGTCA
preceded by two specific flanking nucleotides is favored by some
members which bind as a monomer, such as the steroidogenic factor 1 (2), TR3 orphan receptor (TR3)/NGFI-B/nur77 (3), and the thyroid
receptor (4). Some receptors, i.e. thyroid hormone receptor
and chicken ovalbumin upstream promoter-transcription factors
(COUP-TFs), are shown to be promiscuous for binding to different
arrangements of the AGGTCA half-site (4, 5).
The human and rat TR4 cDNAs were isolated from testis and
hypothalamus by degenerative PCR cloning (6). The TR4 cDNA shows high homology in nucleotide sequence with the TR2 orphan receptor (TR2;
another orphan receptor isolated from our laboratory (7)), suggesting
that these two orphan receptors constitute an unique subfamily within
the steroid receptor superfamily. TR4 mRNA are widely expressed in
the adult rat brain (6). Within the supraoptic nucleus, TR4 is one of
the most abundant steroid receptors expressed there with the order
thyroid hormone receptor > COUP-TFII > TR4 = COUP-TFI
(8). Despite the high abundance of the TR4 transcripts in nervous
tissues, the physiological function of TR4 in neurogenesis remains
unclear.
To understand the role of TR4 in neurogenesis, we sought downstream
regulatory events, namely target genes, which can be regulated by TR4.
Thus, we examined the TR4 expression pattern during mouse embryogenesis
to further confirm its participation in neuronal development, and then
used this information to search for TR4 target genes involved in a
neuronal-specific program. We then sought potential TR4 target genes
based on the binding preference of TR4. In vitro binding
assays suggested that TR4 is capable of binding to 5 Ciliary neurotrophic factor (CNTF) is a member of the cytokine
superfamily. It utilizes a three-component receptor system consisting
of an extracellular CNTF-binding protein, known as CNTFR Because little is known about TR4 targets, we focused study here on a
DR1 element present in CNTFR To clone the DNA
fragment containing the 5th intron of the CNTFR To test for enhancer activity,
pCAT-promoter vector (Promega), which contains an SV40 promoter
upstream from the CAT gene, was used and the human CNTFR-I5 DNA
fragments (from PCR) were inserted into this vector. Plasmids
pCNTFRI5-CATe5+ and pCNTFRI5-CATe5 Plasmid
pCMX-TR4 (21) containing the full-length human TR4 cDNA was
in vitro transcribed and translated by the TNT system according to the manufacturer's instructions (Promega). The product of
TR4 was analyzed in SDS-polyacrylamide gel electrophoresis and showed a
major band with an expected molecular weight of 67.3 kilodaltons as
described previously (21).
EMSA was
carried out as described previously (22). Briefly, 0.05 µl of
TNT-expressed TR4 was included in each reaction. Double-stranded
oligonucleotides corresponding to the human CNTFR-DR1 (5 The DNA-protein binding assay was
performed as described previously with modifications (22). Briefly,
0.05 µl of in vitro translated TR4 was incubated with
various concentrations of the probe. Protein-DNA complexes were
resolved on a 5% non-denaturing polyacrylamide gel (at 4 °C) in
0.5 × TBE. After exposing to a film to localize the free probe
and protein-probe complex, the respective bands were excised and
counted directly in a scintillation counter. The dissociation constant
(Kd) value and Bmax were
calculated using the Ebda program (Biosoft).
Chinese hamster
ovary cells were cultured and transfected by the calcium phosphate
coprecipitation procedure as described previously (22). To normalize
the transfection efficiency, the pCMV To clone the full-length mouse
TR4 cDNA, reverse-transcription followed by PCR (RT-PCR) was
employed, following the standard protocol recommended by the
manufacturer (Perkin-Elmer). Mouse testis total RNA (1 µg) was used
as a template. The N- and C-terminal mouse TR4 cDNA fragments were
separately amplified using the primer sets TR4-16 and TR4-15 or TR4-3
and TR4-42, respectively. The primer sequences are as followings:
TR4-16 (5 The N terminus of the mouse TR4 cDNA
was cloned by RT-PCR. The primers TR4-16 and TR4-23
(5 Total RNAs from selected mouse
tissues were isolated, electrophoresed, and transferred onto a nylon
membrane as described previously (6). The blot was hybridized with the
mouse mTR4N probe, washed, dried, exposed to PhosphorImager
intensifying screen for 16 h. The image was scanned and
printed.
Embryo collection, section
preparation, and in situ hybridization were performed as
described previously (22). Both the antisense and sense TR4 probes were
included for each batch of experiments. Washes were performed with high
stringency (2 × SSC, 50% formamide at 65 °C (1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate,
pH 7.0)) before and after RNase digestion (20 mg/ml for 30 min). Slides
were dipped into Kodak NTB2 emulsion and exposed for 2 weeks.
Subsequently, slides were developed in Kodak D19 developer, fixed,
dehydrated, and mounted for dark field analysis. Some slides were
stained with hematoxylin for light-field analysis.
Based on the
genomic organization and DNA sequence published by Valenzuela et
al. (20), the 5th intron of the human CNTFR
To
examine whether CNTFR-DR1 could bind TR4, we carried out in
vitro DNA binding experiments. Gel retardation assays were performed with in vitro translated TR4 using the
32P-labeled CNTFR-DR1 oligonucleotide as a probe. As shown
in Fig. 2A, a specific DNA-protein complex
was formed in the presence of both probe and TR4 (lane 3,
hollow arrow) but was absent in the reaction containing
probe and the mock-translated control (lane 2). This
TR4-CNTFR-DR1 complex could be essentially abolished by as low as a
10-fold molar excess of unlabeled CNTFR-DR1 oligonucleotide (lane
4), but remained intact in the presence of a 100-fold molar excess
of "mutant" CNTFR-DR1 oligonucleotide (lane 6).
Moreover, this retarded complex could be fully supershifted in the
presence of the anti-TR4 monoclonal antibody (lane 8,
solid arrow). As a negative control, an unrelated monoclonal
antibody with the same IgG2 isotype as that of TR4 showed
no effect on the retarded complex (lane 7). Together, these
data indicate that the CNTFR-DR1 is a specific binding site for
TR4.
To determine the binding affinity of TR4 and CNTFR-DR1, we performed
Scatchard analysis by EMSA. The typical EMSA pattern of protein-DNA
complex formed between increasing amounts of CNTFR-DR1 probe (0.0039-2
ng) and fixed amounts of TR4 was shown (Fig. 2B). The
radioactivity of specific complex (bound) and unbound (free) probe were
quantitated for the subsequent Scatchard plot analysis. The results are
consistent with a single binding population for the specific
DNA-protein complex with a dissociation constant (Kd) of 0.066 nM and
Bmax of 0.089 nM (Fig.
2C). This binding affinity is about 15-45-fold higher than
the Kd range for steroid receptors and their HREs
(4). At very low probe concentrations, the specific protein-DNA complex
was still visible (Fig. 2B, lanes 2-4). That was
further consistent with the calculated high affinity dissociation
constant.
To investigate
whether TR4 could regulate the CNTFR
The entire coding region of
mouse TR4 cDNA was cloned by RT-PCR (for details see "Materials
and Methods") (Fig. 4A). Sequence comparison with other members of the steroid receptor superfamily indicated that the N-terminal mouse TR4 cDNA fragment (mTR4N) covered between primer TR4-16 and TR4-23 is the most divergent; the
homology within this region is around 30% at nucleotide level between
mouse TR4 and TR2.3 To test the specificity
of mTR4N, total RNA samples isolated from mouse testis and kidney were
used as positive and negative controls, respectively. As shown in Fig.
4B, the mTR4N probe hybridized to two bands, 7.8 and 2.8 kilobase in size, in mouse testis (Fig. 4B, lane
1). The sizes of these two TR4-hybridized bands are different from
those of TR2 transcripts, which are 2.4 (major band) and 2.9 kb (minor
band) in size. No hybridization signal was detected in the kidney (Fig.
4B, lane 2). These data are consistent with previous observations (6) supporting that the mTR4N probe is specific
and only hybridizes to TR4 transcripts.
To test whether TR4 could
potentially regulate CNTFR
-Ours
and previous in situ studies suggested that TR4 expression
is related to the neural proliferation status. To further pursue this
possibility, we examined TR4 expression during embryogenesis. In
situ hybridization analysis in mouse embryos revealed that TR4
expression was most prominent in the developing neural structures as
well as in certain non-neuronal regions (Fig. 6,
B-E). TR4 transcripts were detected as early as
embryonic day 9 (E9) (Fig. 6B). This result was confirmed by
RT-PCR with a cycle number at the early amplification phase (data not
shown). Initially within the neural system of E9-E11, TR4 transcripts
were present uniformly throughout the neural tube along both its
rostrocaudal and dorsoventral axes (Fig. 6, B and
C, ne). Subsequently, during E14-E16, expression became
restricted to the ventricular zones of brain vesicles, where cells are
rapidly proliferating (23) (Fig. 6, D-F, arrow and arrowhead). Sense mTR4N probe did not detect specific
signal in E14 embryo (Fig. 6A). Intensive TR4 transcripts in
E16 brain were also observed in the striatum (Fig. 6F,
Sr), active dividing population of the cerebellar primordium
(Fig. 6E, cp), and spinal cord (Fig.
6D, sp). The spinal motor neurons (Fig.
6D, m) were also positive for TR4 expression.
This expression pattern is consistent with a potential role for TR4 in
the proliferation and/or early differentiation of neuronal precursors
within the central nervous system.
TR4 mRNA expression during mouse
embryogenesis. Sagittal sections of embryos were hybridized with
either sense (A) or antisense mTR4N riboprobe
(B-J) and photographed under dark-field illumination.
A-E, low magnification photographs showed the TR4 expression patterns of embryos with the development stages indicated at
the top. The restricted expression of TR4 transcripts to the ventricular zones were indicated as arrows (D)
and arrowhead (E). F-G,
high magnification photographs showed TR4 hybridization signals in
sections of an E16 embryo within regions of the developing forebrain
(F), inner ear (G), spinal cord (H),
eye (I), and the junction between nasal and oral cavities (J). Ventricular zone was indicated
as arrowheads (F). Abbreviations used are:
c, superior cervical ganglion; cp, cerebellum
primordium; cx, cerebral cortex; drg, dorsal root
ganglia; e, otic epithelium; g, ganglion layer of
retina; l, lens; m, motor neuron; ms,
muscle; n, nuclear layer of retina; ne, neural
epithelium; oe, olfactory epithelium; r, retina;
s, sympathetic ganglia; sp, spinal cord;
Sr, striatum; t, tongue epithelium; V,
trigeminal ganglion. The size bars represent 1 and 100 nm for
A-E and F-J, respectively.
During the development of the peripheral nervous system, TR4
transcripts were also actively expressed within the ganglia and the
neural epithelium of many sensory organs. Abundant TR4 transcripts were
detected in the dorsal root (Fig. 6, C-E, dg),
superior cervical (Fig. 6, G-H, c), sympathetic
(Fig. 6H, s), and trigeminal (Fig. 6,
G, V) ganglia. Its abundance within the dorsal
root ganglia through E11-E16 suggests a role for TR4 through the
progression of these neurons from precursors to postmitotic neurons
(24). High levels of TR4 mRNA were also found within all targets of sensory innervation, including the neuronal epithelium of inner ear
(Fig. 6, G, e), retina (Fig. 6I), nasal cavity
(Fig. 6, J, oe), and tongue (Fig. 6,
J, t). The signal intensity of TR4 in the ganglia
layer (neuron bodies) of the retina is stronger than the inner and
outer nuclear layers (photoreceptors) (Fig. 6I, compare
g to n). This pattern of TR4 expression implies
that TR4 could have similar roles on the development and/or early
differentiation of motor, sensory, sympathetic neurons as well as their
precursors.
Widely overlapping expression domains of TR4 and CNTFR Several tissues expressing abundant TR4 transcripts are also known to
respond to CNTF. Examples are the hippocampal (14), retinal (nuclear
and ganglion layers) (15), and motor (neostriatum and spinal motor)
neurons (16), as well as the sympathetic (superior cervical) and
sensory (trigeminal and dorsal root) (25) ganglia. Since CNTFR Outside the developing nervous system, both TR4 and CNTFR During the adult stage, TR4 and CNTFR The DR1 present in CNTFR-I5 represent the first intronic
inducible DR1 response element regulated by TR4. Using in
vitro binding assay, a DR1 sequence has been shown to be the high
affinity binding site for several steroid receptors, including RARs
(28), RXRs (29), peroxisome proliferator-activated receptor (30),
COUP-TFs (31), TR2 (22), and TR4.2 For certain gene
regulation to occur, these DR1-binding factors may compete with each
other for the binding site depending on the affinity and the relative
abundance. The relative affinity among these DR1 binding factors are
unclear. However, our data show a very high affinity of TR4 binding to
the CNTFR-DR1, indicating once TR4 is present, it could dominate over
other DR1 binding factors for occupying the DR1 response element.
Upon binding to a DR1 present in the promoter, these DR1-binding
steroid receptors or their complexes may either activate or repress
gene transcription in reporter gene assays. For example, RAR/RXR
heterodimer mediates retinoic acid response on phosphoenolpyruvate carboxykinase gene induction (28). Peroxisome proliferator-activated receptor alone or complexed with RXR is able to activate the acyl-CoA oxidase gene expression (30). Although COUP-TFs were originally identified as required factors for ovalbumin gene transcription (32),
in contrast, they repress retinoic acid induced transactivation on a
DR1 reporter construct (33). Similar phenomena are also observed for
TR2 (22) and TR42 on a DR1-mediated repression of the
retinoic acid-induced CRBPII promoter activity. As opposed to the
repression effect of TR4 on the CRBPII promoter,2 our data
demonstrate that TR4 could bind to a DR1 sequence and induce
transcriptional activation (Fig. 2, 3). The effects of other
DR1-binding factors on CNTFR-I5 have not been examined yet. These
results indicate that through the binding to a similar DR1 sequence,
whether the transcriptional activity is up or down-regulated may depend
on the receptor, gene context, response element, and the assay system
used. Other studies also have made similar observations (5).
TR4 may be able to interact with other DR1-binding proteins, such as
RAR In addition, the presence of several 5 In addition to the roles of TR4 in
restricting other hormonal signaling pathways2 and in
inhibiting the SV40 promoter (21), our data here suggest that TR4 can
also function as a transcriptional activator with high induction
activity through CNTFR-I5. This TR4-mediated transcriptional activation
is consistent as tested in Chinese hamster ovary or P19 cell lines
(data not shown). In contrast, TR4 repressed the retinoic acid-induced
transcriptional activity with a CAT reporter containing a core DR1
without the flanking sequences and no significant TR4-mediated
induction is observed in the absence of retinoic acid.2
These results suggest that the sequences flanking the DR1 may also
influence the TR4-mediated gene regulation. Finally, this TR4 inducible
system may provide a good assay system for searching the TR4 ligands
and activators.
In summary, our data indicate that human CNTFR We appreciate Pei-Wen Hsiao for assistance in
the cloning and DNA sequencing. We also thank Yi-Fen Lee for providing
the pCMX-TR4 plasmid and anti-TR4 monoclonal antibody.
Endocrinology-Reproductive Physiology
Program,
-
G
GG-3
), has been identified in
the 5th intron of the
component of the ciliary neurotrophic factor
receptor (CNTFR
) gene for the human TR4 orphan receptor (TR4).
Electrophoretic mobility shift assay showed a specific binding with
high affinity (Kd = 0.066 nM) between
TR4 and the CNTFR-DR1. A reporter gene assay using chloramphenicol
acetyltransferase demonstrated that the 5th intron of CNTFR
has an
enhancer activity which could be induced by TR4 in a
dose-dependent manner. Furthermore, our in situ
hybridization data showed that abundant TR4 transcripts were detected
in adult brain, in regions of cortical and hippocampal neurons, as well as in many developing neural structures, including brain, spinal cord,
ganglia (sympathetic and sensory), and neuronal epithelia (retinal,
otic, olfactory, and gustatory). The striking similarities in the
expression patterns of TR4 and CNTFR
in the developing and postnatal
nervous systems further support the potential role of TR4 in
neurogenesis. Collectively, these data suggest that the human CNTFR
gene could represent the first identified neural-specific gene induced
by TR4.
-AGGTCA direct
repeats with 1-6-base pair spacing (DR1-DR6).2 We hypothesized that the
component of ciliary neurotrophic factor receptor (CNTFR
) with DR1
in its 5th intron may be a target gene for TR4.
(9), as
well as two signal transducing
receptor subunits, gp130 and
LIFR
, which it shares with its cytokine relatives (10, 11). CNTFR
is bound to the cell membrane by a glycosylphosphatidylinositol anchor
and its major function is to convey ligand-specificity (9). However,
upon certain stimulation, CNTFR
can also be released from its
glycosylphosphatidylinositol anchor and mimics the effect of CNTF
on target gene activation (12). The expression of CNTFR
is mostly
restricted to nervous tissues and is detected in all neurons that have
been shown to respond to CNTF (13-16). Clinically, CNTF effectively
protects against motor neuron degeneration in human Parkinson's
disease (17). The trophic effect of CNTF is also observed on the
denervated skeletal muscle (18). Consistent with the CNTF effect on
motor neurons, the essential function of the CNTFR
gene has been
proved in mice lacking CNTFR
, which exhibit profound motor neuron
deficits at birth (19). Despite the important function of CNTFR
, the
regulation of the CNTFR
gene remain almost completely unknown.
which can be potentially regulated by
TR4. Thus, the 5th intron of the CNTFR
gene (CNTFR-I5) was cloned to
show the DR1 response element, where TR4 binds, is conserved and
functional. The consequence for TR4 interaction with this DR1 in its
natural gene context was determined using a reporter gene assay.
Moreover, the expression pattern of TR4 during mouse embryogenesis was
examined by in situ hybridization and then compared to that
of CNTFR
. Through these studies, our results suggest TR4 may
function as an inducer in CNTFR
gene regulation and this event could
happen in vivo. Thus, this study provides the first evidence
for TR4 to interact with the neurocytokine signaling pathway during
neurogenesis.
Cloning of the 5th Intron of CNTFR Gene
gene, polymerase
chain reaction (PCR) was employed. Two oligonucleotides (E5:
5
-CACCTTCAATGTGACTGTGC-3
, E6: 5
-GTGCATGTAGCGAATGTGGC-3
) located in
the coding regions flanking the 5th intron of human CNTFR
were
synthesized for PCR amplification. The positions of E5 and E6 primers
are 417-436 and 510-491, respectively (20). Purified genomic DNA (1 µg) isolated from several cell lines (human 1299 and Chinese hamster
ovary) and from mouse tail were used as the templates. The following
thermal cycling program was used to amplify the DNA fragment with the
expected size around 270 base pairs: denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min, for 35 cycles. The amplified DNA fragments (named as CNTFR-I5)
from various template sources were gel purified and subcloned into pT7
blue vector (Novagen). Sequence analysis using the Sequenase kit (U. S. Biochemical Corp.) confirmed the identity of these fragments.
both contain the CNTFR-I5 insert
at the BglII site of the pCAT-promoter vector with the
orientation the same or opposite to CAT expression, respectively.
Plasmid pCNTFRI5-CATe3+ inserted the CNTFR-I5 at the XbaI
site of the pCAT-promoter vector with the orientation the same as CAT
gene expression. The identities of these constructs were confirmed by
DNA sequencing.
-GCCC
C
CTC-3
) were end labeled
by [
-32P]ATP. The mutated CNTFR-DR1 oligonucleotides
(5
-GCCCTGA
CTCTGA
CTCTC-3
) contained two
mutated nucleotides (letters underlined). For competition reactions,
unlabeled double-stranded oligonucleotides were mixed with the labeled
probe prior to addition to the reactions. For antibody supershift
analysis, 1 µl of monoclonal number 29 anti-TR4 antibody2 or 1 µl of unrelated monoclonal antibody
(IgG2 isotype) was incubated with the reactions for 15 min
at room temperature prior to loading on a 5% native gel.
(Clontech) was co-transfected.
Results were plotted as the mean ± S.D. of at least three
independent experiments of CAT expression normalized to
-galactosidase activity.
-AACACGTACACAGACCTCTG-3
), TR4-15
(5
-GCAGACTCACAGATGTAGTG-3
), TR4-3 (5
-CTGAGAAGATCTATATCCGG-3
), and
TR4-42 (5
-CTATAGACTGACTCCGGTGATCTG-3
). The identity of PCR products
was confirmed by sequencing and showed high homology to human
counterparts. These two cDNA fragments were then ligated together
to produce a full-length mouse TR4 cDNA.
-ACACAGTACTCTACCACCTG-3
) were used to amplify a PCR product (around
400 bp in size) from mouse testis total RNA, which was cloned into the
T7 blue vector (Novagen), confirmed by DNA sequencing (U. S. Biochemical Corp.), subcloned into pBluescript SK+ (Stratagene), and
designated as mTR4N-pBS. For Northern blot analysis, radiolabeling was
performed using a PCR reaction including primers (TR4-16 and TR4-23),
0.1 ng of linear template (mTR4N-pBS plasmid), 5 nmol of each dATP,
dGTP, and dTTP, and 0.1 mCi of [
-32P]dCTP (DuPont).
For in situ analysis, radiolabeled antisense and sense TR4
probes (specific activity 1-2 × 109 cpm/µg) were
generated by in vitro transcription as described previously
(22).
Cloning of the 5th Intron of the CNTFR Gene
gene (CNTFR-I5)
contains one perfect DR1 (5
-
C
) and four copies of AGGTCA-like half-sites. It is especially amazing because
so many AGGTCA-like sequences are simutaneously localized within a very
small intron of around 176 base pair. We wondered whether some of these
sites might be HREs regulated by steroid receptors. As shown in Fig.
1A, CNTFR-I5 is located in the middle of the
coding sequence for the first fibronectin-like subdomain within the
cytokine receptor-like domain of the CNTFR
gene (Fig. 1A)
(20). The presence and positions of introns in the cytokine receptor-like domain are conserved among members in the cytokine receptor superfamily (20), implying CNTFR-I5 may contribute a
regulatory role through these response elements. To test this hypothesis, we cloned the CNTFR-I5 from human, hamster, and mouse by
PCR (see "Materials and Methods"). Sequence comparisons between these three species showed that the DR1 and putative thyroid hormone receptor-binding site (TRE-like sequence) (4) were conserved but the
TR3/NGFI-B/nur77-binding site (3) has one mismatch at its end (Fig.
1B). The other two AGGTCA-like sequences were not well
conserved. The appearance of three different types of highly conserved
HREs within a small intron suggests that CNTFR-I5 may be critical for
CNTFR
gene regulation.
Fig. 1.
The genomic position and nucleotide sequence
of the 5th intron of the CNTFR gene. A, the intron-exon
structure of the cytokine receptor-like domain of CNTFR
gene. The
introns are indicated by roman numerals. The position of the
5th intron is indicated by an asterisk (*). B,
alignment of the nucleotide sequence of CNTFR-I5 homologues between
hamster, mouse, and human. Dashes indicate identity and dots represent gaps.
The brackets mark the junction of 5th intron with its
neighboring exons. The box outlines the CNTFR-DR1
response element. The 5
-AGGTCA-like sequences are underlined.
TRE-like and consensus NBRE sequences are as indicated.
The sequence and orientation of E5 and E6 primers are as shown.
[View Larger Version of this Image (32K GIF file)]
Fig. 2.
Binding of the in vitro expressed
TR4 to the CNTFR-DR1 with high affinity. A, analysis of the
DNA binding and antigenic properties of in vitro expressed
TR4 protein by EMSA. TR4 was synthesized in a reticulocyte lysate. The
CNTFR-DR1 oligonucleotides were 32P-end-labeled and used as
the probe. As a negative control, the binding reaction contained either
no lysate (lane 1) or lysate without TR4 (lane
2). Binding reaction mixtures incubated with the probe and the
in vitro expressed TR4 (lanes 3-8) in the
presence of a 10-fold (lane 4) or 100-fold (lane
5) molar excess of unlabeled CNTFR-DR1, or a 100-fold molar excess
of mutated CNTFR-DR1 (mDR1) (lane 6). Supershift
of the specific complex was induced by the presence of anti-TR4
antibody (lane 8) but was not by unrelated monoclonal
antibody (lane 7). The position of the retarded complex and
the supershift band are indicated by the hollow and
solid arrow, respectively. B, the binding profile
of in vitro expressed TR4 to various amounts of the probe
was resolved by EMSA. Constant amounts of TR4 were incubated with
varying concentrations of labeled CNTFR-DR1 probe, as indicated. The
specific DNA-protein complex (arrowhead) and free probe were
detected by autoradiography, excised from the gel, and counted directly
in a scintillation counter. C, Scatchard plot of the result
is shown. The ratio between specific DNA-protein binding and free DNA
probe (bound/free) with respect to specific DNA-protein
binding (bound, nM) was plotted. The
dissociation constant (Kd) and
Bmax values were generated from the Ebda program
(Biosoft).
[View Larger Version of this Image (29K GIF file)]
gene expression through
interaction with CNTFR-I5, we carried out the CAT assay following
co-transfection of expression vectors and CAT reporter constructs into
Chinese hamster ovary cells. Three enhancer-reporter plasmids were
created in the present study to test whether the enhancer activity of
CNTFR-I5 is position- or orientation-dependent (Fig.
3A). As shown in Fig. 3B, in the
presence of the CNTFR-I5, TR4 induced transcriptional activity up to
15-30-fold in a dose-dependent manner (compare lanes
3 and 4 to lane 2; lanes 8 and
9 to lane 7; lanes 13 and
14 to lane 12). In contrast, this induction did
not occur when the antisense TR4 expression vector was transfected
(lanes 5, 6, 10, 11, 15, and
16). Different orientations or positions did not appear to affect
the TR4-mediated transcriptional activity. TR4 does not significantly
induce the CAT reporter in the absence of the DR1(22). Taken together,
these results suggest that TR4 might induce CNTFR
gene expression
via the CNTFR-I5 enhancer.
Fig. 3.
Functional analysis of the CNTFR-I5 gene
fragment as regulated by TR4. A, construction of the
enhancer reporter plasmids containing CNTFR-I5. Names assigned to each
construct are as indicated. CNTFR-I5 DNA fragments were inserted into
either upstream (e"5") or downstream
(e"3") of the vector pCAT-promoter (pCAT-).
Relative orientations of enhancer fragments with respect to the
promoter are indicated by arrows; also indicated as "+"
or as "" for the same or opposite direction to the promoter,
respectively. B, dosage dependent induction of CAT
activities by TR4 via CNTFR-I5. Various reporter constructs without
(lanes 1, 2, 7, and 12) or with expression vector
were transfected into Chinese hamster ovary cells. The reporter
constructs for transfection were either pCATp (lane 1) or
CNTFR-I5-CATe5+ (lanes 2-6) or CNTFR-I5-CATe5
(lanes 7-11) or CNTFR-I5-CAT3+ (lanes 12-16).
Expression plasmids co-transfected include: sense TR4 expression
plasmid, 0.5 µg (lanes 3, 8, 13), 3 µg (lanes 4, 9, 14); or antisense TR4 expression plasmid, 0.5 µg (lanes
5, 10, 15), 3 µg (lanes 6, 11, 16). Chloramphenicol conversion rates were calculated from PhosphorImager quantifiable intensities. Fold induction was normalized relative to the CAT activity
produced by the control plasmid pCATp without the co-transfection with
TR4 expression plasmid. The data are average of at least three
independent experiments with the error bars representing standard
deviation.
[View Larger Version of this Image (48K GIF file)]
Fig. 4.
Cloning and specificity analysis of the mouse
TR4 cDNA fragment. A, the nucleotide and deduced amino
acid sequence of the mouse TR4 cDNA (GenBank accession number
U32939[GenBank]). The putative DNA-binding domain is boxed. The
numbers on the left refers to the nucleotide
(lower) and the amino acid (upper) sequence. The
sequence and orientation of oligonucleotides used to amplify the
N-terminal region (TR4-16 and TR4-23) or to
construct the full-length mouse TR4 cDNA are as indicated (for
details see "Materials and Methods"). B, Northern blot
analysis of probe specificity. Samples of total RNA (20 µg) isolated
from the adult mouse testis (lane 1) and kidney (lane
2) were hybridized with the 32P-labeled mTR4N probe.
The hybridized signal was visualized by PhosphorImager (Molecular
Dynamics). The positions of 28 S and 18 S are indicated on the
left side and the sizes of two hybridized bands are
indicated at the right side of the blot.
[View Larger Version of this Image (35K GIF file)]
gene expression in vivo, we
examined TR4 expression by in situ hybridization. As shown
in Fig. 5A, a coronal section through the
rostral region of the forebrain demonstrated the presence of TR4
transcripts within the hippocampus, habenula nuclei, and thalamus as
well as throughout the cortical layers. The most intense signals were found in the cortex, piriform cortex (Fig. 5, B and
C), regions of Ammon's horn (Fig. 5A, CA1,
CA2, and CA3), and dentate gyrus (Fig. 5, D
and E). Consistent with previous results, the signal within
the dentate gyrus was localized to the granule cells, which undergo
active postnatal neurogenesis (6). No signal was detected with the
sense mTR4N probe (data not shown).
Fig. 5.
Localization of TR4 mRNA in adult mouse
brain by in situ hybridization. Dark field (A,
C, and E) and bright-field (B and
D) of a coronal section at the level of hippocampus hybridized with antisense mTR4N riboprobe. A, low magnification shows
the TR4 transcripts are detected in cortex (cx), dentate
gyrus (dg), Habenular nuclei (hn), piriform
cortex (p), and CA1, CA2, CA3 of Ammon's horn (1, 2, CA3). High magnification views revealing intensive hybridization
signals to the neuron cells of piriform cortex (arrowhead,
p) (B and C), and the granule cells of the dentate gyrus (arrow, g) (D and E).
The size bars represent 1 and 100 nm for A and
B-E, respectively.
[View Larger Version of this Image (91K GIF file)]
Fig. 6.
[View Larger Version of this Image (50K GIF file)]
Striking Similarities in the Expression Patterns of TR4 and
CNTFR within the Developing and Postnatal Nervous
Systems
(25) further strengthen our hypothesis that the regulation of CNTFR
by TR4 may happen in vivo. Both TR4 and CNTFR
transcripts
were localized within the developing nervous system. In addition, the timing of initial expression are tightly correlated. CNTFR
expression is most notably in the neuroepithelial lining of the brain
vesicles as well as in the developing dorsal root ganglia and spinal
cord during E11-E15 of rat embryos with its transcripts restricted in
the mitotically active populations (25). Our in situ data clearly show that TR4 have a similar expression pattern in E9-E14 mouse
embryos (correlate to E10-E15 rat embryos) and our RT-PCR assay (not
shown) show that TR4 transcripts are present as early as E9.
is
essential for the motor neuron development (19), our in situ
data showing abundant TR4 transcripts there indicate that TR4-mediated
CNTFR
induction could be an important process for motor neuron
survival. An interesting coincidence is that the neurotransmitter
dopamine has been proposed as an activator for TR2 and, possibly, for
TR4 (26). Assuming dopamine activates TR4 which may then induce
CNTFR
expression, it is possible that the reduced dopamine level, in
the case of Parkinson's disease, can affect the activation of TR4 and
CNTFR
. Thus, a higher CNTF concentration may be needed to maintain
neuron integrity. This hypothesis reasonably explains the action of
CNTF and proposes TR4 as a bridge between the dopamine and the CNTF
signal transduction pathways.
transcripts are detected in the skeletal muscle (18), liver (27), and
in the neural crest-derived precursors invading the nasal region,
second pharyngeal pouch, heart structures, and intestinal areas (25).
Significant amounts of TR4 transcripts are also present in many
proliferating cell populations where the expression of CNTFR
is
unclear, such as in the pericartilage, the brown fat, and the
developing organs (e.g. lung, kidney, and intestine). This
widespread but specific expression pattern suggest TR4 may be involved
in the normal development of the whole embryo. Gene knock-out
experiments would probably be helpful to understand the TR4 function
and the significance of TR4 to CNTFR
gene induction.
transcripts are co-expressed
within neuron cell bodies of many brain regions, including cortex,
hippocampus, and cerebellum. Outside the adult brain regions, whether
both are co-expressed is unknown.
, RAR
, RXR
, COUP-TFI, COUP-TFII, and TR2, and exert combinational effects on CNTFR
regulation. Among these DR1-binding protein, RAR
(34), RAR
(34), and RXR
(35) are detected in the
more differentiated neuronal types, which is consistent with the roles
of retinoids in neuron differentiation and their expression patterns do
not match that of CNTFR
. Since TR4, TR2,3 COUP-TFI, and
COUP-TFII (33) are localized mainly in the actively mitotic neuron
populations which also express CNTFR
, the chance for them to
regulate CNTFR
transcription is much higher. Especially, COUP-TFII
has been demonstrated to be involved in spinal motor neuron development
in chicken (36). How to coordinate these DR1-binding proteins for the
regulation of CNTFR
will be an intriguing question to follow.
-AGGTCA-like half-sites within
CNTFR-I5 indicates some steroid receptors which bind as a monomer may
regulate the CNTFR
expression. The candidates include thyroid
hormone receptor and TR3/NGFI-B/nur77. This idea is supported by their
localization in adult brain (8, 37). However, much lower protein-DNA
affinity between these receptors and the half-site may not support
these factors that would play major roles on CNTFR
regulation.
is the first
identified neuron-specific target gene regulated by TR4. Induction of
CNTFR
gene expression can be mediated through a perfect DR1 response
element located in its 5th intron. In addition, the expression patterns
of both genes correlated well during the developmental and postnatal
neurogenesis. These findings may extend our views to study the
interactions between steroid receptors and cytokine/cytokine receptors.
*
This work was supported by Grants CA 55639 and DK 47258 from
the National Institutes of Health. 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.
¶
To whom correspondence should be addressed: Comprehensive
Cancer Center, University of Wisconsin, 600 Highland Ave., K4/632, Madison, WI 53792. Tel.: 608-263-0899; Fax: 608-263-8613.
1
The abbreviations used are: HRE, hormone
response element; CNTFR,
receptor of ciliary neurotrophic
factor; TR2, TR2 orphan receptor; TR3, TR3 orphan receptor; TR4, TR4
orphan receptor; COUP-TFI and II, chicken ovalbumin upstream
promoter-transcription factor I and II; RAR, retinoic acid receptor;
RXR, retinoid X receptor; CNTF, ciliary neurotrophic factor; CNTFR-I5,
the 5th intron of CNTFR
gene; RT-PCR, reverse
transcription-polymerase chain reaction; DR1, direct repeat with one
spacing; TRE, thyroid hormone response element; NBRE, TR3/NGFI-B/nur77
response element; mTR4N, mouse N-terminal TR4 cDNA; EMSA,
electrophoretic mobility shift assay; CAT, chloramphenicol
acetyltransferase; E16, embryonic day 16.
2
Y.-F. Lee and C. Chang, manuscript in
preparation.
3
W.-J. Young, S. M. Smith, and C. Chang,
manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.