Mammalian Elongin A Is Not Essential for Cell Viability but Is Required for Proper Cell Cycle Progression with Limited Alteration of Gene Expression

Abstract

Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To investigate the function of Elongin A in vivo, the two alleles of the Elongin A gene have been disrupted by homologous recombination in murine embryonic stem (ES) cells. The Elongin A-deficient ES cells are viable, but show a slow growth phenotype because they undergo a delayed mitosis. The cDNA microarray and RNase protection assay using the wild-type and Elongin A-deficient ES cells indicate that the expression of only a small subset of genes is affected in the mutant cells. Taken together, our results suggest that Elongin A regulates transcription of a subset but not all of genes and reveal a linkage between Elongin A function and cell cycle progression. Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To investigate the function of Elongin A in vivo, the two alleles of the Elongin A gene have been disrupted by homologous recombination in murine embryonic stem (ES) cells. The Elongin A-deficient ES cells are viable, but show a slow growth phenotype because they undergo a delayed mitosis. The cDNA microarray and RNase protection assay using the wild-type and Elongin A-deficient ES cells indicate that the expression of only a small subset of genes is affected in the mutant cells. Taken together, our results suggest that Elongin A regulates transcription of a subset but not all of genes and reveal a linkage between Elongin A function and cell cycle progression. RNA polymerase II embryonic stem transcription factor von Hippel-Lindau neomycin resistance gene herpes simplex virus thymidine kinase reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase fluorescence-activated cell sorting TATA-binding protein Eukaryotic messenger RNA synthesis by RNA polymerase II (pol II)1 is regulated by the concerted action of a set of general transcription factors that control the activity of pol II during the initiation and elongation stages of transcription. At least six general transcription initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) have been identified in eukaryotic cells and found to promote the selective binding of pol II to promoters and to support a basal level of transcription (1Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar). In addition to the general initiation factors, at least 17 elongation factors have been defined biochemically and found to promote efficient elongation of transcripts by pol II (2Aso T. Conaway J.W. Conaway R.C. FASEB J. 1995; 9: 1419-1428Crossref PubMed Scopus (55) Google Scholar, 3Reines D. Conaway J.W. Conaway R.C. Trends Biochem. Sci. 1996; 21: 351-355Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 4Uptain S.M. Kane C.M. Chamberlin M.J. Annu. Rev. Biochem. 1997; 66: 117-172Crossref PubMed Scopus (397) Google Scholar, 5Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). These elongation factors fall into two broad functional classes based on their abilities to either reactivate arrested pol II or suppress transient pausing of pol II. The first class is solely composed of SII (6Wind M. Reines D. Bioessays. 2000; 22: 327-336Crossref PubMed Scopus (167) Google Scholar). The rest of the elongation factors, including TFIIF (7Price D.H. Sluder A.E. Greenleaf A.L. Mol. Cell. Biol. 1989; 9: 1465-1475Crossref PubMed Scopus (133) Google Scholar), Elongin A (8Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (289) Google Scholar), Elongin A2 (9Aso T. Yamazaki K. Amimoto K. Kuroiwa A. Higashi H. Matsuda Y. Kitajima S. Hatakeyama M. J. Biol. Chem. 2000; 275: 6546-6552Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), Elongin A3 (10Yamazaki K. Guo L. Sugahara K. Zhang C. Enzan H. Nakabeppu Y. Kitajima S. Aso T. J. Biol. Chem. 2002; 277: 26444-26451Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and Cockayne syndrome B (CSB) protein (11Selby C.P. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11205-11209Crossref PubMed Scopus (248) Google Scholar), all act to increase the overall rate of mRNA chain elongation by decreasing the frequency and/or duration of transient pausing of pol II at sites along the DNA template. Elongin was initially identified as a heterotrimer composed of A, B and C subunits of ∼770, 118, and 112 amino acids, respectively (8Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (289) Google Scholar,12Bradsher J.N. Jackson K.W. Conaway R.C. Conaway J.W. J. Biol. Chem. 1993; 268: 25587-25593Abstract Full Text PDF PubMed Google Scholar, 13Garrett K.P. Tan S. Bradsher J.N. Lane W.S. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5237-5241Crossref PubMed Scopus (65) Google Scholar, 14Garrett K.P. Aso T. Bradsher J.N. Foundling S.I. Lane W.S. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7172-7176Crossref PubMed Scopus (91) Google Scholar). Elongin A is the transcriptionally active subunit of the Elongin complex, whereas Elongins B and C are positive regulatory subunits. Biochemical studies have shown that Elongins B and C form a stable Elongin BC complex that binds to Elongin A and strongly induces its transcriptional activity (8Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (289) Google Scholar, 15Aso T. Haque D. Barstead R.J. Conaway R.C. Conaway J.W. EMBO J. 1996; 15: 5557-5566Crossref PubMed Scopus (89) Google Scholar). Elongin C functions as the inducing ligand and activates transcription through interaction with a short conserved motif (consensus sequence (T, S)(LXXXCXXX)(V,L,I)) in the elongation activation domain of Elongin A (15Aso T. Haque D. Barstead R.J. Conaway R.C. Conaway J.W. EMBO J. 1996; 15: 5557-5566Crossref PubMed Scopus (89) Google Scholar). Elongin B, a member of the ubiquitin homology protein family, appears to play a chaperone-like role in the assembly of the Elongin complex by binding to Elongin C and facilitating its interaction with Elongin A (8Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (289) Google Scholar, 14Garrett K.P. Aso T. Bradsher J.N. Foundling S.I. Lane W.S. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7172-7176Crossref PubMed Scopus (91) Google Scholar). Notably, Elongins B and C are also found as integral components of a multisubunit complex containing the von Hippel-Lindau (VHL) tumor suppressor protein (16Duan D.R. Pause A. Burgess W.H. Aso T. Chen D.Y.T. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (506) Google Scholar, 17Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (566) Google Scholar). Elongin A and the VHL protein share the Elongin BC binding site motif; and consistent with the assumption of a role for Elongin BC in tumor suppression, more than 70% of VHL mutations found in VHL families and sporadic clear cell renal carcinomas are associated with a mutation or deletion at this site (16Duan D.R. Pause A. Burgess W.H. Aso T. Chen D.Y.T. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (506) Google Scholar, 18Kishida T. Stackhouse T.M. Chen F. Lerman M.I. Zbar B. Cancer Res. 1995; 55: 4544-4548PubMed Google Scholar). To gain more insight into the role of Elongin A in vivo, we have inactivated both alleles of the Elongin A gene in mouse ES cells. We found that Elongin A is not essential for the viability of ES cells. Though, surprisingly, such Elongin A-deficient ES cells display several abnormalities, in cell size, cell growth, and cell cycle distribution, which are similar to those of cells with a delayed onset of mitosis. Furthermore, the absence of Elongin A protein affected the expression of only a subset of class II genes, indicating that mammalian Elongin A seemed to be required for pol II transcription of a subset but not all of genes. A 15-kb genomic fragment spanning the sequence encoding exons 2–11 and the 3′-UTR of murine Elongin A was isolated from a 129/Sv mouse genomic library, using a mouse Elongin A cDNA probe (19Aso T. Amimoto K. Takebayashi S. Okumura K. Hatakeyama M. Cytogenet. Cell Genet. 1999; 86: 259-262Crossref PubMed Google Scholar). Genomic fragments of 4.5 and 1.4 kb flanking exons 8–10 of Elongin A were used to generate a targeting construct in which exons 8–10 were replaced by a pol II-neo-poly(A) cassette (20Deng C. Thomas K.R. Capecchi M.R. Mol. Cell. Biol. 1993; 13: 2134-2140Crossref PubMed Scopus (84) Google Scholar). To increase the frequency of gene targeting, a pair of herpes simplex virus (HSV)-1 and HSV-2 thymidine kinase (TK) cassettes (TK1 and TK2, both under control of the MC1 promoter) was placed flanking the Elongin A genomic sequence in the targeting vector (21Mansour S.L. Thomas K.R. Capecchi M.R. Nature. 1988; 336: 348-352Crossref PubMed Scopus (1308) Google Scholar, 22Rancourt E.D. Tsuzuki T. Capecchi M.R. Genes Dev. 1995; 9: 108-122Crossref PubMed Scopus (232) Google Scholar). CCE ES cells were electroporated with the SalI-linearized targeting vector as described previously (23Robertson E.J. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, NY1987Google Scholar, 24Joyner A.L. Joyner A.L. Gene Targeting: A Practical Approach. IRL Press, NY1993Google Scholar), and heterozygous Elongin A-deficient clones were selected in the presence of 0.3 mg/ml G418 (Sigma) and 5 μm ganciclovir (Japan Syntechs). Among 200 ES cell colonies, three correctly targeted clones were identified by Southern blot analysis, using 5′ and 3′ external probes located outside the homologous regions of the targeting vector. Subsequently, homozygous Elongin A-deficient clones were obtained by selection in higher concentrations of G418 (from 1.5 to 2.0 mg/ml). Genomic DNA was isolated from ES cells, digested with either XbaI or BssHII, separated on a 0.65% agarose gel, and transferred to nylon membranes (Amersham Biosciences) by capillary blotting. The membrane was hybridized with random-primed 32P-labeled external probe (Fig. 1A) by using standard methods. Total RNA was prepared from cultured cells using ISOGEN (Nippon Gene). First-strand cDNA was synthesized using a first-strand cDNA synthesis kit (Amersham Biosciences) as described in the manufacturer's instructions. Subsequently, PCR was performed with primers A1 (5′-GCGTCCCAGAGATGCTCTTCAG-3′) and A2 (5′-TTATCGCCGGGAGAATCTGTTCTTGAATG-3′) to confirm the deletion of Elongin A. RT-PCR analysis of GAPDH mRNA was also performed as a control with primers mGA1 (5′-CTGCCATTTGCAGTGGCAAAG-3′) and mGA2 (5′-TGGTATTCAAGAGAGTAGGGA-3′). In vitro cell growth was assessed by plating 1 × 104 cells per well in six-well tissue culture dishes in triplicate. Cells were harvested at 24-h intervals by trypsinization and counted using a hemocytometer. FACS analysis was performed as described previously (25Oda S. Nishida J. Nakabeppu Y. Sekiguchi M. Oncogene. 1995; 10: 1343-1351PubMed Google Scholar, 26Tominaga Y. Tsuzuki T. Shiraishi A. Kawate H. Sekiguchi M. Carcinogenesis. 1997; 18: 889-896Crossref PubMed Scopus (86) Google Scholar). Briefly, ES cells were harvested and fixed with 70% ethanol. After being washed with ice-cold phosphate-buffered saline, fixed cells were stained for cellular DNA with propidium iodide (Sigma). The cell suspension was passed through a nylon mesh membrane and DNA content and cell numbers were analyzed with a FACS Calibur (Becton Dickinson). For each sample, 1 × 104 cells were analyzed, and the results were processed through Cell Quest software (BD Biosciences). Chromosome slides were prepared according to the standard cytogenetic method. Briefly, cultured cells were treated with 1.5 μg/ml TN-16 (3-(1-anilinoethylidene)-5-benzylpyrolidine-2,4-dione, Wako) for 2 h to collect metaphase cells. Cells were harvested using 0.25% trypsin-EDTA and centrifuged at 1000 rpm for 5 min. Cells were treated with hypotonic solution (75 mm KCl) for 20 min at room temperature and fixed with Carnoy's fixative (methanol:acetic acid = 3:1). Cell suspensions were dropped onto slide glass, then air-dried. Chromosome slides were stained with 3% Giemsa in phosphate-buffered saline for 10–20 min. Chromosome numbers were evaluated using a Zeiss Axiophot microscope under a ×100 objective. Poly(A)+ RNA was isolated according to the manufacturer's protocol using a MACS mRNA isolation kit (Miltenyi Biotec). cDNA generation, hybridization, and data collection were performed by Incyte Genomics. In brief, alterations in gene expression were evaluated by reverse transcription of poly(A)+ RNAs in the presence of Cy3 or Cy5 fluorescent labeling dyes followed by hybridization to a mouse GEM 2 microarray chip. Each chip carries a total of 9509 cDNAs. The data were analyzed using GEM Tools 2.5 software (Incyte Pharmaceuticals, Inc.). The RNase protection assay was performed using the mouse cell cycle regulator multiprobe template sets, mCYC-1, mCYC-2, and mCC-1 (BD Biosciences). [32P]UTP-labeled antisense RNA probe was synthesized using T7 RNA polymerase and was purified by phenol-chloroform extractions and ethanol precipitation. Purified probe was mixed with 10 μg of total RNA from ES cells in hybridization buffer and incubated for 16 h at 56 °C. Free probe and single-stranded RNA molecules were digested with a mixture of RNase A and T1. The RNase-protected molecules were purified and resolved on a denaturing polyacrylamide gel and dried. Autoradiographic signal was scanned on a BAS2000 image analyzer (Fuji), and the signal intensity of each band was quantified using MacBAS software (Fuji) and normalized to the corresponding GAPDH levels. To understand the physiological role of Elongin A in vivo, we disrupted the Elongin A gene in mouse ES cells. We first isolated and analyzed the structure of the mouse Elongin A gene (19Aso T. Amimoto K. Takebayashi S. Okumura K. Hatakeyama M. Cytogenet. Cell Genet. 1999; 86: 259-262Crossref PubMed Google Scholar). A gene-targeting vector was then constructed by replacing exons 8–10 of the Elongin A gene, which encode the domain essential for its transcriptional elongation activity (15Aso T. Haque D. Barstead R.J. Conaway R.C. Conaway J.W. EMBO J. 1996; 15: 5557-5566Crossref PubMed Scopus (89) Google Scholar), with a pol II-neo-poly(A) cassette (Fig.1A). Three independent heterozygous Elongin A-deficient ES cell lines were obtained by homologous recombination, and subsequently, homozygous Elongin A-deficient ES cell lines were generated by culturing these heterozygous ES cells in higher concentrations of G418. Genotyping of the isolated clones was determined by Southern blot analysis with flanking 5′- and 3′-UTR probes (Fig. 1B) and confirmed by RT-PCR (Fig. 1C). To investigate the phenotype of the Elongin A-deficient ES cells, we first compared growth rates of Elongin A-deficient and wild-type ES cells in vitro. As shown in Fig.2A, homozygous Elongin A-deficient ES cells grew about three times as slowly as wild type ES cells, whereas heterozygous Elongin A-deficient ES cells grew at rates indistinguishable from those of wild-type cells. Moreover, microscopic analysis revealed that the cellular and nuclear sizes of homozygous Elongin A-deficient ES cells were significantly larger than those of wild type and heterozygous deficient ES cells (Fig. 2B and data not shown). To clarify the cause of the growth defects of the homozygous Elongin A-deficient ES cells, we first used FACS analysis to determine cell cycle profiles. In contrast to wild type and heterozygous Elongin A-deficient ES cells, significantly more G2/M phase cells (or cells with a DNA content of 4N) than G1 phase cells were present in the homozygous Elongin A-deficient population. We also observed that homozygous Elongin A-deficient ES cells, but none of the other cells, included a substantial fraction with a DNA content of 8N (Fig. 3A). The increase of cells with a DNA content of 4N and the appearance of cells with that of 8N in the homozygous Elongin A-deficient population were confirmed by cytogenetic analysis (Fig. 3, B and C). These results imply that the G2/M phase is prolonged in homozygous Elongin A-deficient ES cells and that homozygous Elongin A-deficient ES cells go through DNA rereplication, even after completion of one cycle or DNA replication. Together, the observed abnormalities in homozygous Elongin A-deficient ES cells, such as increased cell size, more G2/M phase cells, and the presence of products of DNA rereplication, correspond to those of cells with delayed mitosis, which can be caused either by inhibition of G2/M transition or by stimulation of the initiation of DNA replication. We next intended to determine the mechanism responsible for the observed delayed mitosis. Thus, a cDNA microarray analysis was applied to compare populations and levels of mRNA in ES cells carrying or lacking functional Elongin A. Wild-type or homozygous Elongin A-deficient ES cells were grown to log phase, then poly(A)+ RNA was isolated and used for cDNA synthesis and subsequently hybridized to a microarray chip. Under our experimental conditions, mRNAs whose levels were decreased or increased by 2-fold or greater were considered as differentially expressed transcripts. The expression of the vast majority of genes was unaffected by disruption of the Elongin A gene. However, 241 (2.5%) of the genes assayed had a significantly down-regulated expression in Elongin A-deficient ES cells. Interestingly, a roughly equivalent number of genes was up-regulated by Elongin A inactivation (Fig.4A and see Supplemental Tables). However, we presently do not know whether this is a consequence of secondary effects due to loss of Elongin A function as a transcription elongation factor or primary effects caused by loss of Elongin A function, which the expression of a subset of genes. To the results of the microarray analysis and also to genes responsible for the delayed RNase protection assay was for of the expression of cell genes in ES cells (Fig. data not shown). with the microarray analysis, but not and were down-regulated by inactivation of Elongin A. However, the expression of the other cell genes, including the mRNAs of of G2/M such as and was either or was only in Elongin A-deficient ES cells. in vitro a for transcription elongation factors in transcriptional the functions of these factors in have not been we that mammalian cells in which a gene encoding one of the transcription elongation factors, Elongin A, was disrupted by homologous recombination had a delayed phenotype and but defects in RNA polymerase transcription. we have not been to a of Elongin A to delayed mitosis, we can several is that Elongin A is required for the transcription of genes in G2/M and a in those transcripts caused by Elongin A inactivation results in a delayed onset of mitosis. In this Elongin A be a for transcriptional and to the regions of genes. both a action and delayed phenotype inactivation of general transcription factors have been M. J. S. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar, J. J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, M.R. Genes Dev. 1996; 10: PubMed Scopus Google Scholar, F. M.R. Nature. 2000; PubMed Scopus Google Scholar). TATA-binding protein is for the transcription of and the of expression by heterozygous deletion of the of which in to delayed M. J. S. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). Furthermore, factor or is also for the transcription of and A, or of the and of the and its deletion results in G2/M cell cycle J. J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, F. M.R. Nature. 2000; PubMed Scopus Google Scholar). Thus, is that Elongin A is required for one or more genes in through the G2/M of the cell our not identified such genes. The other is that be a functional between transcription elongation by Elongin A and DNA recombination, as was found in transcription elongation factors, and S. A. Genes Dev. 1997; PubMed Scopus Google Scholar, A. EMBO J. PubMed Scopus Google Scholar, S. T. H. T. A. EMBO J. 2000; 21: Scopus Google Scholar). in these two genes have been to a which results from of the recombination to the of RNA polymerase II elongation this is also the in mammalian Elongin A, of functional Elongin A a phenotype and genomic which in being delayed by the we presently do not have of interaction between Elongin A and recombination However, is also that Elongin A carries other cellular functions to transcription elongation in In Elongin A been shown to form a stable complex with the components of the ubiquitin and T. D. S.L. Lane W.S. Shilatifard A. Conaway R.C. Conaway J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we presently do not know whether this complex ubiquitin is that Elongin A with and the of factors, such as of the factor and positive of the initiation of DNA by a Thus, Elongin A function in the cell cycle by a mechanism that not through a transcription In our results that mammalian Elongin A is essential for transcription of a subset but not all of genes and is for cell cycle progression. in vitro be to understand the by which loss of functional Elongin A results in delayed onset of mitosis. with