From the Department of Veterinary Molecular Biology,
Marsh Laboratories, Montana State University, Bozeman, Montana 59717 and the
Howard Hughes Medical Institute, Eccles Institute of
Human Genetics, University of Utah, Salt Lake City, Utah
84112-5331
Received for publication, November 3, 2002
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ABSTRACT |
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The 180-amino acid core of the
TATA-binding protein
(TBPCORE) is conserved from Archae bacteria to man.
Vertebrate TBPs contain, in addition, a large and highly conserved
N-terminal region that is not found in other phyla. We have generated a
line of mice in which the tbp allele is replaced with a
version, tbp Gene expression is a fundamental property of all living systems.
Archae bacteria use only a single RNA
pol1 to transcribe mRNA,
rRNA, and tRNA. Eukaryotes evolved three separate RNA pols with
different specialties to perform these functions. TBP acts in promoter
recognition for transcription initiation by the Archaea RNA pol as well
as by all three eukaryotic RNA pols (1-3). In accordance with having
such an important and conserved function, the 180-amino acid
TBPCORE from Archaea and from all eukaryotes share both a
high degree of amino acid similarity and very similar crystal
structures (3-5).
The differences in complexity observed between Archaea and eukaryotes,
or between single-celled and higher eukaryotes, are coincident with
differences both in genomic complexity and in the complexity of the
gene expression machinery. Similarly, although the general role of TBP
in transcription initiation has been conserved, this function has
become more complex. In Archaea TBP appears to work as a single-subunit
entity (6); in eukaryotes TBP is at the core of obligate large
multiprotein complexes (3). The complex SL1 functions in initiation by
RNA pol I, TFIID is required for production of mRNA by RNA pol II,
TFIIIB is used during transcription of tRNAs and some other small RNAs
by RNA pol III. In mammals, TBP also interacts with SNAPc to
direct production of small nuclear RNAs (snRNAs) by either pol II or
pol III (3).
Attached to the TBPCORE, vertebrate species share a large
N-terminal domain that differs from TBP domains in all other phyla (3,
7-11). Previous studies have implicated the vertebrate N terminus in
general processes that are important for fundamental cellular
activities. For example, biochemical analyses have demonstrated a role
of the vertebrate N terminus in the function of the SNAPc complex (3,
12-14). Kinetic studies indicate that the N-terminal domain influences
TATA-binding and DNA-bending by TBP (15). Another study suggests that
the N terminus plays a role in determining rates of cell proliferation
(16).
Because these fundamental cellular functions are common to most
eukaryotes, including non-vertebrate metazoans, they evolved long
before the vertebrate TBP N
terminus.2 Therefore, we
hypothesized that this domain must have, in addition to any general
functions, a vertebrate-specific role. To test this, we created a line
of mice in which the wild type tbp allele was replaced with
a version, entitled tbp Even though the defects in homozygous mutant mice are manifested as a
highly specific and restricted phenotype, it is untested whether the
mutation actually compromises general cell functions in a sublethal
manner. For example, one might hypothesize that all cells bearing this
mutation would be physiologically compromised; however, only in the
placenta are such defects critical to the function of the organ and the
survival of the organism. To test this possibility, we analyzed
fundamental properties of cells bearing this mutation. We show that
tbp Mouse and MEF Production--
The targeting and mouse production
strategy for producing the TBP- RNase Protection, RT-PCR, and Primer Extension Assays--
RNA
harvests and RNase protection and primer extension assays were
preformed as described previously (18, 19). Probes for DHFR (18),
TFIIB, L7, GAPDH, Oct-1, and NF-Yb (19) have been described previously.
For TBP-related factor-2 (TRF-2), a probe hybridizing to sequences between positions 705 and 833 of the
mouse TRF-2 cDNA (20) was transcribed from a clone that we isolated
by RT-PCR on mouse testis
RNA.3 A probe that hybridized
to mouse U6 RNA sequences extending from the transcription initiation
site to a position 92 bases downstream of the initiation site (21) was
transcribed from a genomic clone that we isolated by PCR of mouse
genomic DNA.3 For
For RT-PCR reactions, 2.5 µg of CsCl-purified DNA-free RNA (19) was
used as template for oligo(dT)-primed reverse transcriptase reactions
as described previously (25). PCR with primers spanning from exon 2 to
exon 3 of the mouse Serial Analysis of Gene
Expression (SAGE)--
SAGE libraries were prepared as
described previously (26). Clones of individual plasmids containing
SAGE tag concatenates were screened by PCR and clones containing
inserts of >500 bp (roughly 30 SAGE tags) were arbitrarily
chosen for sequence analysis. Data were ordered by SAGE2000
software (www.sagenet.org). Data in the article represent all tags that
appeared mRNA and Protein Expression from the tbp
To investigate the molecular consequences of removing the N terminus of
TBP on basal cellular functions, we produced primary MEFs from g.d.
8.5-11.5 embryos. RNase protection assays using a probe that spanned
the site of the
Previously, a mammalian tbp-gene family member, named
TBP-like protein (TLP) (30) or
TRF-2 (31, 32) was identified. The TRF-2CORE is far less
closely related to the homologous region of mouse TBP (39% amino acid
identity) than mouse TBPCORE is to yeast
TBPCORE (81% identity; Fig.
2, Ref. 30). Different annotations of the
human genome suggest that this may be the only other tbp family member in mammals (33) or that there may be one other more
distantly related protein in the genome (34). We measured TRF-2
mRNA levels in adult mouse tissues and in MEFs of all three genotypes. Results showed that TRF-2 mRNA is expressed in wild-type MEFs (Fig. 2). TRF-2 was also expressed in all wild-type somatic mouse
tissues examined at levels about 4-fold higher than those of TBP
mRNA in each tissue and, consistent with previously published reports (32), TRF-2 mRNA was particularly enriched in testis. TRF-2
mRNA levels were unaffected by the TBP- Basal Properties of Cells Lacking the TBP N Terminus--
Because
we have shown that tbp
We first asked whether we could measure defects in
SNAPc-dependent processes. In mammals, SNAPc is involved in
transcription initiation on genes encoding snRNAs by pol III from
TATA-containing promoters, and by pol II from TATA-less promoters (3).
A classic example is its role in transcription of the human U2 gene by
RNA pol II, and the U6 gene by pol III (35). RNase protection assays showed that U6 RNA levels in MEFs of the three genotypes were indistinguishable (Fig. 3A).
Splicing requires an elaborate ribonucleoprotein system that, in
addition to U6 and U2, requires other snRNAs (36-39). As a potentially
more global assay for whether any defects in snRNA expression resulted
from the TBP-
In one study, chicken DT40 cells that were heterozygous-null for TBP
were shown to have lower proliferation rates than did the wild-type
progenitor cell line, which correlates to reduced levels of mRNA
and protein encoding cdc25B (16). Upon stably transfecting these cells
with vectors that express wild-type TBP, proliferation rates match the
progenitor cells. However, clones transfected with vectors that express
a mutant version of TBP lacking all except the first 24 amino acids of
the N terminus exhibit an intermediate proliferation rate (16). These
results suggest that the N terminus is required for full proliferative potential of the cells. Importantly, all of the cells in the study by
Um et al. (16) contained one intact copy of the
endogenous tbp gene including the entire N-terminal region,
and thus, the anti-proliferative effect was manifested as a
haploid-insufficiency.
We established MEF cultures from 139 embryos of heterozygous crosses,
but we were unable to detect any significant correlation between
genotypes and proliferation rates by either growth curves or thymidine
labeling (data not shown). Even in comparing homozygous mutant cells,
where both copies of tbp lacked the N terminus, and
homozygous wild-type cells, proliferation rates were indistinguishable. As a potentially more sensitive molecular assay of proliferation, levels of the mRNA encoding DHFR, which is transcribed only during S-phase of the cell cycle (18, 41, 42), were measured. MEFs of all
three genotypes exhibited similar levels of DHFR mRNA (Fig. 4A).
Previous studies have shown that cdc25B mRNA is expressed only
during G2 phase of the cell cycle (43, 44) and that
expression of this mRNA may be particularly sensitive to mutations
in the tbp gene (16). Therefore, as another molecular marker
of proliferation rates, we compared relative cdc25B mRNA levels in
tbp+/+,
tbp Transcription and RNA Processing in
tbp
Although TBP, TRF-2, U6, TBP is required by all eukaryotes for gene expression as well as
for production of ribosomal RNAs and small RNAs. Consistent with this,
the core of this protein is one of the most highly conserved proteins
known. However, outside of this core, on the N-terminal side of the
protein, different phylogenetic lineages exhibit vastly different
embellishments (3). To test the function of the vertebrate-specific TBP
N terminus, we developed a line of mice in which these sequences had
been largely deleted (11). In this article, we show that this mutation
had no measurable effects on basal functions in cultured primary fibroblasts.
Previous biochemical and cellular studies on the function of the
vertebrate-specific TBP N terminus have shown that this polypeptide domain has strong effects on DNA binding by TBP (15), on how TBP
interacts with SNAPc and with certain transcription factors (13, 14,
48-50), and on the function of TBP in transcribing genes encoding RNA
components of the splicing machinery (14, 50). In addition, one study
has shown that the TBP N terminus can affect rates of cell
proliferation (16). By contrast, we show that MEFs lacking most of the
TBP N terminus show no general defects in transcription, splicing, gene
expression, or proliferation. Results presented here, indicating that
removing most of the TBP N terminus has no basal physiological effects
on fibroblasts, are corroborated by the observation that, even though
few tbp One interesting property of living systems is that they are robust. In
other words, they generally tolerate or rebound from perturbations of
the system. Genetic studies and modeling experiments indicate that
robustness can be accomplished by having parallel pathways that can
independently accomplish the same task (51, 52). Conversely, fragments
of biological systems, such as purified in vitro reactions,
are generally optimized for a single reaction, and are relatively
intolerant of perturbations. One might consider the possibility that
the N terminus of TBP functions in a robust system. As examples,
transcription factor Oct-1 may stabilize SNAPc on snRNA promoters (50)
in the absence of the TBP N terminus, and the ability of the N terminus
to affect DNA binding by the TBPCORE (15) may be partially
redundant with activities of other proteins assembling at promoters.
Elimination of the N terminus may be compensated for by the other
back-up proteins. One might envisage a model where the transcriptional
assembly at a promoter forms a three-dimensional jigsaw puzzle that
does not necessarily fall apart or lose activity as a result of
removing this single piece. By such a model, cells lacking the TBP N
terminus may be physiologically normal, but might also be more prone to
failure in the event that the back-up system fails.
The previous study showing a role for the TBP N terminus in cell
proliferation (16) is harder to reconcile with our data because that
study, like ours, was performed in living cells. Differences in
experimental design are likely responsible for the different results.
For example, whereas both studies required several generations of
in vitro culture to generate a line of cells bearing the
respective targeted mutations, cells in our study were rejuvenated by
turning them into viable mice, breeding the mutant animals, and then
harvesting our experimental cells as primary cultures from the
resultant fetuses. Moreover, all modified tbp alleles in our
study were within the normal tbp locus, and none of our
cells contained selectable marker genes or vector sequences that might
have affected mutant cells (11). Conversely, event though the
tbp-null allele in the study by Um et al. (16) was targeted into the tbp locus, it retained an expressed
hygromycin resistance gene, and the rescuing expression cassettes were
random insertions that brought with them vector sequences, including an
expressed puromycin resistance gene. Our data strongly suggest that the
N terminus of TBP can be deleted without measurably altering cell proliferation.
Vertebrate and non-vertebrate metazoans universally share the basal
properties that have been attributed to the vertebrate TBP N terminus
in previous studies (see above). Thus, if this domain indeed
participates in such fundamental processes in vivo, they are
likely accomplished equally well by the N termini of nematodes,
dipterans, lepidopterans, and echinoderms, even though the N terminus
in each group shows neither similarity to the vertebrate N terminus nor
to each other (NCBI/GenBankTM). The natural diversity in
TBP N-terminal sequences between metazoan clades suggests that, in
terms of accomplishing these general functions, this domain should
exhibit large amino acid sequence plasticity. Conversely, we have found
that natural selection has been particularly intolerant of mutations
that would modify the amino acid sequence of TBP N terminus in
vertebrates.2 Thus, we suspected that, whether or not this
domain participates in any fundamental cellular functions, the
vertebrate TBP N terminus must have a truly vertebrate-specific
activity, and it is this function that has been so highly conserved by
natural selection in vertebrates. In designing the TBP- In conclusion, we report that removing the TBP N terminus is
inconsequential for general physiological properties of mouse fibroblasts. As such, it is unlikely that this mutation, alone, compromises any vital basal cellular functions in vivo,
leading us to dismiss the possibility that the mutation results in
sublethally sick cells. Rather, we posit that all activities of this
polypeptide domain likely fall within two classes. Thus, all TBP N
terminus-dependent functions must be either functionally
redundant components of robust systems or they will be functionally
restricted activities that are not manifested in primary embryonic
fibroblast cultures.
N, which lacks 111 of 135 N-terminal amino acid residues. Most tbp
N/
N fetuses die in midgestation. To
test whether a disruption of general cellular processes contributed to
this fetal loss, primary fibroblast cultures were established from +/+,
N/+, and
N/
N fetuses. The cultures exhibited no
genotype-dependent differences in proliferation or in
expression of the proliferative markers dihydrofolate reductase (DHFR)
mRNA (S phase-specific) and cdc25B mRNA
(G2-specific). The mutation had no effect on transcription initiation site fidelity by either RNA polymerase II (pol II) or pol
III. Moreover, the mutation did not cause differences in levels of U6
RNA, a pol III-dependent component of the splicing machinery, in mRNA splicing efficiency, in expression of
housekeeping genes from either TATA-containing or TATA-less promoters,
or in global gene expression. Our results indicated that general
eukaryotic cell functions are unaffected by deletion of these
vertebrate-specific sequences from TBP. Thus, all activities of
this polypeptide domain must either be compensated for by redundant
activities or be restricted to situations that are not represented by
primary fibroblasts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N, in which 111 of the
135 vertebrate-specific N-terminal amino acids were deleted (11). At
gestational day (g.d.) 9.5, tbp
N/
N fetuses are generally
normal (11). Over 90% of these mutant fetuses die between g.d. 10.5 and 12.5; loss results from a failure of their placenta to evade a
maternal immune rejection response (11). Importantly, although less
than 3% of the tbp
N/
N animals
survive to weaning, these survivors are healthy and fertile (11).
N/+ and
tbp
N/
N primary mouse embryo
fibroblasts (MEFs) are normal in all measured general functions. Our
data indicate that, if the TBP N terminus truly participates in any
fundamental functions in vivo, these processes must be
functionally redundant.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N mice has been reported elsewhere
(11). The selectable neo cassette used for gene targeting
was removed from the line by Cre/loxP, and for all samples
in this article, the mutation had been back-crossed onto a C57Bl/6
background for
5 generations (11). For establishing primary
fibroblast cell lines, whole uteri were harvested from females with
timed pregnancies and were surface-sterilized with 70% ethanol.
Deciduae were opened and embryonic surface tissue was harvested and
dissociated with trypsin. Explanted cells were cultured in Dulbecco's
modified Eagle's medium + 10% newborn calf serum + 1×
penicillin/streptomycin/fungizone (BioWhittaker). Cultures were
trypsinized and passed to fresh dishes when confluent, after 5-14
days. By this time under these conditions, fibroblasts had outgrown
other surviving cell types to the point that only cells with
fibroblastic morphology could be readily found under the microscope.
These cultures were split in experiments.
-actin, mouse genomic sequence
was not available. A mouse genomic fragment,3 extending
from a position in exon 2 corresponding to base 89 of the mouse
cDNA (22) to a position 51 base pairs into intron 2, was amplified
from mouse genomic DNA using one primer designed from mouse exon 2, and
a second primer designed from the genomic sequence of rat intron 2 (23), and this fragment was cloned into a plasmid vector. The
-actin
probe hybridizes to 146 bases of the mouse pre-mRNA and 95 bases of
the mouse mRNA (Fig. 3B). For TBP, a probe that was
complementary to sequences extending from the BglII site in
exon 2 (position 37 of the original published cDNA sequence) to
position 168 in exon 3 (24) was transcribed from a subclone of the
mouse TBP cDNA. This probe hybridized to 131 bases of wild-type TBP
mRNA, and 81 bases of TBP-
N mRNA (Fig. 1B). For
primer extensions, the following primers were used: U6 primer, 5'-ATC
GAA TTC ACG AAT TTG CTG GTC ATC C-3';
-actin primer, 5'-TGG GGT ACT
TCA GGG TCA GGA TAC-3'.
-actin gene confirmed that all cDNAs had
uniform levels of
-actin cDNA and lacked detectable genomic DNA
(data not shown). PCR was performed using 0.2% of each RT reaction per
PCR reaction, as described in the figure legends, using mouse
cdc25B-specific primers having the following sequences: 5' primer,
5'-ATT CCA GCT CTG CCC AAG CTT TGG C-3'; 3' primer, 5'-TCC ACA AAT CCG
TCA TCT TCT TCA-3'. The PCR product spans from positions 626 to 821 of
the mouse cdc25B mRNA (GenBankTM XM_123867; translation
initiation is at position 607), which contains a centrally located
splice junction for a 621-base pair intron at position 712 (GenBankTM NW_000178). Thus, genomic DNA gives a 816-base
pair band that is clearly discernable from the 195-base pair cDNA
signal (data not shown).
10 times in the combined libraries and represent all 46 genes present at an average of
1.5 parts per thousand. Summation of
tag frequencies indicated that 77% of all tags sequenced were
represented by these 46. The identities of mRNAs represented by
individual tags was determined using the tag-to-gene mapper
program on the NCBI SAGEmap homepage (www.ncbi.nlm.nih.gov/SAGE/SAGEtag.cgi).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N
Allele--
The tbp
N allele retains all
known transcriptional and post-transcriptional regulatory signals of
the wild-type tbp allele (25, 27, 28), but it produces a
version of TBP protein lacking 111 of 135 vertebrate-specific
N-terminal amino acids (Fig.
1A, described in Ref. 11).
Retention of the first 24 amino acids was predicted to preserve the
in vivo stability of the mutant protein (11, 29). Thus, the
allele is identical to the native tbp allele except that it
encodes an epitope tag in place of amino acids 25-135 of the protein,
and it contains a loxP-containing oligonucleotide insertion
roughly 2.5-kb upstream of the gene (11).
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Fig. 1.
Primary structure and expression of TBP and
TBP- N in MEFs. A, alignment of
wild-type TBP and TBP-
N, showing the universal C terminus
(black box) and the vertebrate-specific N terminus
(white box). The mutant version retains amino acids 1-24
and 136-316 of the wild-type allele, and contains two copies of the
9-amino acid FLAG (Kodak) epitope tag (gray box labeled
E) (11). B, expression of TBP mRNA in MEFs.
Total RNA (10 µg) from two cultures each of
tbp+/+,
tbp
N/+, and
tbp
N/
N MEFs was analyzed by
RNase protection using a probe that differentiates between wild-type
and mutant TBP mRNA. All samples were supplemented to contain a
total of 50 µg of RNA with yeast RNA. MEF genotypes are indicated at
top. At right is indicated the positions of
undigested probe and of the protected fragments for wild-type
(TBP+) and mutant (TBP
N) mRNA.
Abbreviations: M, molecular size markers; P,
1:100 dilution of undigested probe; C, control containing
probe hybridized to 50 µg of yeast RNA. C, Western blot of
TBP and TBP-
N protein expression in MEFs. Whole cells of the
indicated genotypes were harvested by scraping in 1% SDS, and lysates
were sonicated and boiled. Nucleic acid content was estimated by
reading the A260 on each lysate, and 20 µg of
nucleic acid equivalents was boiled with an equal volume of Laemmli
buffer for each determination (17). Western blots were probed with
commercial antibody against the C-terminal domain of human TBP (BD
PharMingen), which shows 100% amino acid identity with this region of
mouse TBP and was visualized by ECL (Amersham Biosciences).
N mutation were used to compare levels of wild-type
and mutant TBP mRNA accumulation in tbp+/+,
tbp
N/+, and
tbp
N/DN MEFs. Results showed that
homozygous wild-type and homozygous mutant cells had similar levels of
TBP or TBP-
N mRNA, respectively, and, in heterozygous cells,
mRNA from each allele was equally represented (Fig. 1B).
Western blots using either whole cell (Fig. 1C) or nuclear
(data not shown) extracts indicated that MEFs of all three genotypes
contained similar levels of total and nuclear TBP and TBP-
N protein;
heterozygous MEFs exhibited similar amounts of both TBP and TBP-
N
protein. Based on mRNA level, protein level, and subcellular
distribution, TBP and TBP-
N proteins had no significant differences
in synthesis/turnover rates or nuclear localization.
N mutation in both the
MEFs and in adult tissues (Fig. 2). Because TRF-2 contains only a short
N-terminal region fused to the core (30), we consider it highly
unlikely that TRF-2 compensated for deletion of the N-terminal region
of TBP in tbp
N/
N fibroblasts or
mice.
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Fig. 2.
Mouse TRF-2 expression in MEFs. At
top is diagramed the primary structure and the C-terminal
(shaded regions) amino acid conservation between yeast TBP
(yTBP), mouse TBP (mTBP), and mTRF-2. Below is an RNase
protection assay on dilutions of total RNA from wild-type mouse testis
(10, 3.0, 1.0, 0.3, 0.1, and 0.03 µg from left to
right), and on 20 µg each of total RNA from wild-type
mouse kidney and from MEFs of the indicated genotypes. All samples were
supplemented to 50 µg with yeast RNA. The TRF-2-specific RNase
protection fragment is indicated at the right. Abbreviations
as in the legend to Fig. 1.
N/
N
mutant animals occasionally survive to become healthy fertile adults
(11), it is unlikely that large global defects result from the
mutation. However, previous studies had implicated this domain in basal
processes (see the Introduction). These studies led us to ponder
whether, rather than only functioning in a particular situation
(pregnancy), the N terminus functioned ubiquitously, but defects
resulting from its deletion were sublethal in most cell types.
Therefore, we tested whether defects in basal functions could be
detected in cells bearing the TBP-
N mutation.
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Fig. 3.
Expression of U6 RNA and splicing efficiency
in MEFs. A, U6 RNA expression. 5 µg of total RNA from
MEFs of the indicated genotypes was supplemented to 50 µg with yeast
RNA and assayed by RNase protection. The positions of undigested probe
and U6 RNA are indicated at left. B, -actin
pre-mRNA splicing efficiency. At top is diagrammed the
single-probe internally controlled RNase protection strategy for
differentiating prespliced and spliced
-actin RNA. 5 µg each of
cytoplasmic (Cy) and nuclear (Nu) RNA was
supplemented to 50 µg with yeast RNA as above. The positions of
undigested probe, unspliced pre-mRNA and spliced mRNA are
indicated at the left. Abbreviations as in the legend to
Fig. 1.
N mutation, we examined the splicing efficiency of
pre-mRNA encoding
-actin by RNase protection (Fig.
3B). Levels of correctly spliced cytoplasmic mRNA
(95-base band) were unaffected by the mutation, and there was no
evidence for increased nuclear accumulation of unspliced pre-mRNA
(146-base band in lanes designated Nu). This indicated that
splicing efficiency was similar between MEFs of the three genotypes. We
concluded that deletion of TBP amino acids 25-135 was inconsequential
for U6 RNA expression and mRNA splicing in the MEFs. Previous
studies have shown that the first 55 amino acids of human TBP are
sufficient for SNAPc functions in vitro, including U6
transcription (40). These data, in combination with our results,
might suggest that SNAPc-dependent functions of the TBP N
terminus reside entirely in the first 24 amino acids of the protein.
Alternatively, it is possible that other compensatory interactions at
the snRNA promoters stabilize SNAPc interactions in cells lacking the
TBP N terminus (see "Discussion").
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Fig. 4.
Expression of proliferative cell markers in
MEFs and in tissues. A, DHFR mRNA expression. 30 µg of total RNA from MEFs of the indicated genotypes was supplemented
with yeast RNA to 50 µg and assayed by RNase protection using the
DHFR-specific probe. B, cdc25B mRNA expression. Total
RNA from four independent cultures each of wild-type
(tbp+/+), heterozygous
(tbp N/+), and homozygous mutant
(tbp
N/
N) MEFs, two individual
g.d. 10.5 whole fetuses of each genotype, and one g.d. 10.5 whole
placenta of each genotype (indicated at left; 21 independent
tissue/cell samples shown) was assayed by RT-PCR. Reactions were
terminated at the indicated PCR cycles (top) to generate an
expression curve for each, showing that the reactions were not
saturated. The PCR product spans a 621-base pair intron. Only the
195-base pair cDNA-derived sequence, not the 816-base genomic
product, was detected, which confirmed that samples were not
contaminated by genomic DNA. Control reactions using primers that
spanned from exon 2 to exon 3 of mouse
-actin showed equivalent
signals from all samples (not shown).
N/+, and
tbp
N/
N MEFs, as well as in g.d.
10.5 fetuses and placentas of all three genotypes. Results showed that
cdc25B mRNA levels were similar between MEFs of all three
genotypes, between fetuses of all three genotypes, and between
placentas of all three genotypes (Fig. 4B). The
genotype-independent reduced level of cdc25B mRNA in placentas as
compared with those in MEFs and fetuses likely represents a lower
overall rate of proliferation in this organ. These data suggest that
the TBP N terminus does not play a significant role, either as a
dominant (i.e. haploid-insufficient) or recessive trait, in
determining proliferation rates in MEFs.
N/
N and
tbp
N/+ Cells--
Because our study
examines a targeted mutation in a fundamental component of the basal
transcription machinery in vivo, we examined whether the
mutation had measurable effects on initiation site fidelity or RNA
accumulation. Transcription initiation site fidelity was assessed by
primer extension assays on MEFs of all three genotypes. Results
indicated that our mutation had no quantitative or qualitative effects
on initiation site fidelity of RNAs transcribed by either pol II
(
-actin mRNA) or by pol III (U6 RNA)(Fig.
5).
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Fig. 5.
Transcription initiation site fidelity in
MEFs. Poly(A+)-selected mRNA (panel A)
or total RNA (panel B) was analyzed by primer-extension
assays using either the -actin primer (the
-actin gene is
transcribed by pol II; panel A) or the U6 primer (the
u6 gene is transcribed by pol III; panel B).
Positions of free primers and primer extension products are indicated
at the left of each panel.
-actin, DHFR, and cdc25B mRNA levels
were unaffected by the
N mutation (Figs. 1-5), we wished to determine whether there were effects on a broader range of mRNAs. Two additional highly abundant housekeeping mRNAs from genes with TATA-containing promoters, encoding ribosomal protein L7 and GAPDH, and
three low abundance housekeeping mRNAs from TATA-less promoters, encoding TFIIB, Oct-1, and NF-Yb, were chosen for individual analysis (45-47). Expression of all of these mRNAs were unaffected by the TBP mutation (Fig. 6A). As an
unbiased representation of global gene expression, we used SAGE to
estimate the relative abundance of all mRNAs in a sample based on
the frequency that unique 3'-end sequence tags are present in a
cDNA library made from that mRNA sample (26). SAGE libraries
were created from +/+ and
N/
N MEFs and we sequenced 6890 tags,
giving resolution of all mRNAs present at
1.5 parts per thousand
in each sample (77% of all of the mRNA in the cells; Fig.
6B). The SAGE data were externally corroborated because two
of the tags, nos. 21 and 22, represented mRNAs that we had
independently assayed by quantitative RNase protection and primer
extension assays (
-actin, Fig. 3B, and GAPDH, Fig.
6A, respectively). None of the abundant tags showed a strong
effect of the mutation (
10-fold difference between cell types,
dark shaded zones in Fig. 6B) and only three
differed by 3-fold or more (light shaded zones). These three
mRNAs may have been differentially regulated as a direct
consequence of our mutation; however, they may also have simply
represented stochastic differences between the cell lines that were
independent of the TBP mutation. Further study will be required to
distinguish these possibilities. Importantly, the data indicated that
overall gene expression was not different in the two cell lines. We
conclude that the TBP-
N mutation does not affect basal gene
expression, and we infer that any gene expression defects arising from
this mutation must be highly cell type- and/or gene-specific.
View larger version (40K):
[in a new window]
Fig. 6.
Global gene expression. A,
expression of selected housekeeping genes. RNase protection
assays for the mRNAs indicated at the right were
performed on total RNA from MEFs of the indicated genotypes as
described in previous figures. B, expression of abundant
mRNAs. SAGE was performed on libraries from +/+ and N/
N MEFs
at a resolution of 1.5 parts per 1000. Data are presented for the most
abundant 46 tags. The top panel shows the relative
expression profiles presented as fold-difference between the two cell
lines, where up-regulation (positive values) are tags that are more
abundant in the mutant than in wild-type MEFs, and down-regulation
(negative values) are less abundant in the mutants. The bottom
panel shows the abundance of each tag in both libraries combined,
presented as parts per thousand. After exclusion of redundant di-tags,
summation of all iterations of the 46 most abundant tags indicates
that, of the 6890 tags in this analysis, 5872 (77%) were one of these
46. Tag frequencies for representative mRNAs, indicated by their
numerical designation on the bar graphs, are as follows: tag
5, ribosomal S28 subunit; tag 7, ribosomal L23 subunit; tag 11, ferritin light chain mRNA; tag 13, ribosomal L36 subunit; tag 15, EST Mm.108736; tag 21,
-actin; tag 22, GAPDH; tag 24, EST Mm.154997;
tag 28, ribosomal L28a subunit; tag 30, eTEF1
; tag 32, HMG box-2
protein mRNA; tag 34, EST Mm.194288; tag 45, cytochrome
c.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N/
N mice survive to
weaning, survivors are healthy and fertile (11). Moreover,
physiological processes that depend on proliferation are unaffected in
survivors. For example, homozygous mutant fetuses grow and develop at
normal rates in immune-compromised mothers (11), and homozygous mutant
adults are normal size, they exhibit normal peripheral blood leukocyte
counts, normal hair growth, and normal male fertility even after over a
year of continuous mating (data not shown). What might be the reasons
that our in vivo results on TBP-
N mutant mice do not show
defects in any of the processes in which this domain has been
implicated by in vitro studies?
N mutation,
we hypothesized that this function would be involved in a specific gene
regulation process. Consistent with this, we have found that mice
homozygous for this mutation suffer from a highly specific defect that
compromises a
2-microglobulin-dependent component of the
interaction between the placenta of the developing fetus and the
mother's immune system (11).
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ACKNOWLEDGEMENTS |
---|
We thank K. Thomas, C. Lenz, D. Taylor, J. Prigge, T. Frerck, N. Hobbs, L. Eng, K. Lustig, N. Meisner, T. Larson, D. Schwartzenberger, T. Tucker, A. Sealey, J. Kundert, and the members of the Schmidt and Capecchi laboratories for suggestions and contributions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the March of Dimes Foundation, National Institutes of Health, National Science Foundation, and USDA Animal Health Funds (to E. E. S.) and by awards from the Howard Hughes Medical Institute and the Mathers Charitable Foundation (to M. R. C.).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.
§ Funded as a Basil O'Connor New Investigator of the March of Dimes Foundation, a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation, and as an Investigator of the Montana Agricultural Experiment Station. To whom correspondence should be addressed. E-mail: eschmidt@montana.edu.
¶ Supported by a postdoctoral fellowship from the USDA and by an appointment in the laboratory of M. W. White.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211205200
2 A. A. Bondareva and E. E. Schmidt, submitted for publication.
3 E. E. Schmidt, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: pol, polymerase; TBP, TATA-binding protein; MEF, mouse embryo fibroblast; RT, reverse transcriptase; SAGE, serial analysis of gene expression; EST, expressed sequence tag; g.d., gestational day; DHFR, dihydrofolate reductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRF, TBP-related factor.
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