From the Department of Molecular Biosciences and § ARC Special Research Centre for Molecular Genetics of Development, University of Adelaide, Adelaide, 5005 South Australia, Australia
Received for publication, September 7, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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CP2-related proteins comprise a family of
DNA-binding transcription factors that are generally activators of
transcription and expressed ubiquitously. We reported a differential
display polymerase chain reaction fragment, Psc2, which was
expressed in a regulated fashion in mouse pluripotent cells in
vitro and in vivo. Here, we report further
characterization of the Psc2 cDNA and function. The
Psc2 cDNA contained an open reading frame homologous to
CP2 family proteins. Regions implicated in DNA binding and oligomeric
complex formation, but not transcription activation, were conserved.
Psc2 expression in vivo during
embryogenesis and in the adult mouse demonstrated tight spatial and
temporal regulation, with the highest levels of expression in the
epithelial lining of distal convoluted tubules in embryonic and adult
kidneys. Functional analysis demonstrated that PSC2 repressed
transcription 2.5-15-fold when bound to a heterologous promoter in ES,
293T, and COS-1 cells. The N-terminal 52 amino acids of PSC2
were shown to be necessary and sufficient for this activity and did not
share obvious homology with reported repressor motifs. These results
represent the first report of a CP2 family member that is expressed in
a developmentally regulated fashion in vivo and that acts
as a direct repressor of transcription. Accordingly, the protein has
been named CP2-Related Transcriptional Repressor-1
(CRTR-1).
The mouse transcription factor CP2 was identified as an activator
of the mouse Consistent with the ability of mouse CP2 to activate transcription,
human CP2/LBP-1c has been shown to activate transcription from an SV40
promoter (6) and from cellular promoters such as those directing
expression of the serum amyloid A3 gene (11). The ability to activate
transcription is conserved among other family members; LBP-1b activates
transcription from the Members of the CP2 family of transcription factors bind a consensus DNA
sequence consisting of a direct bipartite repeat sequence, CNRG-N6-CNRG (3, 10). Binding sites for this family of
proteins have been described in the viral and cellular promoters
described above; binding sites for CP2/LBP-1c have been described in
the Human CP2 has been reported to bind DNA as a dimer (5, 7, 15), although
other reports have shown that LBP-1c (14) and chicken CP2 (10) bind DNA
as tetramers. Truncation studies have localized a region of LBP-1c
required for oligomerization to amino acids 266-403 (14). Formation of
hetero-multimers between CP2 family members LBP-1a, b, and c has also
been reported (8). CP2 family members can form complexes with
nonrelated cellular proteins. For example, LBP-1c interacts with YY1 on
the human immunodeficiency virus, type I promoter (16), an unidentified protein (40-45 kDa) forming the stage selector protein complex that
binds to the Mammalian members of the CP2 family are generally expressed
ubiquitously (4, 5, 10, 18). Whereas LBP-9 expression in cultured cell
lines suggests some regulation of expression (9), the expression of
this gene has not been mapped in vivo. Using differential
display PCR1 analysis we
identified three novel genes that exhibit regulated expression during
pluripotent cell
differentiation.2 Expression
of these genes was temporally regulated during conversion of ES cells
to EPL cells, an in vitro system that recapitulates conversion of inner cell mass to primitive ectoderm in vivo
(19), and in the pluripotent cells of the pregastrulation mouse embryo. In this paper we report further analysis of one of these genes, denoted
Psc2, which was expressed in pluripotent cells in
vivo at 3.5 and 4.5 days post coitum (d.p.c.) and down-regulated
around 4.75 d.p.c.. We demonstrate that the Psc2
cDNA encodes a novel mouse member of the CP2 family, which differs
from the known members in two respects. Firstly, expression of this
gene is tightly regulated in vivo in both temporal and
spatial fashion, with the strongest expression detected in the
epithelial lining of distal convoluted tubules (DCTs) in the embryonic
and adult kidney. Secondly, the protein exhibits a novel
transcriptional repression activity, localized in the N terminus of the
protein, when tethered to a heterologous promoter. Accordingly, we have
renamed the gene CP2-Related Transcriptional Repressor-1 (CRTR-1).
cDNA Isolation and Sequencing--
CRTR-1
cDNA clones were isolated from a DNA Manipulations--
A sequence encoding a complete
CRTR-1 open reading frame was amplified by reverse
transcriptase-PCR on D3 ES cell RNA using the SuperScript
One-Step reverse transcriptase-PCR system (Life Technologies, Inc.)
according to the manufacturer's instructions. Primers used for
amplification were SR1 (5'-ATAGTCGACCAGCCATGCTGTTCTGG-3') and SR2
(5'-ATAAAGCTTGAGCTCAGAGTCCACACTTCAG-3'). The amplified CRTR-1 open reading frame fragment was cloned into
pGemT-easy (Promega, Madison, WI) according to the manufacturer's
instructions to generate pGemT-CRTR-1 and sequenced by automated
sequencing (PE Biosystems).
In-frame fusions between the Gal4 DNA binding domain and
CRTR-1 fragments were generated in the plasmid pGalO
(23-27), which contains amino acids 1-147 of the Gal4 DNA binding
domain (28). A fragment encoding the full-length CRTR-1 open reading
frame was excised from pGemT-CRTR-1 by digestion with SalI
and SacI and cloned into
SalI/SacI-digested pGalO to produce the plasmid pGalO.CRTR-1. The N-terminal 52 amino acids and C-terminal 435 amino acids of CRTR-1 were amplified by PCR on pGemT-CRTR-1 using Pfu Turbo (Stratagene) in accordance with the
manufacturer's instructions and the primer combinations SR1 + SR4
(5'-ATAGTCGACTACAGTATGTGTTGTGT-3') and SR2 + SR3
(5'-ATAGAGCTCACAACACATACTGTAG-3'), respectively. PCR fragments were
digested with SalI and SacI, purified by gel electrophoresis, and cloned into
SalI/SacI-digested pGalO to produce pGalO·CRTR-1(1-52) and pGalO·CRTR-1(47-481), respectively.
CRTR-1-specific riboprobes for in situ hybridization were
synthesized from pCRTR-1-1.2.8, generated by subcloning a 460-bp SmaI/HincII fragment from cDNA clone 1.2 (see
Fig. 1A) into EcoRV-digested pBluescript-KS+.
RNA Isolation, Riboprobe Synthesis, and Ribonuclease Protection
Analysis--
Cytoplasmic RNA was isolated from D3 ES and EPL (19)
cells as described previously (29). Mouse embryos from 10.5 to
17.5 d.p.c., 16.5-d.p.c. embryonic tissues, and adult mouse
tissues were isolated and homogenized, and total RNA was isolated using the guanidinium isothiocyanate method (30).
CRTR-1 antisense riboprobes for use in RNase protections
were synthesized as described previously (31) by transcription of
HincII linearized cDNA clone 1.2 (see Fig.
1A) with T3 RNA polymerase (Roche Molecular
Biochemicals). Ribonuclease protection assays were performed on
10 µg of total RNA as previously described (31, 33) except that
hybridizations were for 14 h at 45 °C. To reduce overexposure
of the loading control, low specific activity mGAP probes were
synthesized using 40 µCi of [
Digoxygenin-labeled riboprobes for wholemount in situ
hybridization were generated from the 736-bp CRTR-1
differential display PCR fragment cloned into
pBluescript-KS+2 (see Fig. 1A). Sense and
antisense transcripts were generated as described (32) by
BamHI digestion and transcription with T7 RNA polymerase and
XhoI digestion and transcription with T3 RNA polymerase, respectively.
33P-labeled sense and antisense CRTR-1 probes
for in situ hybridizations were generated as described
elsewhere (33) by transcription of pCRTR-1-1.2.8 linearized with
BamHI and XhoI and transcribed with T3 and T7 RNA
polymerase, respectively. [ Wholemount in Situ Hybridization and in Situ
Hybridization--
16.5-d.p.c. kidneys were isolated from BALB/c mouse
embryos. Wholemount in situ hybridization was carried out as
described elsewhere (32) except that 16.5-d.p.c. embryonic kidneys were prewashed for 20 min in radioimmune precipitation buffer three times
before post-fixing, prehybridized, hybridized, and washed at 65 °C.
Probed embryonic kidneys were dehydrated at room temperature for 10 min
in 100% methanol, 15 min in isopropanol, and 2 × 15 min in
Histo-Clear (National Diagnostics) before embedding in paraffin
wax. 7-µm serial sections were cut using a Leica microtome, floated
on water at 45 °C, placed onto silanized microscope slides, de-paraffinized in Histo-Clear, and re-hydrated through a methanol series; then the sections were counterstained with methyl green and mounted with DePex and a coverslip. Sections were viewed using a
Nikon Eclipse TE300 inverted microscope and photographed with Ektachrome 100 ASA slide film (Kodak).
Adult kidneys were dissected from BALB/c female mice and fixed as
above. 7-µm serial sections were cut as described for wholemount in situ hybridization. Radiolabeled in situ
hybridization was carried out as described elsewhere (34) with the
following modifications. Sections were heated to 55 °C for 30 min
before de-paraffinization in Histo-Clear. Sections were prehybridized
at 52 °C for 1 h and washed twice in 2× SSPE for 2 min prior
to overnight hybridization with riboprobes. Post-hybridization washes
were carried out prior to RNase digestion as follows: 50% formamide,
2× SSPE, 0.1% SDS, 10 mM Cell Culture and Transfections--
ES and EPL cell culture was
carried out as described previously (19). D3 ES cells, COS-1 cells, and
293T cells were maintained as described previously (35).
Cell transfections were carried out with FuGeneTM6 (Roche
Molecular Biochemicals) transfection reagent according to the
manufacturer's instructions. COS-1, 293T, and ES cells were plated out
in 24-well trays (Falcon) at densities of 35,000, 50,000, and 100,000 cells/well, respectively, and transfected the following day with 200 ng/well Gal4-DBD plasmid + 200 ng/well pTK-MH100x4-LUC (36) + 50 ng/well pRLTK (Promega) or 200 ng/well pGalO·CRTR-1 + 200 ng/well
pHRE-Luc (37, 38) + 50 ng/well pRLTK. Control transfections carried out
with 200 ng/well pTK-MH100x4-LUC or 200 ng/well pHRE-Luc + 50 ng/well
pRLTK were made up to 450 ng DNA/well with
pBluescript-KS+ carrier DNA. Luciferase activity was
assayed on a TD-20/20 luminometer (Turner Designs) using the
Dual-Luciferase® reporter assay system (Promega) according to
the manufacturer's instructions. Firefly luciferase expression was
normalized against Renilla luciferase. Experiments were repeated in triplicate.
CRTR-1 cDNA Isolation and Sequence--
Based on the known
expression in pluripotent cells in vitro and in
vivo, CRTR-1 cDNA clones were isolated from a D3 ES
cell
Both strands of CRTR-1 cDNA fragments were sequenced to
generate the CRTR-1 cDNA sequence (GenBankTM
accession number AF311309). The 9405-bp cDNA contained a poly(A) tail 25 bp downstream of a consensus polyadenylation signal (AATAAA), consistent with the typical positioning of the polyadenylation signal
10-30 bp upstream of the poly(A) tail (39). A 1446-bp open reading
frame extended from nucleotide 92 to nucleotide 1537 and was followed
by a long 3' untranslated region of 7868 bp. The CRTR-1 protein
predicted from this compiled sequence is 481 amino acids long, with a
predicted molecular mass of 54,702 daltons. No other significant
reading frames were identified.
CRTR-1 Sequence Analysis--
Comparison of the predicted CRTR-1
amino acid sequence with entries in protein sequence data bases
revealed considerable similarity to a group of proteins related to the
mouse transcription factor CP2 (2) (Table
I). Fig. 2
shows a multiple sequence alignment of reported CP2 family members with
CRTR-1. Included in Table I and the multiple sequence alignment are the
mouse family members CP2 (2) and NF2d9 (4); human family members
LBP-1a, LBP-1b, LBP-1c, LBP-1d (8), and LBP-9 (9); and the DNA binding
domain of the D. melanogaster protein GRH (40).
Conservation between CRTR-1 and the other mammalian proteins was
extensive (Fig. 2) and extended across the CRTR-1 sequence, with the
exception of amino acids 1-47 and 381-401, which were conserved only
with LBP-9. Furthermore, the 51-amino acid deletion at position 189 specific to LBP-1d and the 37-amino acid insertion (amino acids
274-312) specific to LBP-1b were not found in CRTR-1. Similarity to
GRH was confined to amino acids 632-865, shown to be sufficient
for DNA binding to elements in the Dopa decarboxylase (Ddc) promoter (41). The failure of proteins containing
deletions within this region (LBP-1d (7, 8) and chicken CP2 (10)) or
truncated N-terminally past amino acid 65 or C-terminally past amino
acid 383 (LBP-1c (14)) to bind DNA is consistent with the
identification of this region as a DNA binding sequence that is
conserved in the CRTR-1 protein.
Truncation studies have localized an oligomerization domain within
LBP-1c to amino acids 266-403 (14). This region was well conserved
within CRTR-1, suggesting a potential for formation of homo- and
hetero-oligomeric protein complexes. Within the equivalent regions of
CP2 (amino acids 398-425) and LBP-1c/LBP-1d are located a
glutamine/proline repeat and a polyglutamine repeat, respectively, which have been predicted to form a transcriptional activation domain
(2) but are not conserved in CRTR-1 or LBP-9.
LBP-9 (9) is the CP2 family member that shows the greatest level of
similarity to CRTR-1 (Table I; Fig. 2). Whereas there was considerable
conservation of amino acid sequences between these proteins, similarity
was restricted to the open reading frame and did not extend into the
reported, incomplete 3' untranslated region of LBP-9.
Regulated Expression of CRTR-1 during Mouse
Development--
Expression of CRTR-1 has been shown to be
specifically regulated in pluripotent cell populations in
vitro and in vivo.2 CRTR-1
expression during later mouse development was investigated by
ribonuclease protection analysis using total embryonic RNA isolated
from 10.5-17.5 d.p.c. embryos, tissue-specific total RNA samples
isolated from 16.5-d.p.c. embryos, and tissue-specific total RNA
samples isolated from adult mice. CRTR-1 expression was not
detected in total RNA isolated from 12.5- and 13.5-d.p.c. embryos and
was expressed at low levels in 10.5- and 11.5-d.p.c. embryos (Fig.
3A). CRTR-1
expression was highest between 14.5 and 17.5 d.p.c. Of the
16.5-d.p.c. embryonic tissues analyzed (Fig. 3B),
CRTR-1 was not detected in 16.5-d.p.c. embryonic brain. Low
levels of CRTR-1 expression were detected in 16.5-d.p.c.
embryonic intestine, limb, lung, and skin, with highest expression in
16.5 d.p.c. embryonic kidney. Levels of CRTR-1
expression observed in 16.5-d.p.c. embryonic kidney were comparable
with levels of expression observed in ES cells. Of the tissue-specific
total RNA samples isolated from adult mice, CRTR-1 was not
detected in brain, heart, liver, and spleen (Fig. 3C).
CRTR-1 was expressed at low levels in lung, mesenteric lymph
nodes, muscle, ovary, and thymus; at elevated levels in placenta,
testis, and small intestine; and at high levels in adult kidney and
stomach, which expressed CRTR-1 at levels 7- and 1.5-fold
greater than ES cells, respectively. Expression of CRTR-1
was therefore specifically regulated in a temporal and spatial fashion
both during embryogenesis and in the adult mouse.
Expression of CRTR-1 in Embryonic and Adult Kidney Is Restricted to
the Distal Convoluted Tubules--
Cellular localization of
CRTR-1 expression was investigated in embryonic and adult
kidneys. Wholemount in situ hybridization analysis was
carried out using kidneys isolated from 16.5-d.p.c. embryos where
CRTR-1 expression was demonstrated to be highest (Fig.
3B). Embryonic kidneys were probed with
CRTR-1-specific sense and antisense digoxygenin-labeled
riboprobes prior to embedding, sectioning, and counterstaining. Kidney
sections probed with CRTR-1 sense control probe showed no
specific staining (Fig. 4A).
Kidney sections probed with CRTR-1 antisense probe showed
specific staining representing CRTR-1 expression in the
epithelial monolayer lining a subset of tubules in the embryonic kidney
cortex (Fig. 4, B and C).
CRTR-1-expressing tubules were identified as DCTs because they were located adjacent to glomeruli, consistent with the location of DCTs within the kidney cortex (42). Furthermore, only a small proportion of the tubules present in any cortical section expressed CRTR-1, consistent with the greater relative representation
of proximal convoluted tubules in this region of the kidney (43-46). Finally, the morphology of CRTR-1-expressing tubules was
clear and open, consistent with the morphology of DCTs but distinct from that of proximal convoluted tubules, in which the epithelium forms
a brush border consisting of microvilli that project into the lumen of
the tubule (45, 46). CRTR-1 expression was not detected in
proximal convoluted tubules, glomeruli, or kidney vasculature (Fig. 4,
B and C).
CRTR-1-expressing cells in the adult mouse kidney were
determined by radiolabeled in situ hybridization to kidney
sections because the greater volume of the adult kidney precludes the
use of wholemount in situ hybridization. Adult kidneys were
sectioned and probed with CRTR-1-specific,
[ CRTR-1 Acts as a Transcriptional Repressor in a Variety of Cell
Types--
Members of the CP2 family have been reported to act as
transcriptional activators in both in vitro (1, 3) and
in vivo (3, 8-11) transcription assays. The ability of
CRTR-1 to act as a transcriptional regulator could not be investigated
using target gene expression because the DNA binding sequence for this protein is unknown. The transcriptional activity of CRTR-1 was therefore assessed as a fusion protein with amino acids 1-174 of the
Gal4 DNA binding domain (DBD) (28) in the plasmid pGalO·CRTR-1. pTK-MH100x4-LUC (36), which contains a luciferase gene regulated by the
thymidine kinase (TK) promoter and four upstream tandem copies of the
Gal4 binding site, was used as a reporter.
Plasmids were transfected into COS-1 cells, and levels of luciferase
activity were analyzed in cell extracts 36 h post-transfection. Cotransfection of the Gal4-DBD, pGalO (23-27), with pTK-MH100x4-LUC did not alter the reproducible levels of luciferase activity (Fig. 5A, column 1)
compared with pTK-MH100x4-LUC alone (Fig. 5A, column 2). Cotransfection of pGalO·CRTR-1 with pTK-MH100x4-LUC resulted in a 10-15-fold reduction in luciferase activity (Fig. 5A,
column 3). CRTR-1-mediated transcriptional repression was
also demonstrated in 293T (Fig. 5B) and ES cells (Fig.
5C), where expression of the Gal4-DBD-CRTR-1 fusion protein
reduced luciferase expression 2.5- and 3.5-fold, respectively. This
transcriptional repression was specific for the reporter plasmid
pTK-MH100x4-LUC and not a result of general transcriptional toxicity of
the Gal4-DBD-CRTR-1 fusion protein, because luciferase activity within
these assays was normalized against expression of Renilla luciferase,
expressed from pRLTK under the control of the constitutive thymidine
kinase promoter. Furthermore, expression of luciferase from pHRE-Luc (37, 38), in which expression of luciferase is controlled by the SV40
promoter and three upstream copies of the hypoxia-inducible factor
response element, was not altered by cotransfection with pGalO·CRTR-1
(Fig. 5B, columns 4 and 5). These
results demonstrate that CRTR-1 acts as a transcriptional repressor in
a variety of cell types including ES cells and 293T cells,
representative of in vivo expression.
The Ability of CRTR-1 to Repress Transcription Resides in an
N-terminal Repression Domain--
The N-terminal 40 amino acids of CP2
have been shown to contain the CP2 transcriptional activation
domain.3 This region of
CRTR-1 was not conserved with members of the CP2 family reported to act
as transcriptional activators but was closely related to LBP-9, which
can antagonize LBP-1b-mediated transcriptional activation (9).
PCR was used to amplify the N-terminal 52 amino acids and the
C-terminal 435 amino acids of CRTR-1. PCR products were cloned in frame
with the Gal4-DBD in pGalO to generate pGalO·CRTR-1(1-52) and
pGalO·CRTR-1(47-481), respectively. Cotransfection of COS-1 cells
with pGalO·CRTR-1(47-481) and pTK-MH100x4-LUC did not affect levels
of luciferase activity (Fig. 5A, column 4)
compared with transfection of pTK-MH100x4-LUC alone (Fig.
5A, column 2) or transfection of pGalO and
pTK-MH100x4-LUC (Fig. 5A, column 1). However,
cotransfection of pGalO·CRTR-1(1-52) and pTK-MH100x4-LUC resulted in
10-15-fold reduction in luciferase activity (Fig. 5A,
column 5), consistent with the level of repression resulting
from cotransfection with full-length CRTR-1 (Fig. 5A,
column 3). This demonstrates that the N-terminal 52 amino
acids of CRTR-1 are both necessary and sufficient for the
transcriptional repression exerted through CRTR-1.
CRTR-1 Is a Novel Mouse Member of the CP2 Family of Transcription
Factors--
The CRTR-1 open reading frame was closely related to a
group of proteins including the mouse transcription factor CP2 (Table I
and Fig. 2) (2), the founding member of an expanding group of highly
conserved proteins implicated in transcriptional control. Amino acid
conservation across the CP2 open reading frame suggested conservation
of functionally important regions of the CRTR-1 protein. In particular,
a potential DNA binding domain, distinct from structurally characterized DNA binding domains, was identified between CRTR-1 amino
acids 45 and 260, consistent with conservation of this region with the
DNA binding domain of the D. melanogaster protein GRH (41) and deletion mapping of the LBP-1c DNA binding domain (14). Furthermore, a region implicated by deletion mapping in
homo-oligomerization of LBP-1c (14) was highly conserved with CRTR-1
residues 261-386, suggesting that this protein is likely to support
the formation of protein complexes. Whereas CRTR-1 shared greatest
identity (88%) with the recently reported human protein LBP-9 (9),
conservation was restricted to the open reading frame and did not
extend into the 3' untranslated region. This suggests either that the
reported CRTR-1 and LBP-9 cDNAs are derived from alternative
splicing of a homologous gene in mice and humans or that the proteins
are not products of homologous genes. Sequence analysis therefore identified CRTR-1 as a novel mouse member of the CP2 family, with potential roles in transcriptional control and the formation of protein complexes.
CRTR-1 Is a Novel Transcriptional Repressor--
CRTR-1 was shown
to repress transcription when bound at a heterologous promoter. Whereas
the extent of repression varied from 2.5- to 15-fold in different cell
types (Fig. 5), conservation of this activity in different cell lines
suggests that these results are indicative of normal CRTR-1 activity.
This is supported by the fact that 293T and ES cells, in which
repression was demonstrated, are representative of in vivo
expression sites in kidney and pluripotent cells, respectively. The
transcriptional repression activity of CRTR-1 was found to be localized
to the N-terminal 52 amino acids, a region that does not show strong
homology to other members of the CP2 family, with the exception of
LBP-9, which has been shown to antagonize LBP-1b-mediated
transcriptional activation by an unknown mechanism (9). The results
presented here demonstrate that the N-terminal 52 amino acids of CRTR-1
are both necessary and sufficient for CRTR-1-mediated transcriptional
repression when recruited to the promoter by DNA binding and that the
observed transcriptional repression was not the result of steric
hindrance caused by Gal4-DBD-CRTR-1 fusion proteins.
Activity as a transcriptional repressor distinguishes CRTR-1 from most
other members of the CP2 family, which have been reported to act as
transcriptional activators (1, 3, 8-11). This is consistent with the
lack of amino acid conservation at the N terminus, which contains the
activation domain in CP2,4 and with the lack of
polyglutamine- and glutamine/proline-rich sequences suggested
as activation domains in the LBP-1c and CP2 sequences, respectively
(2). LBP-9, identified as a sequence-specific binding protein on the
Transcriptional repression can be mediated through several different
mechanisms such as interference with assembly of the transcriptional
machinery (47) or recruitment of corepressors including histone
deacetylases (48). Protein sequence motifs present in DNA-binding
transcriptional repressors that mediate interaction with corepressor
proteins include PXDLS in the ikaros protein (49-51), WRPW in
Hairy-related bHLH proteins (52, 53), and a Gly/Arg-rich sequence
present in the transcription factor YY1 (54). Furthermore, a histone
deacetylase-independent mechanism of transcriptional repression has
been described for methyl-CpG-binding protein 2 that is dependent on
the presence of a conserved 30-amino acid sequence that contains two
clusters of basic amino acids (55). These motifs could not be
identified within the N-terminal 52 amino acids of CRTR-1, suggesting a
novel mechanism of transcriptional repression for this protein.
Conservation of repressor activity in cell lines of diverse origin and
properties such as ES cells, 293T cells, and COS-1 cells suggests that
factors required for CRTR-1-mediated repression are widely expressed.
Expression of CRTR-1 Is Spatially and Temporally Regulated during
Mouse Development--
Expression of CRTR-1 was shown to be
spatially and temporally regulated, both during embryogenesis and in
the adult mouse. In vitro, CRTR-1 was expressed
in ES cells and rapidly down-regulated upon differentiation to EPL
cells (Fig. 1, B and C). An equivalent expression
pattern has been described in vivo where CRTR-1
expression in pluripotent cells of 3.5-d.p.c. mouse embryos is
down-regulated at around 4.75 d.p.c.2 Re-expression of
CRTR-1 in the embryo from 10.5 to 12.5 d.p.c. and at
higher levels from 14.5 to 17.5 d.p.c. was demonstrated by
ribonuclease protection analysis (Fig. 3A). This analysis
also indicated differential CRTR-1 expression in various
tissues. For example, in the 16.5-d.p.c. embryo (Fig. 3B),
CRTR-1 expression was greatest in the kidney, at low levels
in the intestine, limb, lung, and skin, and not detected in the brain,
whereas in the adult mouse, CRTR-1 (Fig. 3C) was
expressed at highest levels within adult mouse kidney, at moderate
levels in the stomach, testis, placenta, and small intestine, at low
levels in lung, mesenteric lymph nodes, muscle, ovaries, and thymus,
and not detected in brain, heart, liver, and spleen. Detailed
investigation of CRTR-1 expression in 16.5-d.p.c. embryonic
and adult mouse kidney (Fig. 4) demonstrated restriction of expression
to the epithelium of distal convoluted tubules, identified by
morphological and histological criteria.
Tight spatial and temporal localization of CRTR-1 expression
in vitro and in vivo distinguishes CRTR-1 from
most other CP2 family members that are reported to be expressed
ubiquitously (4, 5, 10, 18). Variable expression of LBP-9 as
detected by reverse transcriptase-PCR in cultured cell lines may also
be indicative of regulated expression. This analysis (9) was limited to
cell lines of placental (JEG-3), adrenal (NCI-H295A), cervical (HeLa),
hepatic (HepG2), and kidney (COS-1) origin and human adrenal tissue.
LBP-9 expression was detected at highest levels in JEG-3 cells, at lower levels in COS-1, HepG2 and HeLa cells, and was not
detected in NCI-H295A cells or human adrenal tissue. CRTR-1 was expressed in embryonic and adult kidney and placenta, but expression was not detected in adult liver. Whereas direct parallels cannot be drawn between expression in vivo and potentially
deregulated expression in cell lines in vitro, the
differential sites of CRTR-1 and LBP-9 expression
support the suggestion, based on sequence conservation, that these
genes may not represent homologues.
The sites of CRTR-1 expression in vivo suggest at
least two functions for CRTR-1 in the mouse: in pluripotent cells
during early mouse development and in the development and function of kidney DCTs. Whereas both pluripotent cells and the DCT lining are
epithelial in origin, other epithelial cells including the lining of
kidney proximal convoluted tubules did not express detectable CRTR-1, excluding a general role for the CRTR-1 protein in
cells of this type. It is of interest that both demonstrated sites of CRTR-1 expression in vivo are associated with
cavitation, within the egg cylinder and DCTs, respectively. A general
model for cavitation, based on integrated action of extracellular
diffusible "death" signals and matrix-localized survival signals,
has been hypothesized, based on mechanistic investigation of
proamniotic cavity formation in pluripotent cell populations (56).
CRTR-1 expression could potentially be responsive to signals
of this nature (57).
DCTs form part of the nephron, the basic filtration unit of the kidney,
and become functional at around 16.0 d.p.c. (58). These tubules
control blood pH through regulated ion channels that direct the
reabsorption of Na+ and HCO3
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin gene, which binds a promoter element overlapping the CCAAT box (1-3). CP2 is the founding member of a group
of highly conserved proteins identified in mice, humans, and chickens
referred to as the CP2 family of transcription factors (4). Human
cDNAs encoding multiple CP2-related proteins have been identified.
These include human CP2 (2, 5) (also referred to as LSF and LBP-1c
(6-8)), LBP-1d (8) (also known as LSF-ID (7)), an alternatively
spliced form of LBP-1c (8), LBP-1a (8), LBP-1b (8) (an alternatively
spliced form of LBP-1a (8)), and LBP-9 (9). The mouse protein NF2d9
shows 94% identity to LBP-1a and is recognized as the homologue (4),
and a chicken CP2 homologue has also been reported (10).
155/
131 region of the human P450scc promoter
(9), LBP-1a activates transcription from a human immunodeficiency
virus, type I promoter (8), and chicken CP2 activates
transcription from the
A-crystallin gene promoter (10).
-fibrinogen (12, 3), synthase kinase-3
(13), and
-globin (5) promoters; binding sites for NF2d9 have been described in the
Cyp 2d-9 (steroid 16
-hydroxylase) promoter (4); and binding sites for LBP-9 have been described in the human P450scc promoter (9). Amino acids 63-270 of CP2 share sequence similarity with
the region required for DNA binding in the Drosophila
melanogaster transcription factor grainyhead
(grh) (2). This region is highly conserved within other CP2
family members and appears to be important for DNA binding because
LBP-1d, which is translated from an alternatively spliced form of
CP2/LBP-1c and lacks amino acids 189-239, is unable to bind the LBP-1c
DNA binding sequence (7, 8). N- and C-terminal truncation studies have
defined the minimum DNA binding region of LBP-1c between amino acids 65 and 383 (14).
-globin promoter (5) and a neuron-specific protein FE65
(17). Protein sequences required for hetero-oligomerization have not
been defined.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ZAP II ES cDNA library
derived from D3 ES cell RNA (CLONTECH Inc.).
Library screening was carried out as described previously (20) using
random primed [
-32P]dATP-labeled (Geneworks Ltd.,
Adelaide, South Australia, Australia) DNA probes (Gigaprime kit,
Geneworks Ltd.). Clones q1, 1.2, 6A, 8.2.1, and 8B were isolated
by successive screening of the library with a 736-bp fragment isolated
by EcoRI digestion of the cloned CRTR-1
differential display PCR fragment,2 a 132-bp fragment
isolated by EcoRI/AccI digestion of cDNA
clone q1, a 190-bp fragment isolated by
EcoRI/NcoI digestion of cDNA clone 1.2, a
384-bp fragment isolated by EcoRI digestion of cDNA clone 6A, and a 500-bp fragment isolated by EcoRI digestion
of cDNA clone 8.2.1, respectively (see Fig. 1A). Third
round duplicate positive plaques from each library screen were
isolated, grown to high titer, and excised by the
ZAP excision
process into pBluescript-SK+ vector as described in
the manufacturer's instructions. CRTR-1 cDNA fragments
were isolated by restriction digestion, subcloned into
pBluescript-KS+, and sequenced by automated DNA sequencing
(PE Biosystems) using the universal sequencing primer and reverse
sequencing primer. CRTR-1 nucleotide and open reading frame
sequences were analyzed using DNASIS version 2.0 computer software
(Hitachi Software Engineering Co.). CRTR-1 nucleotide and
amino acid sequence comparisons were carried out using the
GenBankTM data base and BLAST software (21). Protein
sequence alignments were performed using the ClustalW and Boxshade
software (22).
-32P]rUTP in the
reaction, whereas CRTR-1 probes were synthesized using 125 µCi of [
-32P]rUTP. 37,000 counts/min of mGAP probe
and 150,000 counts/min of all other probes were added to each
hybridization. [
-32P]rUTP was obtained from Geneworks Ltd.
-33P]rUTP was
obtained from Geneworks Ltd.
-mercaptoethanol at
52 °C for 5 min; 50% formamide, 2× SSPE, 10 mM
-mercaptoethanol at 52 °C for 5 min; 50% formamide, 2× SSPE, 10 mM
-mercaptoethanol at 60 °C for 10 min; and twice in
2× SSPE at room temperature for 5 min. Slides were air-dried and
warmed to 37 °C. Sections were counterstained with hematoxylin, mounted with DePex and a coverslip, viewed using light and dark field
condensers on a Zeiss Axioplan microscope, and photographed with
Ektachrome 160T ASA slide film (Kodak).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ZAP II cDNA library (CLONTECH Inc;
(20)) using successively more 5' CRTR-1-specific probes
(Fig. 1A). Clones were
confirmed to be CRTR-1-specific using Southern analysis
(data not shown), sequence data, and expression analysis (Fig.
1B and data not shown). Ribonuclease protection using
riboprobes generated from the CRTR-1 cDNA clone 1.2 demonstrated rapid down-regulation of CRTR-1 expression upon
differentiation of ES cells to EPL cells (Fig. 1, B and
C), consistent with the pattern described previously for the
differential display PCR product using Northern blot and in
situ hybridization.2
View larger version (36K):
[in a new window]
Fig. 1.
Isolation, sequencing, and confirmation of
CRTR-1 cDNA clones. A, schematic
figure outlining the cDNA library screening strategy employed to
isolate CRTR-1 cDNAs. The CRTR-1 transcript, represented
by the shaded box, was predicted to be 9400 bp in length by
Northern analysis (see Footnote 2). cDNA clones, and the probes
used for their isolation, are indicated together with the restriction
sites used for their excision. RI, EcoRI;
A, AccI; N, NcoI;
dd, differential display; kb, kilobase(s).
B, ribonuclease protection analysis of CRTR-1
expression using 10 µg of total RNA isolated from ES and EPL cells
cultured in 50% MEDII in the presence of leukemia inhibitory
factor for 2, 4, 6, and 8 days. An mGAP-specific antisense riboprobe
was used as a loading control. C, quantitation of
ribonuclease protection analysis used to confirm isolated
CRTR-1 cDNAs as represented in B. The
expression of CRTR-1 was normalized against the mGAP loading
control. The standard mean error of three independent ribonuclease
protection assays using three different CRTR-1-specific
antisense riboprobes is shown.
Percentage identity and similarity of CRTR-1 to other proteins from
GenBankTM
View larger version (74K):
[in a new window]
Fig. 2.
Multiple amino acid sequence alignment of
CRTR-1. A, reported members of the CP2 family of
transcription factors and the DNA binding domain (amino acids 632-865)
of the D. melanogaster protein GRH. Dark
shading indicates conservation of identical amino acids, whereas
lighter shading indicates conservation of similar amino
acids. B, schematic summary of conserved regions in CRTR-1
functionally important in LBP-1c (14) (conserved DNA binding domain
(amino acids 45-366) and conserved oligomerization domain (amino acids
248-386)) and GRH (41) (conserved DNA binding domain (amino
acids 45-260)). Also shown are the N-terminal 47 amino acids of CRTR-1
conserved only with LBP-9 (9).
View larger version (69K):
[in a new window]
Fig. 3.
CRTR-1 expression during later mouse
development and in the adult mouse. Ribonuclease protection assays
were carried out on 10 µg of total RNA isolated from (A)
10.5-17.5-d.p.c. mouse embryos, (B) tissues from the
16.5-d.p.c. mouse embryo, and (C) tissues from adult mice.
mGAP antisense riboprobes were used as a loading control.
SI, small intestine.
View larger version (96K):
[in a new window]
Fig. 4.
CRTR-1 expression in embryonic and
adult mouse kidneys. A-C, wholemount in
situ hybridization of 16.5-d.p.c. mouse kidney probed with
CRTR-1-specific sense (A) and antisense
(B and C) digoxygenin-labeled riboprobes.
D-G, radiolabeled in situ hybridization on
7-µm adult kidney sections using CRTR-1 sense (D and
E) and antisense (F and G)
[ -33P]rUTP-labeled riboprobes. Developed
slides were viewed under light (D and F) and dark
field (E and G) condensers. D, distal
convoluted tubule; G, glomerulus; P,
proximal convoluted tubule. Magnifications are as follows: × 20 (A), × 20 (B), × 40 (C), × 10 (D), × 10 (E), × 20 (F), × 20 (G).
-33P]-labeled sense and antisense riboprobes.
Hybridization was not detected using CRTR-1 sense control
probe (Fig. 4, D and E). Adult kidney sections
probed with CRTR-1 antisense probe showed specific localization of CRTR-1 expression to the epithelial
monolayer lining a subset of tubules in the adult kidney cortex (Fig.
4, F and G). Consistent with the expression in
embryonic kidneys, these tubules were identified as DCTs.
CRTR-1 transcripts were not detected in the proximal
convoluted tubules, glomeruli, or kidney vasculature (Fig.
4G). This analysis demonstrates that expression of
CRTR-1 is spatially regulated in at least two distinct sites, the pluripotent cells of the developing mouse
embryo2 and the epithelial cells lining the embryonic and
adult kidney distal convoluted tubules.
View larger version (12K):
[in a new window]
Fig. 5.
CRTR-1 represses transcription from a basal
promoter. COS-1 cells (A), 293T cells (B),
and ES cells (C) were transfected with expression vectors
for Gal4-DBD-CRTR-1 fusion proteins pGalO·CRTR-1,
pGalO·CRTR-1(47-481), and pGalO·CRTR-1(1-52) and the reporter
plasmids pTK-MH100x4-LUC (Gal-Luc Reporter) and
pHRE-Luc (HRE-Luc Reporter), as indicated. Luciferase
expression was normalized against expression of Renilla luciferase
expressed from the cotransfected plasmid pRLTK. The mean and S.D. of
three independent experiments is represented.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
155/
131 region of the P450scc promoter, also exhibits unusual
transcriptional activity. Whereas LBP-1b, which also binds this
sequence, activated transcription of a linked reporter gene 21-fold in
JEG-3 cells, LBP-9 did not activate transcription in the same system
(9). Transfection of cells with increasing amounts of LBP-9 suppressed
the LBP-1b-mediated reporter activation to basal levels. The mechanism
of inhibition was not resolved and could result from direct repression
of transcription, steric exclusion of LBP-1b from the DNA binding site,
or displacement of LBP-1b from the promoter by formation of complexes
with LBP-9. By contrast, CRTR-1 is the first reported CP2 family member
that represses transcription directly from a heterologous promoter. Conservation of the 52-amino acid region of CRTR-1, responsible for
transcriptional repression, with the equivalent region of LBP-9 may
provide a mechanistic explanation for the suppression of
LBP-1b-mediated transcription activation by LBP-9. In particular, if
heteromeric complexes including LBP-9 can be localized at the P450scc
promoter, this protein and possibly CRTR-1 may be capable of acting as
a dominant repressor of promoters that are activated by CP2 family
proteins. Resolution of this possibility requires identification of the
CRTR-1 DNA binding sequence and binding partners.
ions
from kidney filtrates and the secretion of K+ and
H+ ions (59, 60). This process is regulated by the
signaling molecule aldosterone, a ligand for the mineralcorticoid
receptor that is associated with regulation of genes required for
Na+ and H+ exchange (61, 62). By RNase
protection, CRTR-1 expression in the embryonic and adult
kidney was at similar or higher levels to that in ES cells. This points
to an extremely high level of expression in kidney DTCs, which comprise
only a small proportion of the cells within the kidney, suggesting
important CRTR-1 function at this location. DCTs arise from the
metanephric mesenchyme, which is located near the cortical periphery
after 13 d.p.c. Although CRTR-1 expression in the
embryo may be associated with induction of these tubules during
development, continued high level expression of the gene at later
stages of embryogenesis and in the adult is suggestive of a role for
CRTR-1 in DCT function and physiology. Resolution of the functional
relevance of CRTR-1 expression at different locations and developmental
stages awaits functional investigation.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Dan Peet and Murray Whitelaw for helpful discussions and technical advice about transcription assays, Dr. Steve Jane for communication of results before publication, Kelly Loffler for assistance with in situ hybridizations, Tricia Pelton for assistance with wholemount in situ hybridizations, Drs. Tom Schulz and Roger Voyle for provision of RNA samples, and Dr. Julie Haynes for assistance with kidney sections.
![]() |
FOOTNOTES |
---|
* This research was supported by grants from the Australian Research Council (ARC) and by the ARC Special Research Center for Molecular Genetics of Development.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF311309.
Current address: Dept. of Ophthalmology, Flinders University of
South Australia, Bedford Park, 5042 South Australia, Australia.
¶ To whom correspondence should be addressed. Tel.: 61 8 8303 5354; Fax: 61 8 8303 4348; E-mail: peter.rathjen@adelaide.edu.au.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M008167200
2 T. Pelton, S. Sharma, T. Schulz, J. Rathjen, and P. Rathjen, submitted for publication.
3 S. Jane, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; ES, embryonic stem; EPL, early primitive ectoderm-like; d.p.c., day(s) post coitum; DCT, distal convoluted tubule; bp, base pair(s); mGAP, mouse glyceraldehyde-3-phosphate dehydrogenase; SSPE, saline/sodium phosphate/EDTA; DBD, DNA binding domain; TK, thymidine kinase.
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