Fundamental Cellular Processes Do Not Require Vertebrate-specific Sequences within the TATA-binding Protein*

Edward E. SchmidtDagger §, Alla A. BondarevaDagger , Jay R. RadkeDagger , and Mario R. Capecchi||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, tbpDelta N, which lacks 111 of 135 N-terminal amino acid residues. Most tbpDelta N/Delta 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 +/+, Delta N/+, and Delta N/Delta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 tbpDelta N, in which 111 of the 135 vertebrate-specific N-terminal amino acids were deleted (11). At gestational day (g.d.) 9.5, tbpDelta N/Delta 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 tbpDelta N/Delta N animals survive to weaning, these survivors are healthy and fertile (11).

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 tbpDelta N/+ and tbpDelta N/Delta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse and MEF Production-- The targeting and mouse production strategy for producing the TBP-Delta 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.

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 beta -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 beta -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-Delta 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'; beta -actin primer, 5'-TGG GGT ACT TCA GGG TCA GGA TAC-3'.

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 beta -actin gene confirmed that all cDNAs had uniform levels of beta -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).

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 >= 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

mRNA and Protein Expression from the tbpDelta N Allele-- The tbpDelta 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-Delta N in MEFs. A, alignment of wild-type TBP and TBP-Delta 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+/+, tbpDelta N/+, and tbpDelta N/Delta 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 (TBPDelta 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-Delta 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).

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 Delta N mutation were used to compare levels of wild-type and mutant TBP mRNA accumulation in tbp+/+, tbpDelta N/+, and tbpDelta N/DN MEFs. Results showed that homozygous wild-type and homozygous mutant cells had similar levels of TBP or TBP-Delta 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-Delta N protein; heterozygous MEFs exhibited similar amounts of both TBP and TBP-Delta N protein. Based on mRNA level, protein level, and subcellular distribution, TBP and TBP-Delta N proteins had no significant differences in synthesis/turnover rates or nuclear localization.

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-Delta 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 tbpDelta N/Delta 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.

Basal Properties of Cells Lacking the TBP N Terminus-- Because we have shown that tbpDelta N/Delta 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-Delta N mutation.

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).


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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, beta -actin pre-mRNA splicing efficiency. At top is diagrammed the single-probe internally controlled RNase protection strategy for differentiating prespliced and spliced beta -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.

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-Delta N mutation, we examined the splicing efficiency of pre-mRNA encoding beta -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").

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).


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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 (tbpDelta N/+), and homozygous mutant (tbpDelta N/Delta 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 beta -actin showed equivalent signals from all samples (not shown).

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+/+, tbpDelta N/+, and tbpDelta N/Delta 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.

Transcription and RNA Processing in tbpDelta N/Delta N and tbpDelta 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 (beta -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 beta -actin primer (the beta -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.

Although TBP, TRF-2, U6, beta -actin, DHFR, and cdc25B mRNA levels were unaffected by the Delta 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 Delta N/Delta 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 (beta -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-Delta 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.


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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 Delta N/Delta 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, beta -actin; tag 22, GAPDH; tag 24, EST Mm.154997; tag 28, ribosomal L28a subunit; tag 30, eTEF1alpha ; 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

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 tbpDelta N/Delta 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-Delta N mutant mice do not show defects in any of the processes in which this domain has been implicated by in vitro studies?

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-Delta 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 beta 2-microglobulin-dependent component of the interaction between the placenta of the developing fetus and the mother's immune system (11).

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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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