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Chromosomal position, structure, expression, and requirement of genes for chicken transcription factor IIA
Gene 397 (2007) 94 – 100 www.elsevier.com/locate/gene
Chromosomal position, structure, expression, and requirement of genes for chicken transcription factor IIA ☆ Tomoko Mabuchi a , Toshifumi Wakamatsu a,1 , Tomoyoshi Nakadai a,2 , Miho Shimada a,2 , Kazuhiko Yamada b , Yoichi Matsuda b , Taka-aki Tamura a,⁎ a
Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan b Creative Research Initiative, Hokkaido University, Sapporo 060-0810, Japan Received 21 September 2006; received in revised form 14 March 2007; accepted 13 April 2007 Available online 21 April 2007
Abstract Transcription factor IIA (TFIIA) is one of the general transcription factors for RNA polymerase II and composed of three subunits, TFIIAα, TFIIAβ and TFIIAγ. TFIIAα and TFIIAβ are encoded by a single gene (TFIIAαβ) and mature through internal cleavage of TFIIAαβ. In this study, we found that structures of TFIIAαβ and TFIIAγ are highly homologous with each mammalian counterpart. Exon–intron organizations of the human and chicken TFIIA genes were also homologous. The sequence of the cleavage region of the chicken TFIIAαβ precursor protein was fitted to the consensus cleavage recognition site. It was thus demonstrated that TFIIA is conserved in vertebrates. TFIIA proteins are present ubiquitously in chicken tissues. Fluorescent in situ hybridization revealed that TFIIAαβ and TFIIAγ genes are located in chromosome 5 and a mini-chromosome, respectively. We generated semi-knockout chicken DT40 cells for TFIIAαβ and TFIIAγ genes with high homologous recombination efficiencies, whereas we failed to establish double-knockout cells for each gene. It is thought that both genes for TFIIA are required in vertebrates. TFIIA siRNA resulted in deceleration of cell growth rate, suggesting that, consistent with those of knockout assays, TFIIA is associated with cell growth regulation. © 2007 Elsevier B.V. All rights reserved. Keywords: TFIIA; Transcription factor; DT40; Gene disruption; General transcription factor
1. Introduction In transcriptional regulation of RNA polymerase II-dependent genes, various kinds of general transcription factors (GTFs), including TFIIA (transcription factor IIA), TFIIB, Abbreviations: GTF, general transcription factor; TFIIA, transcription factor IIA; TBP, TATA-binding protein; CRS, cleavage recognition site; AA, amino acid; FISH, fluorescence in situ hybridization; RNAi, RNA interference; siRNA, small interference RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ☆ GenBank accession numbers. chicken TFIIAab: AB264607, chicken TFIIAg: AB264606. ⁎ Corresponding author. Fax: +81 43 290 2824. E-mail address: [email protected] (T. Tamura). 1 Present address: Institute of Medical Molecular Design Incorporated, 5-24-5 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2 Present address: Department of Molecular Biology, Saitama Medical School, Moroyama, Saitama 350-0495, Japan. 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.04.015
TFIID, TFIIE, TFIIF and TFIIH are required for correct and efficient transcription in addition to RNA polymerase II. TFIID is composed of TBP and multiple TBP-associated factors. RNA polymerase II and GTFs assemble on a promoter for initiation of transcription. At the first step in this pre-initiation complex formation, TBP (TATA-binding protein) or TFIID binds to the promoter sequence. TFIIA binds to TBP and is incorporated into the TBP-DNA complex. TFIIA works to stabilize the complex (Weideman et al., 1997; Ranish et al., 1999). TFIIA has additional functions in transcriptional regulation. For example, TFIIA dissociates a TBP dimer and inhibits dimer formation of TBP (Taggart and Pugh, 1996; Coleman et al., 1999). TFIIA suppresses repressor proteins that interfere with DNA binding of TBP/TFIID (Inostroza et al., 1992; Merino et al., 1993; Ge and Roeder, 1994; Auble et al., 1994; Kirov et al., 1996; Ozer et al., 1998b). TFIIA also functions as a coactivator for sequence-specific transcription factors (Ozer et al.,
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1994; Sun et al., 1994; Kang et al., 1995; Kobayashi et al., 1995; Chi and Carey, 1996). TFIIA that has several functions, is thus considered to be an activator for the formation of the preinitiation complex. Requirement of TFIIA in transcription regulation depends on the structure of a core promoter and existence of a regulatory element (Ma et al., 1996; Solow et al., 1999). In a reconstituted in vitro transcription system composed of highly purified GTFs including TBP but not TFIID, TFIIA does not exhibit its transcription activation function (Sun et al., 1994; Ma et al., 1993). Moreover, it is thought that TFIIA is dispensable for some activator-dependent transcriptional stimulation (Ozer et al., 1994; Sun et al., 1994; Ma et al., 1996; Ranish et al., 1999). Accordingly, TFIIA is not always a necessary factor, at least in vitro, although its in vivo function has not been fully elucidated. Human TFIIA consists of three subunits: TFIIA α (34 kDa), TFIIA β (19 kDa) (Ma et al., 1993; DeJong and Roeder, 1993), and TFIIA γ (12 kDa) (Ozer et al., 1994; DeJong et al., 1995). TFIIAα and TFIIAβ are encoded by a single gene (TFIIAαβ) and generated from the TFIIAαβ precursor via intramolecule cleavage. Amino acid sequence of QVDG located just upstream from a junction of the cleavage site is conserved in higher eukaryotes and called cleavage recognition site (CRS) (Hoiby et al., 2004). Processing of the TFIIAαβ precursor occurs between glycine and aspartic acid just two amino acids downstream from the CRS (Hoiby et al., 2004). Unprocessed TFIIAαβ can functionally substitute with mature TFIIAα and TFIIAβ in an in vitro transcription system (DeJong et al., 1995). Since the mature TFIIAβ subunit is sensitive to proteasome-dependent degradation (Hoiby et al., 2004), processing of TFIIAαβ may be involved in regulation of the level of TFIIA. In S. cerevisiae, TFIIA is composed of two subunits, TOA1 and TOA2 (Ranish et al., 1992), which correspond to human TFIIAαβ and TFIIAγ, respectively. The N-terminal and Cterminal halves of TOA1 are analogous to those of mammalian TFIIAα and TFIIAβ, respectively (DeJong and Roeder, 1993). TOA2 is an ortholog of mammalian TFIIAγ (DeJong et al., 1995). TOA1 and TOA2 are essential for yeast cell growth (Ranish et al., 1992; Kang et al., 1995; Ozer et al., 1998a), though TFIIA does not play a critical role but plays an accessory role in transcriptional regulation as described above. There has been no investigation so far of the requirement of TFIIA for growth of vertebrate cells. In this study, we determined structures of the chicken TFIIA and their chromosomal loci. We disrupted a single allele of TFIIAαβ and TFIIAγ genes. However, we were unable to establish a double-knockout cell for both genes. Knock-down of TFIIA resulted in decreased cell growth rate. The present study suggests that TFIIA is involved in growth of vertebrate cells. 2. Materials and methods 2.1. Cell culture and transfection DT40 cells derived from chicken B lymphoma were cultured at 39.5 °C in RPMI1640 medium supplemented with 10% fetal
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calf serum and 1% chicken serum (JRH Bioscience). Electroporation for DNA transfection of 107 cells was carried out at 550 V and 25 μF using Gene Pulser II (Bio-Rad). The transfected cells were maintained in 10 ml medium as previously described (Lahti, 1999). NIH3T3 cells were maintained in a low-glucose DMEM with 10% fetal calf serum. 2.2. Protein extraction and Western blotting Chicken tissues were homogenized in an extraction buffer (20 mM Tris–HCl [pH 7.4], 0.1% SDS, and 1% Triton X-100) supplemented with a protein inhibitor mixture composed of 1 mM benzamidine-HCl, 1 μg/ml pepstatin A, 1 mM PMSF, and 1 μg/ml leupeptin. Ten million cells were lysed in buffer C (50 mM HEPES-KOH [pH 7.8], 0.42 M KCl, 0.1 mM EDTA, 5 mM MgCl2, and 20% glycerol) supplemented with the protein inhibitor mixture. Following a brief centrifugation, supernatant fractions were collected as a whole cell extract. Protein concentration was determined with a BCA protein assay kit (Pierce). Cellular proteins in the SDS sampling buffer were separated by a 15% SDS-PAGE and transferred onto a PVDF membrane (Millipore) by electroblotting. TFIIA polypeptides were detected by Western blotting with rabbit polyclonal antibodies against human TFIIAαβ and TFIIAγ and ECL Plus (GE Healthcare Bioscience). Antibodies against β-actin (Sigma), α-tubulin, and GAPDH (glyceraldehyde-3phosphate dehydrogenase) (Ambion) were commercial products. Beta-actin was detected by an alkaline phosphatase method, whereas others were detected by ECL Plus. 2.3. Antibodies against TFIIA The open reading frame of human TFIIAαβ and TFIIAγ was connected to an oligo-histidine (His) 6-tag at the N-terminus and inserted into pET16b vector (Nakadai et al., 2004). Recombinant protein was expressed in BL21(DE3) pLysS strain of E. coli as previously described (Nakadai et al., 2004). Bacterially expressed proteins were dissolved with a lysis buffer containing 6 M guanidine-HCl (Ma et al., 1993). The lysate was centrifuged at 42 krpm for 1 h, and recombinant protein in the supernatant fraction was purified with Ni-NTA agarose (Qiagen) as described previously (Nakadai et al., 1999). Rabbits were immunized with SDS-PAGE-purified proteins, and polyclonal antibodies were generated. Antibodies were affinity-purified from sera by using recombinant TFIIA-immobilized NHS-activated Sepharose beads (Hitrap; GE Healthcare Bioscience). 2.4. Disruption of chicken TFIIA genes The fifth exon of TFIIAαβ and the second exon of TFIIAγ in DT40 cell were disrupted by homologous recombination with a drug-resistant gene-carrying targeting vector. For the first allele disruption, a histidinol-resistant gene was used as a selection marker. Cells were transfected with 25 μg of the linearized plasmid. Histidinol-resistant cells were selected in a culture medium with 1 mg/ml histidinol (Shimada et al., 2003). After resistant cells had been obtained, one representative
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heterozygous knockout cell was subjected to a secondary disruption procedure using a puromycin-resistant gene-carrying targeting vector and 0.5 μg/ml of puromycin (Lahti, 1999). 2.5. Southern blot analysis DT40 cells in an extraction buffer (10 mM Tris–HCl [pH 8.0], 50 mM EDTA, 0.5% SDS, 0.1 M NaCl, 50 μg/ml RNase A, and 100 μg/ml proteinase K) were incubated at 55 °C for 1 h. The lysate was dialyzed against TE buffer after phenol treatment. DNA (20 μg) was digested with restriction enzymes. KpnI plus ApaI and BglII were used for analysis of TFIIAαβ and TFIIAγ loci, respectively. DNA separated by agarose gel electrophoresis was transferred to a Hybond N+ membrane (GE Healthcare Bioscience). DNA was hybridized with a 32P-labeled gene-specific probe at 42 °C overnight in a hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's solution, 0.1 M sodium phosphate buffer [pH 6.5], 0.5% SDS, and 100 μg/ml salmon sperm DNA).
to the manufacturer's instructions. Sequence for targeting the mouse TFIIAαβ is 5′-AACAGCAAGCTCCGTTGGTGC. siRNA for GAPDH as a negative control was supplied as a reagent in the kit. Two 29-mer oligodeoxynucleotides for TFIIAαβ (sense: 5′-AACAGCAAGCTCCGTTGGTGCCCTGTCTC and antisense: 5′-AAGCACCAACGGAGCTTGCTGCCTGTCTC) contain corresponding siRNA sequences (underlined) and flanking sequences complementary to the T7 primer. Templates for siRNA were hybridized with the primer sequence and extended with the Klenow enzyme. The extended sense and corresponding antisense siRNA templates were transcribed with T7 RNA polymerase, and transcripts were hybridized to generate double-stranded siRNA according to the manufacturer's instructions. Cells were transfected with 100 nM siRNA for each gene using XtremeGENE siRNA transfection Reagent (Roche). 3. Results
2.6. Determination of TFIIA loci by FISH
3.1. Structure of chicken TFIIA genes
Chromosome preparation and FISH (fluorescence in situ hybridization) detection were performed as described previously (Matsuda and Chapman, 1995; Suzuki et al., 1999). Probes for chicken TFIIAαβ and TFIIAγ genes were prepared from cDNAs of TFIIAαβ and TFIIAγ, respectively, and labeled with biotin 16dUTP (Boehringer) following the manufacturer's protocol.
We first isolated cDNAs for chicken TFIIAαβ and TFIIAγ. Numbers of predicted amino acid (AA) residues of chicken TFIIAαβ and TFIIAγ were 377 and 109, respectively (Fig. 1A and B). Identities between human sequences of TFIIAαβ (NCBI Gene Database) and TFIIAγ (Upadhyaya et al., 2002) and their chicken counterparts were 90% and 96%, respectively (Fig. 1A and B). Lengths of chicken and human TFIIAγ (109 AAs) were identical, and those of TFIIAαβ were almost the same (i.e., 377 and 376 AAs for chicken and human proteins, respectively) (Fig. 1A and B). The amino acid sequence of QXDG in the CRS for the TFIIAαβ precursor was located at the
2.7. RNA interference (RNAi) Small interference RNA (siRNA) oligonucleotide was prepared with a Silencer siRNA Construction kit (Ambion) according
Fig. 1. Homology between chicken and human TFIIA Amino acid sequences of chicken TFIIAαβ [cTFIIAαβ] (A) and TFIIAγ [cTFIIAγ] (B) were aligned with human counterparts (i.e., NCBI Gene Database for TFIIAαβ and Upadhyaya et al. (2002) for TFIIAγ). Identical, similar, and related amino acid residues between these orthologs are indicated with asterisks, double dots, and single dots, respectively. (C) Sequences of the cleavage recognition site (CRS) and cleavage position (arrowhead) of chicken and human TFIIAαβ are shown.
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Fig. 2. FISH detection for chicken TFIIA genes Chromosomal sites of TFIIAαβ (A) and TFIIAγ (B) genes in chicken cells were determined by FISH. Arrowheads show the hybridizing FISH signals. Panels a and b show the staining patterns for R band and G band, respectively.
273rd AA from the first methionine and was identical to AA sequences in other vertebrate CRSs (Fig. 1C) (Hoiby et al., 2004). Hence, lengths of human and chicken TFIIAβ were the same. Taken together, the results demonstrated that TFIIA is highly conserved among vertebrates. Through PCR-assisted cloning of TFIIA gene-containing genomic DNAs from chicken DT40 cells, we determined Exon–intron junctions of the TFIIA genes. The chicken TFIIAαβ gene contained nine coding exons same as the human gene (data not shown). On the other hand, coding region of the chicken TFIIAγ gene was composed of four exons (data not shown). Human TFIIAγ gene has one 5' non-coding exon and four coding exons (Upadhyaya et al., 2002). In conclusion, Exon–intron organizations of the TFIIA genes were also conserved in vertebrates.
3.3. Amounts of TFIIA proteins in chicken tissues Amounts of TFIIA proteins in chicken tissues were measured by Western blotting (Fig. 3). Purified antibodies against human TFIIAαβ and TFIIAγ were prepared as described in Materials and methods section . A small amount of TFIIAαβ precursor protein was detected in some tissues (data not shown). As seen in Fig. 3, both TFIIAβ (panel A) and TFIIAγ (panel B) were present ubiquitously in all tissues examined. Considerable amounts of TFIIAβ were present in the kidney and ovary, whereas TFIIAγ was present in abundance in the lung, spleen, and testis (Fig. 3). The testes contained a particularly large amount of TFIIAγ. On the other hand, the pancreas and liver contained small amounts of both TFIIA
3.2. Chromosomal positions of TFIIA genes We determined chromosomal positions of chicken TFIIAαβ and TFIIAγ genes by FISH detection using each cDNA probe. It is generally known that chicken cells have seven large chromosomes and a number of mini-chromosomes that can not be precisely designated. Moreover, chromosome 2 exists as a trisomy in DT40 cells. Data obtained by FISH analysis revealed that signals for TFIIAαβ are mapped on chromosome 5 at q2.3–q2.5 (Fig. 2A). On the other hand, signals of TFIIAγ were detected on a couple of mini-chromosomes (Fig. 2B). These data imply that TFIIA genes exist as diploids in DT40 cells.
Fig. 3. Tissue distribution of TFIIA proteins in chicken tissues. Amounts of TFIIAβ (A) and TFIIAγ (B) were determined by Western blotting using 50 μg of proteins and anti-TFIIAαβ and anti-TFIIAγ antibodies, respectively. Betaactin was also analyzed (C).
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proteins. The anti-TFIIAαβ antibody also detected mature TFIIAα protein. The tissue distribution profile of TFIIAα was almost the same as that of TFIIAβ (data not shown). 3.4. Disruption of TFIIA genes in chicken DT40 cells In order to investigate the role of TFIIA in growth of vertebrate cells, the TFIIA gene in DT40 cells was disrupted because homologous recombination occurs in these cells. Targeting vectors for TFIIAαβ and TFIIAγ genes were constructed using a histidinol-resistant gene (Fig. 4A). If homologous recombination occurs between the targeting vector and endogenous gene, exons V and II of TFIIAαβ and TFIIAγ, respectively, will be interrupted by a drug-resistant gene. For each TFIIA gene, 100 histidinol-resistant cell clones were analyzed by Southern blotting for integrated histidinol-resistant genes (Fig. 4B). Among cells having a histidinol-resistant phenotype, efficiencies of homologous recombination were 76% for TFIIAαβ and 26% for TFIIAγ. It was confirmed that homologous recombination-derived knockout was accomplished efficiently as for TFIIA genes. A relatively low recombination efficiently for the TFIIAγ gene could be attributed
to relatively short DNA stretches flanking the drug-resistant gene (Fig. 4A). Although, the amounts of TFIIAαβ and TFIIAγ proteins in these single allele-disrupted cells (i.e., semi-knockout cells) were decreased slightly, both semi-knockout cells showed almost the same growth property as that of wild-type cells (data not shown). Next, we tried to generate double-knockout cells from histidinol-resistant TFIIA semi-knockout cells using another targeting vector. To construct a targeting vector for the second allele disruption, we used a puromycin-resistant gene. After transfection of semi-knockout cells for each gene with the targeting vector followed by drug selection, we obtained 90 clones of TFIIAαβ+/−derived cells and 30 clones of TFIIAγ+/− derived cells that are resistant to both histidinol and puromycin. Southern analysis revealed, however, that no clone had an expected secondary disrupted allele for either TFIIA gene (Fig. 4C, as for TFIIAαβ). As described above, homologous recombination efficiencies of the targeting vector for the first primary disruption were considerably high for both genes in DT40 cells. Hence, we thought that it is not feasible to establish a double-knockout cell for the TFIIA gene.
Fig. 4. Disruption of TFIIA genes of DT40 cells (A) Structure of the insert in the targeting vector for primary disruption of TFIIAαβ (a) and TFIIAγ (b) genes. The histidinol-resistant gene (3.4 kbp) in inserts is shown by a box with oblique lines. Restriction sites used in this study are shown by arrowheads. P: ApaI. K: KpnI. G: BglII. Short bars with terminal arrowheads represent position of the probe sequence. (B) Southern analysis of semi-knockout cells. Genomic DNA was digested with ApaI+KpnI and BglII for TFIIAαβ and TFIIAγ genes, respectively. As for TFIIAβ, expected lengths of Southern signals for the normal allele and mutated allele are 12 kbp and 10 kbp, respectively. As for TFIIAγ, expected lengths of Southern signals for the normal allele and mutated allele are 7.4 kbp and 10.8 kbp, respectively. Lane 1: wild-type cell. Lanes 2–6: histidinol-resistant clones. Asterisks and arrows depict positions of DNA fragments derived from normal and mutated alleles, respectively. (C) Genomic analysis of histidinol and puromycin (hisD/puro)-resistant cells. Representative results of Southern analysis of cells subjected to TFIIAαβ gene disruption for the second allele are presented. The targeting vector for secondary disruption is similar to that described in the panel A but contains a 2.7-kbp puromycin-resistant gene. Genomic DNA was digested with ApaI and KpnI and subjected to Southern analysis using the same probe as that described in the panel B. Asterisk and arrow show normal allele-and mutated allele-derived signals, respectively.+/−: parental TFIIAαβ+/−cell.
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Fig. 5. Effect of TFIIA siRNA on cell growth (A) NIH3T3 cells were transfected with siRNAs for TFIIAαβ and GAPDH. Forty-eight hours after transfection, amounts of TFIIAαβ and TFIIAα in 10 μg of cellular proteins were detected by Western blotting and determined densitometrically. GAPDH and α-tubulin were also analyzed. (B) Growth profiles of transfected cells. Cells were grown in the medium and cell numbers were counted at indicated time points.
3.5. Role of TFIIA in cell growth We investigated roles of TFIIA in mouse NIH3T3 cells by RNAi. A siRNA derived from mouse TFIIAαβ mRNA sequence decreased intracellular proteins of TFIIAαβ and TFIIAβ to 45% and 77% of the control, respectively (Fig. 5A). In these conditions, cell growth profiles were assays. It was demonstrated that numbers of the knock-down cells were nearly 60% of those of control cells at any time points analyzed (Fig. 5B). These results suggest that TFIIA participates in cell growth regulation. 4. Discussion In this study, we identified genes for chicken TFIIAαβ and TFIIAγ. Identities of chicken and mammalian TFIIAs were higher than 90% (Fig. 1A and B). Sequences around Exon–intron junctions were also conserved in chicken and mammalian TFIIA. TFIIAαβ is restrictedly cleaved at a point just downstream of the CRS to produce mature TFIIAα and TFIIAβ. We identified QVDG in the middle of TFIIAαβ that entirely coincides with the consensus CRS sequence (Fig. 1C). It was also revealed that AAs at Exon–intron junctions of chicken TFIIAs are homologous to those of mammalian counterparts (data not shown). From these findings, we concluded that TFIIA genes are highly conserved among vertebrates. We measured TFIIA protein levels by Western blotting, and found that both TFIIAβ and TFIIAγ exist ubiquitously in chicken tissues (Fig. 5). This finding is reasonable since TFIIA works in basal transcription. It is known that mammalian testes contain large amounts of TFIIAγ compared with other tissues (Upadhyaya et al., 1999). In the chicken, TFIIAγ was present in the testis abundantly, too (Fig. 3). We tried to establish TFIIA-null cells. As for the TFIIA gene, a knockout study has been performed only in yeast. Yeast TFIIA genes, TOA1 and TOA2, are indispensable for cell growth
(Ranish et al., 1992; Kang et al., 1995; Ozer et al., 1998a). In this study, we were able to establish semi-knockout chicken cells for both TFIIAαβ and TFIIAγ genes (Fig. 4B). The semiknockout cells exhibited a similar growth rate with that of wildtype cells. However, we were unable to establish a doubleknockout cell for either TFIIA allele even though we obtained multiple cells that are resistant to both histidinol and puromycin (Fig. 4C).It is thought that the puromycin-resistant genes were integrated randomly into chromosomes during a secondary disruption procedure. It is known that homologous recombination efficiency of secondary disruption of the Rad52 gene that participates in the homologous recombination declined to 20– 50% of that of primary disruption in DT40 cells (YamaguchiIwai et al., 1998). Therefore, efficiency of secondary disruption may be significantly high (e.g., probably over 20–50%) unless recombination-related genes are targeted. In the present study, recombination frequencies of the primary disruption for TFIIAαβ and TFIIAγ genes were considerably high (76% and 30% for TFIIAαβ and TFIIAγ, respectively). Therefore, we expected that we were able to establish significant numbers of double-knockout cells for TFIIA genes if these genes are dispensable. However, we failed to establish a double-knockout cell for both genes. This situation might indicate that both TFIIAαβ and TFIIAγ genes are indispensable for vertebrate cells, though a possibility that plenty of TFIIAαβ and TFIIAγ are critical for adequate recombination efficiency in DT40 cells can not be completely ruled out. In knock-down assays using TFIIAαβ siRNA, it was suggested that TFIIA is involved in propagation of NIH3T3 cells (Fig. 5), since both TFIIAαβ and TFIIAγ are needed for TFIIA function. This phenomenon is consistent with our idea described above that TFIIA can be an essential gene for vertebrate cell growth. TFIIA-semi-knockout DT40 cells grew well like native cells even the level of intracellular TFIIA in those mutant
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cells decreased slightly (data now shown). This situation seems to be inconsistent with the knock-down assay of Fig. 5. Compared to DT40 cells, amount of TFIIA may be limited in NIH3T3 cells, or NIH3T3 may be sensitive to concentration of TFIIA. Alternatively, transfection-triggered stress might confer NIH3T3 cells susceptibility to TFIIA. Actually, yeast TFIIA is known to be involved in stress response (Kraemer et al., 2006). It has been clearly demonstrated that TFIIA facilitates DNA binding of TFIID in vitro as holo-TFIIA and sometimes functions as a co-activator for a sequence-specific transcriptional activator. In contrast to in vitro characterizations, little has been known about the in vivo role of TFIIA. The present study hypothesizes that TFIIA is an essential gene for all of the eukaryotes. TBP-associated factors are not essential for cell growth even though some of them are responsible for development. In the case of TFIIA, it also plays as an accessory role in transcriptional regulation. If TFIIA is indispensable for cell growth, the TFIIA requirement can be explained by several hypotheses. First, TFIIA may play a critical role in transcriptional regulation of limited numbers of essential genes. Second, TFIIA may function globally in transcription regulation, and the total sum of TFIIA-regulated gene expression enables cells to grow. Alternatively, TFIIA might be involved in a certain essential non-transcriptional cellular process. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology of Japan and by a grant from Futaba Electric Foundation. References Auble, D.T., Hansen, K.E., Mueller, C.G., Lane, W.S., Thorner, J., Hahn, S., 1994. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8, 1920–1934. Chi, T., Carey, M., 1996. Assembly of the isomerized TFIIA-TFIID-TATA ternary complex is necessary and sufficient for gene activation. Genes Dev. 10, 2540–2550. Coleman, R.A., Taggart, A.K., Burma, S., Chicca II, J.J., Pugh, B.F., 1999. TFIIA regulates TBP and TFIID dimers. Mol. Cell 4, 451–457. DeJong, J., Roeder, R.G., 1993. A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA. Genes Dev. 7, 2220–2234. DeJong, J., Bernstein, R., Roeder, R.G., 1995. Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription. Proc. Natl. Acad. Sci. U. S. A. 92, 3313–3317. Ge, H., Roeder, R.G., 1994. The high mobility group protein HMG1 can reversibly inhibit class II gene transcription by interaction with the TATAbinding protein. J. Biol. Chem. 269, 17136–17140. Hoiby, T., Mitsiou, D.J., Zhou, H., Erdjument-Bromage, H., Tempst, P., Stunnenberg, H.G., 2004. Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA. EMBO J. 23, 3083–3091. Inostroza, J.A., Mermelstein, F.H., Ha, I., Lane, W.S., Reinberg, D., 1992. Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell 70, 477–489. Kang, J.J., Auble, D.T., Ranish, J.A., Hahn, S., 1995. Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase IIspecific role in basal and activated transcription. Mol. Cell. Biol. 15, 1234–1243.
Kirov, N.C., Lieberman, P.M., Rushlow, C., 1996. The transcriptional corepressor DSP1 inhibits activated transcription by disrupting TFIIATBP complex formation. EMBO J. 15, 7079–7087. Kobayashi, N., Boyer, T.G., Berk, A.J., 1995. A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly. Mol. Cell. Biol. 15, 6465–6473. Kraemer, S.M., et al., 2006. TFIIA plays a role in the response to oxidative stress. Eukaryot. Cell 5, 1081–1090. Lahti, J.M., 1999. Use of gene knockouts in cultured cells to study apoptosis. Methods 17, 305–312. Ma, D., et al., 1993. Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription. Genes Dev. 7, 2246–2257. Ma, D., Olave, I., Merino, A., Reinberg, D., 1996. Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA. Proc. Natl. Acad. Sci. U. S. A. 93, 6583–6588. Matsuda, Y., Chapman, V.M., 1995. Application of fluorescence in situ hybridization in genome analysis of the mouse. Electrophoresis 16, 261–272. Merino, A., Madden, K.R., Lane, W.S., Champoux, J.J., Reinberg, D., 1993. DNA topoisomerase I is involved in both repression and activation of transcription. Nature 365, 227–232. Nakadai, T., et al., 1999. HP33: hepatocellular carcinoma-enriched 33-kDa protein with similarity to mitochondrial N-acyltransferase but localized in a microtubule-dependent manner at the centrosome. J. Cell Sci. 112, 1353–1364. Nakadai, T., Shimada, M., Shima, D., Handa, H., Tamura, T.A., 2004. Specific interaction with transcription factor IIA and localization of the mammalian TATA-binding protein-like protein (TLP/TRF2/TLF). J. Biol. Chem. 279, 7447–7455. Ozer, J., Moore, P.A., Bolden, A.H., Lee, A., Rosen, C.A., Lieberman, P.M., 1994. Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription. Genes Dev. 8, 2324–2335. Ozer, J., Lezina, L.E., Ewing, J., Audi, S., Lieberman, P.M., 1998a. Association of transcription factor IIA with TATA binding protein is required for transcriptional activation of a subset of promoters and cell cycle progression in Saccharomyces cerevisiae. Mol. Cell. Biol. 18, 2559–2570. Ozer, J., Mitsouras, K., Zerby, D., Carey, M., Lieberman, P.M., 1998b. Transcription factor IIA derepresses TATA-binding protein (TBP)-associated factor inhibition of TBP-DNA binding. J. Biol. Chem. 273, 14293–14300. Ranish, J.A., Lane, W.S., Hahn, S., 1992. Isolation of two genes that encode subunits of the yeast transcription factor IIA. Science 255, 1127–1129. Ranish, J.A., Yudkovsky, N., Hahn, S., 1999. Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13, 49–63. Shimada, M., Nakadai, T., Tamura, T.A., 2003. TATA-binding protein-like protein (TLP/TRF2/TLF) negatively regulates cell cycle progression and is required for the stress-mediated G(2) checkpoint. Mol. Cell. Biol. 23, 4107–4120. Solow, S.P., Lezina, L., Lieberman, P.M., 1999. Phosphorylation of TFIIA stimulates TATA binding protein-TATA interaction and contributes to maximal transcription and viability in yeast. Mol. Cell. Biol. 19, 2846–2852. Sun, X., Ma, D., Sheldon, M., Yeung, K., Reinberg, D., 1994. Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIIDmediated transcription. Genes Dev. 8, 2336–2348. Suzuki, T., et al., 1999. Cytogenetic assignment of 29 functional genes to chicken microchromosomes by FISH. Cytogenet. Cell. Genet. 87, 233–237. Taggart, A.K., Pugh, B.F., 1996. Dimerization of TFIID when not bound to DNA. Science 272, 1331–1333. Upadhyaya, A.B., Lee, S.H., DeJong, J., 1999. Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis. J. Biol. Chem. 274, 18040–18048. Upadhyaya, A.B., Khan, M., Mou, T.C., Junker, M., Gray, D.M., DeJong, J., 2002. The germ cell-specific transcription factor ALF: structural properties and stabilization of the TATA-binding protein (TBP)-DNA complex. J. Biol. Chem. 277, 34208–34216. Weideman, C.A., et al., 1997. Dynamic interplay of TFIIA, TBP and TATA DNA. J. Mol. Biol. 271, 61–75. Yamaguchi-Iwai, Y., et al., 1998. Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52. Mol. Cell. Biol. 18, 6430–6435.
Chromosomal position, structure, expression, and requirement of genes for chicken transcription factor IIA ☆ Tomoko Mabuchi a , Toshifumi Wakamatsu a,1 , Tomoyoshi Nakadai a,2 , Miho Shimada a,2 , Kazuhiko Yamada b , Yoichi Matsuda b , Taka-aki Tamura a,⁎ a
Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan b Creative Research Initiative, Hokkaido University, Sapporo 060-0810, Japan Received 21 September 2006; received in revised form 14 March 2007; accepted 13 April 2007 Available online 21 April 2007
Abstract Transcription factor IIA (TFIIA) is one of the general transcription factors for RNA polymerase II and composed of three subunits, TFIIAα, TFIIAβ and TFIIAγ. TFIIAα and TFIIAβ are encoded by a single gene (TFIIAαβ) and mature through internal cleavage of TFIIAαβ. In this study, we found that structures of TFIIAαβ and TFIIAγ are highly homologous with each mammalian counterpart. Exon–intron organizations of the human and chicken TFIIA genes were also homologous. The sequence of the cleavage region of the chicken TFIIAαβ precursor protein was fitted to the consensus cleavage recognition site. It was thus demonstrated that TFIIA is conserved in vertebrates. TFIIA proteins are present ubiquitously in chicken tissues. Fluorescent in situ hybridization revealed that TFIIAαβ and TFIIAγ genes are located in chromosome 5 and a mini-chromosome, respectively. We generated semi-knockout chicken DT40 cells for TFIIAαβ and TFIIAγ genes with high homologous recombination efficiencies, whereas we failed to establish double-knockout cells for each gene. It is thought that both genes for TFIIA are required in vertebrates. TFIIA siRNA resulted in deceleration of cell growth rate, suggesting that, consistent with those of knockout assays, TFIIA is associated with cell growth regulation. © 2007 Elsevier B.V. All rights reserved. Keywords: TFIIA; Transcription factor; DT40; Gene disruption; General transcription factor
1. Introduction In transcriptional regulation of RNA polymerase II-dependent genes, various kinds of general transcription factors (GTFs), including TFIIA (transcription factor IIA), TFIIB, Abbreviations: GTF, general transcription factor; TFIIA, transcription factor IIA; TBP, TATA-binding protein; CRS, cleavage recognition site; AA, amino acid; FISH, fluorescence in situ hybridization; RNAi, RNA interference; siRNA, small interference RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ☆ GenBank accession numbers. chicken TFIIAab: AB264607, chicken TFIIAg: AB264606. ⁎ Corresponding author. Fax: +81 43 290 2824. E-mail address: [email protected] (T. Tamura). 1 Present address: Institute of Medical Molecular Design Incorporated, 5-24-5 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2 Present address: Department of Molecular Biology, Saitama Medical School, Moroyama, Saitama 350-0495, Japan. 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.04.015
TFIID, TFIIE, TFIIF and TFIIH are required for correct and efficient transcription in addition to RNA polymerase II. TFIID is composed of TBP and multiple TBP-associated factors. RNA polymerase II and GTFs assemble on a promoter for initiation of transcription. At the first step in this pre-initiation complex formation, TBP (TATA-binding protein) or TFIID binds to the promoter sequence. TFIIA binds to TBP and is incorporated into the TBP-DNA complex. TFIIA works to stabilize the complex (Weideman et al., 1997; Ranish et al., 1999). TFIIA has additional functions in transcriptional regulation. For example, TFIIA dissociates a TBP dimer and inhibits dimer formation of TBP (Taggart and Pugh, 1996; Coleman et al., 1999). TFIIA suppresses repressor proteins that interfere with DNA binding of TBP/TFIID (Inostroza et al., 1992; Merino et al., 1993; Ge and Roeder, 1994; Auble et al., 1994; Kirov et al., 1996; Ozer et al., 1998b). TFIIA also functions as a coactivator for sequence-specific transcription factors (Ozer et al.,
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1994; Sun et al., 1994; Kang et al., 1995; Kobayashi et al., 1995; Chi and Carey, 1996). TFIIA that has several functions, is thus considered to be an activator for the formation of the preinitiation complex. Requirement of TFIIA in transcription regulation depends on the structure of a core promoter and existence of a regulatory element (Ma et al., 1996; Solow et al., 1999). In a reconstituted in vitro transcription system composed of highly purified GTFs including TBP but not TFIID, TFIIA does not exhibit its transcription activation function (Sun et al., 1994; Ma et al., 1993). Moreover, it is thought that TFIIA is dispensable for some activator-dependent transcriptional stimulation (Ozer et al., 1994; Sun et al., 1994; Ma et al., 1996; Ranish et al., 1999). Accordingly, TFIIA is not always a necessary factor, at least in vitro, although its in vivo function has not been fully elucidated. Human TFIIA consists of three subunits: TFIIA α (34 kDa), TFIIA β (19 kDa) (Ma et al., 1993; DeJong and Roeder, 1993), and TFIIA γ (12 kDa) (Ozer et al., 1994; DeJong et al., 1995). TFIIAα and TFIIAβ are encoded by a single gene (TFIIAαβ) and generated from the TFIIAαβ precursor via intramolecule cleavage. Amino acid sequence of QVDG located just upstream from a junction of the cleavage site is conserved in higher eukaryotes and called cleavage recognition site (CRS) (Hoiby et al., 2004). Processing of the TFIIAαβ precursor occurs between glycine and aspartic acid just two amino acids downstream from the CRS (Hoiby et al., 2004). Unprocessed TFIIAαβ can functionally substitute with mature TFIIAα and TFIIAβ in an in vitro transcription system (DeJong et al., 1995). Since the mature TFIIAβ subunit is sensitive to proteasome-dependent degradation (Hoiby et al., 2004), processing of TFIIAαβ may be involved in regulation of the level of TFIIA. In S. cerevisiae, TFIIA is composed of two subunits, TOA1 and TOA2 (Ranish et al., 1992), which correspond to human TFIIAαβ and TFIIAγ, respectively. The N-terminal and Cterminal halves of TOA1 are analogous to those of mammalian TFIIAα and TFIIAβ, respectively (DeJong and Roeder, 1993). TOA2 is an ortholog of mammalian TFIIAγ (DeJong et al., 1995). TOA1 and TOA2 are essential for yeast cell growth (Ranish et al., 1992; Kang et al., 1995; Ozer et al., 1998a), though TFIIA does not play a critical role but plays an accessory role in transcriptional regulation as described above. There has been no investigation so far of the requirement of TFIIA for growth of vertebrate cells. In this study, we determined structures of the chicken TFIIA and their chromosomal loci. We disrupted a single allele of TFIIAαβ and TFIIAγ genes. However, we were unable to establish a double-knockout cell for both genes. Knock-down of TFIIA resulted in decreased cell growth rate. The present study suggests that TFIIA is involved in growth of vertebrate cells. 2. Materials and methods 2.1. Cell culture and transfection DT40 cells derived from chicken B lymphoma were cultured at 39.5 °C in RPMI1640 medium supplemented with 10% fetal
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calf serum and 1% chicken serum (JRH Bioscience). Electroporation for DNA transfection of 107 cells was carried out at 550 V and 25 μF using Gene Pulser II (Bio-Rad). The transfected cells were maintained in 10 ml medium as previously described (Lahti, 1999). NIH3T3 cells were maintained in a low-glucose DMEM with 10% fetal calf serum. 2.2. Protein extraction and Western blotting Chicken tissues were homogenized in an extraction buffer (20 mM Tris–HCl [pH 7.4], 0.1% SDS, and 1% Triton X-100) supplemented with a protein inhibitor mixture composed of 1 mM benzamidine-HCl, 1 μg/ml pepstatin A, 1 mM PMSF, and 1 μg/ml leupeptin. Ten million cells were lysed in buffer C (50 mM HEPES-KOH [pH 7.8], 0.42 M KCl, 0.1 mM EDTA, 5 mM MgCl2, and 20% glycerol) supplemented with the protein inhibitor mixture. Following a brief centrifugation, supernatant fractions were collected as a whole cell extract. Protein concentration was determined with a BCA protein assay kit (Pierce). Cellular proteins in the SDS sampling buffer were separated by a 15% SDS-PAGE and transferred onto a PVDF membrane (Millipore) by electroblotting. TFIIA polypeptides were detected by Western blotting with rabbit polyclonal antibodies against human TFIIAαβ and TFIIAγ and ECL Plus (GE Healthcare Bioscience). Antibodies against β-actin (Sigma), α-tubulin, and GAPDH (glyceraldehyde-3phosphate dehydrogenase) (Ambion) were commercial products. Beta-actin was detected by an alkaline phosphatase method, whereas others were detected by ECL Plus. 2.3. Antibodies against TFIIA The open reading frame of human TFIIAαβ and TFIIAγ was connected to an oligo-histidine (His) 6-tag at the N-terminus and inserted into pET16b vector (Nakadai et al., 2004). Recombinant protein was expressed in BL21(DE3) pLysS strain of E. coli as previously described (Nakadai et al., 2004). Bacterially expressed proteins were dissolved with a lysis buffer containing 6 M guanidine-HCl (Ma et al., 1993). The lysate was centrifuged at 42 krpm for 1 h, and recombinant protein in the supernatant fraction was purified with Ni-NTA agarose (Qiagen) as described previously (Nakadai et al., 1999). Rabbits were immunized with SDS-PAGE-purified proteins, and polyclonal antibodies were generated. Antibodies were affinity-purified from sera by using recombinant TFIIA-immobilized NHS-activated Sepharose beads (Hitrap; GE Healthcare Bioscience). 2.4. Disruption of chicken TFIIA genes The fifth exon of TFIIAαβ and the second exon of TFIIAγ in DT40 cell were disrupted by homologous recombination with a drug-resistant gene-carrying targeting vector. For the first allele disruption, a histidinol-resistant gene was used as a selection marker. Cells were transfected with 25 μg of the linearized plasmid. Histidinol-resistant cells were selected in a culture medium with 1 mg/ml histidinol (Shimada et al., 2003). After resistant cells had been obtained, one representative
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heterozygous knockout cell was subjected to a secondary disruption procedure using a puromycin-resistant gene-carrying targeting vector and 0.5 μg/ml of puromycin (Lahti, 1999). 2.5. Southern blot analysis DT40 cells in an extraction buffer (10 mM Tris–HCl [pH 8.0], 50 mM EDTA, 0.5% SDS, 0.1 M NaCl, 50 μg/ml RNase A, and 100 μg/ml proteinase K) were incubated at 55 °C for 1 h. The lysate was dialyzed against TE buffer after phenol treatment. DNA (20 μg) was digested with restriction enzymes. KpnI plus ApaI and BglII were used for analysis of TFIIAαβ and TFIIAγ loci, respectively. DNA separated by agarose gel electrophoresis was transferred to a Hybond N+ membrane (GE Healthcare Bioscience). DNA was hybridized with a 32P-labeled gene-specific probe at 42 °C overnight in a hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's solution, 0.1 M sodium phosphate buffer [pH 6.5], 0.5% SDS, and 100 μg/ml salmon sperm DNA).
to the manufacturer's instructions. Sequence for targeting the mouse TFIIAαβ is 5′-AACAGCAAGCTCCGTTGGTGC. siRNA for GAPDH as a negative control was supplied as a reagent in the kit. Two 29-mer oligodeoxynucleotides for TFIIAαβ (sense: 5′-AACAGCAAGCTCCGTTGGTGCCCTGTCTC and antisense: 5′-AAGCACCAACGGAGCTTGCTGCCTGTCTC) contain corresponding siRNA sequences (underlined) and flanking sequences complementary to the T7 primer. Templates for siRNA were hybridized with the primer sequence and extended with the Klenow enzyme. The extended sense and corresponding antisense siRNA templates were transcribed with T7 RNA polymerase, and transcripts were hybridized to generate double-stranded siRNA according to the manufacturer's instructions. Cells were transfected with 100 nM siRNA for each gene using XtremeGENE siRNA transfection Reagent (Roche). 3. Results
2.6. Determination of TFIIA loci by FISH
3.1. Structure of chicken TFIIA genes
Chromosome preparation and FISH (fluorescence in situ hybridization) detection were performed as described previously (Matsuda and Chapman, 1995; Suzuki et al., 1999). Probes for chicken TFIIAαβ and TFIIAγ genes were prepared from cDNAs of TFIIAαβ and TFIIAγ, respectively, and labeled with biotin 16dUTP (Boehringer) following the manufacturer's protocol.
We first isolated cDNAs for chicken TFIIAαβ and TFIIAγ. Numbers of predicted amino acid (AA) residues of chicken TFIIAαβ and TFIIAγ were 377 and 109, respectively (Fig. 1A and B). Identities between human sequences of TFIIAαβ (NCBI Gene Database) and TFIIAγ (Upadhyaya et al., 2002) and their chicken counterparts were 90% and 96%, respectively (Fig. 1A and B). Lengths of chicken and human TFIIAγ (109 AAs) were identical, and those of TFIIAαβ were almost the same (i.e., 377 and 376 AAs for chicken and human proteins, respectively) (Fig. 1A and B). The amino acid sequence of QXDG in the CRS for the TFIIAαβ precursor was located at the
2.7. RNA interference (RNAi) Small interference RNA (siRNA) oligonucleotide was prepared with a Silencer siRNA Construction kit (Ambion) according
Fig. 1. Homology between chicken and human TFIIA Amino acid sequences of chicken TFIIAαβ [cTFIIAαβ] (A) and TFIIAγ [cTFIIAγ] (B) were aligned with human counterparts (i.e., NCBI Gene Database for TFIIAαβ and Upadhyaya et al. (2002) for TFIIAγ). Identical, similar, and related amino acid residues between these orthologs are indicated with asterisks, double dots, and single dots, respectively. (C) Sequences of the cleavage recognition site (CRS) and cleavage position (arrowhead) of chicken and human TFIIAαβ are shown.
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Fig. 2. FISH detection for chicken TFIIA genes Chromosomal sites of TFIIAαβ (A) and TFIIAγ (B) genes in chicken cells were determined by FISH. Arrowheads show the hybridizing FISH signals. Panels a and b show the staining patterns for R band and G band, respectively.
273rd AA from the first methionine and was identical to AA sequences in other vertebrate CRSs (Fig. 1C) (Hoiby et al., 2004). Hence, lengths of human and chicken TFIIAβ were the same. Taken together, the results demonstrated that TFIIA is highly conserved among vertebrates. Through PCR-assisted cloning of TFIIA gene-containing genomic DNAs from chicken DT40 cells, we determined Exon–intron junctions of the TFIIA genes. The chicken TFIIAαβ gene contained nine coding exons same as the human gene (data not shown). On the other hand, coding region of the chicken TFIIAγ gene was composed of four exons (data not shown). Human TFIIAγ gene has one 5' non-coding exon and four coding exons (Upadhyaya et al., 2002). In conclusion, Exon–intron organizations of the TFIIA genes were also conserved in vertebrates.
3.3. Amounts of TFIIA proteins in chicken tissues Amounts of TFIIA proteins in chicken tissues were measured by Western blotting (Fig. 3). Purified antibodies against human TFIIAαβ and TFIIAγ were prepared as described in Materials and methods section . A small amount of TFIIAαβ precursor protein was detected in some tissues (data not shown). As seen in Fig. 3, both TFIIAβ (panel A) and TFIIAγ (panel B) were present ubiquitously in all tissues examined. Considerable amounts of TFIIAβ were present in the kidney and ovary, whereas TFIIAγ was present in abundance in the lung, spleen, and testis (Fig. 3). The testes contained a particularly large amount of TFIIAγ. On the other hand, the pancreas and liver contained small amounts of both TFIIA
3.2. Chromosomal positions of TFIIA genes We determined chromosomal positions of chicken TFIIAαβ and TFIIAγ genes by FISH detection using each cDNA probe. It is generally known that chicken cells have seven large chromosomes and a number of mini-chromosomes that can not be precisely designated. Moreover, chromosome 2 exists as a trisomy in DT40 cells. Data obtained by FISH analysis revealed that signals for TFIIAαβ are mapped on chromosome 5 at q2.3–q2.5 (Fig. 2A). On the other hand, signals of TFIIAγ were detected on a couple of mini-chromosomes (Fig. 2B). These data imply that TFIIA genes exist as diploids in DT40 cells.
Fig. 3. Tissue distribution of TFIIA proteins in chicken tissues. Amounts of TFIIAβ (A) and TFIIAγ (B) were determined by Western blotting using 50 μg of proteins and anti-TFIIAαβ and anti-TFIIAγ antibodies, respectively. Betaactin was also analyzed (C).
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proteins. The anti-TFIIAαβ antibody also detected mature TFIIAα protein. The tissue distribution profile of TFIIAα was almost the same as that of TFIIAβ (data not shown). 3.4. Disruption of TFIIA genes in chicken DT40 cells In order to investigate the role of TFIIA in growth of vertebrate cells, the TFIIA gene in DT40 cells was disrupted because homologous recombination occurs in these cells. Targeting vectors for TFIIAαβ and TFIIAγ genes were constructed using a histidinol-resistant gene (Fig. 4A). If homologous recombination occurs between the targeting vector and endogenous gene, exons V and II of TFIIAαβ and TFIIAγ, respectively, will be interrupted by a drug-resistant gene. For each TFIIA gene, 100 histidinol-resistant cell clones were analyzed by Southern blotting for integrated histidinol-resistant genes (Fig. 4B). Among cells having a histidinol-resistant phenotype, efficiencies of homologous recombination were 76% for TFIIAαβ and 26% for TFIIAγ. It was confirmed that homologous recombination-derived knockout was accomplished efficiently as for TFIIA genes. A relatively low recombination efficiently for the TFIIAγ gene could be attributed
to relatively short DNA stretches flanking the drug-resistant gene (Fig. 4A). Although, the amounts of TFIIAαβ and TFIIAγ proteins in these single allele-disrupted cells (i.e., semi-knockout cells) were decreased slightly, both semi-knockout cells showed almost the same growth property as that of wild-type cells (data not shown). Next, we tried to generate double-knockout cells from histidinol-resistant TFIIA semi-knockout cells using another targeting vector. To construct a targeting vector for the second allele disruption, we used a puromycin-resistant gene. After transfection of semi-knockout cells for each gene with the targeting vector followed by drug selection, we obtained 90 clones of TFIIAαβ+/−derived cells and 30 clones of TFIIAγ+/− derived cells that are resistant to both histidinol and puromycin. Southern analysis revealed, however, that no clone had an expected secondary disrupted allele for either TFIIA gene (Fig. 4C, as for TFIIAαβ). As described above, homologous recombination efficiencies of the targeting vector for the first primary disruption were considerably high for both genes in DT40 cells. Hence, we thought that it is not feasible to establish a double-knockout cell for the TFIIA gene.
Fig. 4. Disruption of TFIIA genes of DT40 cells (A) Structure of the insert in the targeting vector for primary disruption of TFIIAαβ (a) and TFIIAγ (b) genes. The histidinol-resistant gene (3.4 kbp) in inserts is shown by a box with oblique lines. Restriction sites used in this study are shown by arrowheads. P: ApaI. K: KpnI. G: BglII. Short bars with terminal arrowheads represent position of the probe sequence. (B) Southern analysis of semi-knockout cells. Genomic DNA was digested with ApaI+KpnI and BglII for TFIIAαβ and TFIIAγ genes, respectively. As for TFIIAβ, expected lengths of Southern signals for the normal allele and mutated allele are 12 kbp and 10 kbp, respectively. As for TFIIAγ, expected lengths of Southern signals for the normal allele and mutated allele are 7.4 kbp and 10.8 kbp, respectively. Lane 1: wild-type cell. Lanes 2–6: histidinol-resistant clones. Asterisks and arrows depict positions of DNA fragments derived from normal and mutated alleles, respectively. (C) Genomic analysis of histidinol and puromycin (hisD/puro)-resistant cells. Representative results of Southern analysis of cells subjected to TFIIAαβ gene disruption for the second allele are presented. The targeting vector for secondary disruption is similar to that described in the panel A but contains a 2.7-kbp puromycin-resistant gene. Genomic DNA was digested with ApaI and KpnI and subjected to Southern analysis using the same probe as that described in the panel B. Asterisk and arrow show normal allele-and mutated allele-derived signals, respectively.+/−: parental TFIIAαβ+/−cell.
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Fig. 5. Effect of TFIIA siRNA on cell growth (A) NIH3T3 cells were transfected with siRNAs for TFIIAαβ and GAPDH. Forty-eight hours after transfection, amounts of TFIIAαβ and TFIIAα in 10 μg of cellular proteins were detected by Western blotting and determined densitometrically. GAPDH and α-tubulin were also analyzed. (B) Growth profiles of transfected cells. Cells were grown in the medium and cell numbers were counted at indicated time points.
3.5. Role of TFIIA in cell growth We investigated roles of TFIIA in mouse NIH3T3 cells by RNAi. A siRNA derived from mouse TFIIAαβ mRNA sequence decreased intracellular proteins of TFIIAαβ and TFIIAβ to 45% and 77% of the control, respectively (Fig. 5A). In these conditions, cell growth profiles were assays. It was demonstrated that numbers of the knock-down cells were nearly 60% of those of control cells at any time points analyzed (Fig. 5B). These results suggest that TFIIA participates in cell growth regulation. 4. Discussion In this study, we identified genes for chicken TFIIAαβ and TFIIAγ. Identities of chicken and mammalian TFIIAs were higher than 90% (Fig. 1A and B). Sequences around Exon–intron junctions were also conserved in chicken and mammalian TFIIA. TFIIAαβ is restrictedly cleaved at a point just downstream of the CRS to produce mature TFIIAα and TFIIAβ. We identified QVDG in the middle of TFIIAαβ that entirely coincides with the consensus CRS sequence (Fig. 1C). It was also revealed that AAs at Exon–intron junctions of chicken TFIIAs are homologous to those of mammalian counterparts (data not shown). From these findings, we concluded that TFIIA genes are highly conserved among vertebrates. We measured TFIIA protein levels by Western blotting, and found that both TFIIAβ and TFIIAγ exist ubiquitously in chicken tissues (Fig. 5). This finding is reasonable since TFIIA works in basal transcription. It is known that mammalian testes contain large amounts of TFIIAγ compared with other tissues (Upadhyaya et al., 1999). In the chicken, TFIIAγ was present in the testis abundantly, too (Fig. 3). We tried to establish TFIIA-null cells. As for the TFIIA gene, a knockout study has been performed only in yeast. Yeast TFIIA genes, TOA1 and TOA2, are indispensable for cell growth
(Ranish et al., 1992; Kang et al., 1995; Ozer et al., 1998a). In this study, we were able to establish semi-knockout chicken cells for both TFIIAαβ and TFIIAγ genes (Fig. 4B). The semiknockout cells exhibited a similar growth rate with that of wildtype cells. However, we were unable to establish a doubleknockout cell for either TFIIA allele even though we obtained multiple cells that are resistant to both histidinol and puromycin (Fig. 4C).It is thought that the puromycin-resistant genes were integrated randomly into chromosomes during a secondary disruption procedure. It is known that homologous recombination efficiency of secondary disruption of the Rad52 gene that participates in the homologous recombination declined to 20– 50% of that of primary disruption in DT40 cells (YamaguchiIwai et al., 1998). Therefore, efficiency of secondary disruption may be significantly high (e.g., probably over 20–50%) unless recombination-related genes are targeted. In the present study, recombination frequencies of the primary disruption for TFIIAαβ and TFIIAγ genes were considerably high (76% and 30% for TFIIAαβ and TFIIAγ, respectively). Therefore, we expected that we were able to establish significant numbers of double-knockout cells for TFIIA genes if these genes are dispensable. However, we failed to establish a double-knockout cell for both genes. This situation might indicate that both TFIIAαβ and TFIIAγ genes are indispensable for vertebrate cells, though a possibility that plenty of TFIIAαβ and TFIIAγ are critical for adequate recombination efficiency in DT40 cells can not be completely ruled out. In knock-down assays using TFIIAαβ siRNA, it was suggested that TFIIA is involved in propagation of NIH3T3 cells (Fig. 5), since both TFIIAαβ and TFIIAγ are needed for TFIIA function. This phenomenon is consistent with our idea described above that TFIIA can be an essential gene for vertebrate cell growth. TFIIA-semi-knockout DT40 cells grew well like native cells even the level of intracellular TFIIA in those mutant
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cells decreased slightly (data now shown). This situation seems to be inconsistent with the knock-down assay of Fig. 5. Compared to DT40 cells, amount of TFIIA may be limited in NIH3T3 cells, or NIH3T3 may be sensitive to concentration of TFIIA. Alternatively, transfection-triggered stress might confer NIH3T3 cells susceptibility to TFIIA. Actually, yeast TFIIA is known to be involved in stress response (Kraemer et al., 2006). It has been clearly demonstrated that TFIIA facilitates DNA binding of TFIID in vitro as holo-TFIIA and sometimes functions as a co-activator for a sequence-specific transcriptional activator. In contrast to in vitro characterizations, little has been known about the in vivo role of TFIIA. The present study hypothesizes that TFIIA is an essential gene for all of the eukaryotes. TBP-associated factors are not essential for cell growth even though some of them are responsible for development. In the case of TFIIA, it also plays as an accessory role in transcriptional regulation. If TFIIA is indispensable for cell growth, the TFIIA requirement can be explained by several hypotheses. First, TFIIA may play a critical role in transcriptional regulation of limited numbers of essential genes. Second, TFIIA may function globally in transcription regulation, and the total sum of TFIIA-regulated gene expression enables cells to grow. Alternatively, TFIIA might be involved in a certain essential non-transcriptional cellular process. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology of Japan and by a grant from Futaba Electric Foundation. References Auble, D.T., Hansen, K.E., Mueller, C.G., Lane, W.S., Thorner, J., Hahn, S., 1994. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8, 1920–1934. Chi, T., Carey, M., 1996. Assembly of the isomerized TFIIA-TFIID-TATA ternary complex is necessary and sufficient for gene activation. Genes Dev. 10, 2540–2550. Coleman, R.A., Taggart, A.K., Burma, S., Chicca II, J.J., Pugh, B.F., 1999. TFIIA regulates TBP and TFIID dimers. Mol. Cell 4, 451–457. DeJong, J., Roeder, R.G., 1993. A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA. Genes Dev. 7, 2220–2234. DeJong, J., Bernstein, R., Roeder, R.G., 1995. Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription. Proc. Natl. Acad. Sci. U. S. A. 92, 3313–3317. Ge, H., Roeder, R.G., 1994. The high mobility group protein HMG1 can reversibly inhibit class II gene transcription by interaction with the TATAbinding protein. J. Biol. Chem. 269, 17136–17140. Hoiby, T., Mitsiou, D.J., Zhou, H., Erdjument-Bromage, H., Tempst, P., Stunnenberg, H.G., 2004. Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA. EMBO J. 23, 3083–3091. Inostroza, J.A., Mermelstein, F.H., Ha, I., Lane, W.S., Reinberg, D., 1992. Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell 70, 477–489. Kang, J.J., Auble, D.T., Ranish, J.A., Hahn, S., 1995. Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase IIspecific role in basal and activated transcription. Mol. Cell. Biol. 15, 1234–1243.
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