Transcriptional Regulation of the Cytosolic Chaperonin θ Subunit Gene, Cctq, by Ets Domain Transcription Factors Elk-1, Sap-1a, and Net in the Absence of Serum Response Factor
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m212242200
ISSN1083-351X
AutoresYuji Yamazaki, Hiroshi Kubota, Masami Nozaki, Kazuhiro Nagata,
Tópico(s)RNA Research and Splicing
ResumoThe chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone that facilitates protein folding in eukaryotic cytosol, and the expression of CCT is highly dependent on cell growth. We show here that transcription of the gene encoding the θ subunit of mouse CCT, Cctq, is regulated by the ternary complex factors (TCFs), Elk-1, Sap-1a, and Net (Sap-2). Reporter gene assay using HeLa cells indicated that the Cctq gene promoter contains a cis-acting element of the CCGGAAGT sequence (CQE1) at –36 bp. The major CQE1-binding proteins in HeLa cell nuclear extract was recognized by anti-Elk-1 or anti-Sap-1a antibodies in electrophoretic mobility shift assay, and recombinant Elk-1, Sap-1a, or Net specifically recognized CQE1. The CQE1-dependent transcriptional activity in HeLa cells was virtually abolished by overexpression of the DNA binding domains of TCFs. Overexpression of full-length TCFs with Ras indicated that exogenous TCFs can regulate the CQE1-dependent transcription in a Ras-dependent manner. PD98059, an inhibitor of MAPK, significantly repressed the CQE1-dependent transcription. However, no serum response factor was detected by electrophoretic mobility shift assay using the CQE1 element. These results indicate that transcription of the Cctq gene is regulated by TCFs under the control of the Ras/MAPK pathway, probably independently of serum response factor. The chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone that facilitates protein folding in eukaryotic cytosol, and the expression of CCT is highly dependent on cell growth. We show here that transcription of the gene encoding the θ subunit of mouse CCT, Cctq, is regulated by the ternary complex factors (TCFs), Elk-1, Sap-1a, and Net (Sap-2). Reporter gene assay using HeLa cells indicated that the Cctq gene promoter contains a cis-acting element of the CCGGAAGT sequence (CQE1) at –36 bp. The major CQE1-binding proteins in HeLa cell nuclear extract was recognized by anti-Elk-1 or anti-Sap-1a antibodies in electrophoretic mobility shift assay, and recombinant Elk-1, Sap-1a, or Net specifically recognized CQE1. The CQE1-dependent transcriptional activity in HeLa cells was virtually abolished by overexpression of the DNA binding domains of TCFs. Overexpression of full-length TCFs with Ras indicated that exogenous TCFs can regulate the CQE1-dependent transcription in a Ras-dependent manner. PD98059, an inhibitor of MAPK, significantly repressed the CQE1-dependent transcription. However, no serum response factor was detected by electrophoretic mobility shift assay using the CQE1 element. These results indicate that transcription of the Cctq gene is regulated by TCFs under the control of the Ras/MAPK pathway, probably independently of serum response factor. The chaperonin-containing t-complex polypeptide 1 (CCT) 1The abbreviations used are: CCT, chaperonin-containing t-complex polypeptide 1; TCF, ternary complex factor; SRE, serum response element; SRF, serum response factor; CQE, CCT θ subunit gene transcription-activating element; E74-EBS, Ets binding site in E74 gene; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; WT, wild type.1The abbreviations used are: CCT, chaperonin-containing t-complex polypeptide 1; TCF, ternary complex factor; SRE, serum response element; SRF, serum response factor; CQE, CCT θ subunit gene transcription-activating element; E74-EBS, Ets binding site in E74 gene; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; WT, wild type. is a molecular chaperone assisting in the folding of newly synthesized proteins in the cytosol upon ATP hydrolysis (1Kubota H. Hynes G. Willison K. Eur. J. Biochem. 1995; 230: 3-16Crossref PubMed Scopus (256) Google Scholar, 2Valpuesta J.M. Martin-Benito J. Gomez-Puertas P. Carrascosa J.L. Willison K.R. FEBS Lett. 2002; 26423: 1-6Google Scholar). CCT belongs to the chaperonin family that includes mitochondrial HSP60, bacterial GroEL, plastid ribulosebisphosphate carboxylase/oxygenase subunit-binding protein, and archea group II chaperonins. Tubulin and actin are major substrates of CCT (3Tian G. Huang Y. Rommeaere H. Vandekerckhove J. Ampe C. Cowan N.J. Cell. 1996; 86: 287-296Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 4Farr G.W. Scharl E.C. Schumacher R.J. Sondek S. Horwich A.L. Cell. 1997; 89: 927-937Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 5Thulasiraman V. Yang C.-F. Frydman J. EMBO J. 1999; 18: 85-95Crossref PubMed Scopus (264) Google Scholar, 6Llorca O. Martin-Benito J. Grantham J. Ritco-Vonsovici M. Willison K.R. Carrascosa J.L. Valpuesta J.M. EMBO J. 2001; 20: 4065-4075Crossref PubMed Scopus (115) Google Scholar), and many other proteins, including cyclin E (7Won K.-A. Schumacher R.J. Farr G.W. Horwich A.L. Reed S.I. Mol. Cell. Biol. 1998; 18: 7584-7589Crossref PubMed Scopus (133) Google Scholar), transducin (4Farr G.W. Scharl E.C. Schumacher R.J. Sondek S. Horwich A.L. Cell. 1997; 89: 927-937Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), myosin (8Srikakulam R. Winkelmann D.A. J. Biol. Chem. 1999; 274: 27265-27273Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), VHL tumor suppressor (9Feldman D.E. Thulasiraman V. Ferreyra R.G. Frydman J. Mol. Cell. 1999; 4: 1051-1061Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and luciferase (10Frydman J. Hartl F.U. Science. 1996; 272: 1497-1502Crossref PubMed Scopus (215) Google Scholar, 11Gebauer M. Melki R. Gehring U. J. Biol. Chem. 1998; 273: 29475-29480Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), are reported to be CCT substrates. In vivo, ∼15% of newly synthesized proteins are estimated to be folded with the assistance of CCT (5Thulasiraman V. Yang C.-F. Frydman J. EMBO J. 1999; 18: 85-95Crossref PubMed Scopus (264) Google Scholar). Double torus-like structure of CCT with 8-fold symmetry was revealed by electron microscopy (12Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (228) Google Scholar), and CCT is composed of eight different subunits of ∼60 kDa each: α, β, γ, δ, ϵ, ζ–1, η, and θ (13Kubota H. Hynes G. Carne A. Ashworth A. Willison K. Curr. Biol. 1994; 4: 89-99Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 14Kubota H. Hynes G. Willison K. Gene (Amst.). 1995; 154: 231-236Crossref PubMed Scopus (75) Google Scholar). These subunits are encoded by independent genes (15Kubota H. Yokota S. Yanagi H. Yura T. Eur. J. Biochem. 1999; 262: 492-500Crossref PubMed Scopus (51) Google Scholar) and share 30% identity at the amino acid sequence level (1Kubota H. Hynes G. Willison K. Eur. J. Biochem. 1995; 230: 3-16Crossref PubMed Scopus (256) Google Scholar). We determined entire nucleotide sequences of all of the genes encoding CCT subunits and showed that many of them exhibit strong transcriptional activity comparable with the combination of SV40 promoter and enhancer (15Kubota H. Yokota S. Yanagi H. Yura T. Eur. J. Biochem. 1999; 262: 492-500Crossref PubMed Scopus (51) Google Scholar). The expression levels of CCT subunits are strongly correlated with growth rate and markedly up-regulated at G1/S transition through early S phase in cultured cells (16Yokota S. Yanagi H. Yura T. Kubota H. J. Biol. Chem. 1999; 274: 37070-37078Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). CCT subunits are significantly increased in tumor tissues, and the expression levels of CCT subunits are highly correlated with that of proliferating cell nuclear antigen (17Yokota S. Yamamoto Y. Shimizu K. Momoi H. Kamikawa T. Yamaoka Y. Yanagi H. Yura T. Kubota H. Cell Stress Chaperones. 2001; 6: 345-350Crossref PubMed Scopus (79) Google Scholar). The expression patterns of different subunits are basically similar but distinct in synthesis and degradation rates, leading to differential turnover (18Yokota S. Yanagi H. Yura T. Kubota H. Eur. J. Biochem. 2001; 268: 4664-4673Crossref PubMed Scopus (44) Google Scholar). Although CCT is up-regulated during recovery from chemical stress (19Yokota S.I. Yanagi H. Yura T. Kubota H. Eur. J. Biochem. 2000; 267: 1658-1664Crossref PubMed Scopus (64) Google Scholar), and heat shock transcription factors may mediate the up-regulation (20Kubota H. Matsumoto S. Yokota S. Yanagi H. Yura T. FEBS Lett. 1999; 461: 125-129Crossref PubMed Scopus (26) Google Scholar), this stress response is unrelated to cell growth. Recently, we found that transcription of the gene encoding the α subunit of CCT, Ccta, is regulated by selenocysteine tRNA gene transcription-activating factor family transcription factors ZNF143 and ZNF76 (21Kubota H. Yokota S. Yanagi H. Yura T. J. Biol. Chem. 2000; 275: 28641-28648Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The level of ZNF143 is regulated by the mitogen-activated protein kinase (MAPK) pathway (22Adachi K. Tanaka T. Saito H. Oka T. Endocrinology. 1999; 140: 618-623Crossref PubMed Scopus (15) Google Scholar). Although these results suggested that a transcriptional control mechanism related to cell growth plays a role in CCT subunit regulation, little is known about common transcriptional mechanisms controlling the expression of different CCT subunits. Elk-1 (Ets-like transcription factor), Sap-1a (SRF accessory protein), and Net (new Ets transcription factor; also called Sap-2) comprise the ternary complex factor (TCF) subfamily of the Ets domain transcription factors (23Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 24Wasylyk B. Hagman J. Gutierrez-Hartmann A. Trends Biochem. Sci. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 25Sharrocks A.D. Nat. Rev. Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (803) Google Scholar). Ets family proteins specifically recognize DNA sequences containing the core trinucleotide GGA using a highly conserved DNA-binding domain (23Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 26Sementchenko V.I. Watson D.K. Oncogene. 2000; 19: 6533-6548Crossref PubMed Scopus (309) Google Scholar). The core sequence is essential for Ets factor binding, and its flanking sequences are important for determining the different sequence specificity of these proteins. Elk-1, Sap-1a, or Net forms ternary complexes with serum response factor (SRF) on the serum response elements (SREs) of immediate early gene promoters, including c-fos (27Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (532) Google Scholar) and egr-1 (28Clarkson R.W. Shang C.A. Levitt L.K. Howard T. Waters M.J. Mol. Endocrinol. 1999; 13: 619-631Crossref PubMed Scopus (61) Google Scholar, 29Ayadi A. Zheng H. Sobieszczuk P. Buchwalter G. Moerman P. Alitalo K. Wasylyk B. EMBO J. 2001; 20: 5139-5152Crossref PubMed Scopus (111) Google Scholar), and play important roles in the transcriptional regulation of these genes. These three TCF subfamily proteins share three conserved domains: the DNA-binding domain localized at the amino terminus (30Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar), the SRF-binding domain in the middle portion (31Shore P. Sharrocks A.D. Mol. Cell. Biol. 1994; 14: 3283-3291Crossref PubMed Scopus (135) Google Scholar), and the transcriptional activation domain whose activity is stimulated by phosphorylation by MAPK at the carboxyl terminus (32Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (504) Google Scholar, 33Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar, 34Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar). Although the sequence specificity and transcriptional activity of Elk-1 and Sap-1a are similar each other, Net differs from these proteins by containing two additional domains, the Net-inhibitory domain (35Maira S.M. Wurtz J.M. Wasylyk B. EMBO J. 1996; 15: 5849-5865Crossref PubMed Scopus (77) Google Scholar) and the CtBP interaction domain of Net (36Criqui-Filipe P. Ducret C. Maira S.M. Wasylyk B. EMBO J. 1999; 18: 3392-3403Crossref PubMed Scopus (135) Google Scholar), which strongly inhibit the transcription of target genes. Although the TCF subfamily proteins alone show no significant binding to SREs (33Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar), the Drosophila E74 gene promoter contains a DNA element recognized by these factors independently of SRF (37Mo Y. Vaessen B. Johnston K. Marmorstein R. Mol. Cell. 1998; 2: 201-212Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 38Rao V.N. Reddy E.S.P. Oncogene. 1992; 7: 65-70PubMed Google Scholar). However, little is known about SRF-independent target sequence of Elk-1, Sap-1a, or Net in mammalian genes. Here, we examined transcriptional regulation of the gene encoding the mouse CCT θ subunit, Cctq, and found that the Cctq gene promoter contains a cis-acting element (CQE1) recognized by Elk-1, Sap-1a, and Net independently of SRF. By overexpression of the TCF subfamily proteins with Ras, CQE1-dependent transcriptional activity was regulated in response to Ras activity. CQE1-dependent transcriptional activity of the Cctq gene, as well as activity of a reporter construct containing the Ccta gene promoter, was significantly affected by a MAPK inhibitor. We discuss the role of TCF subfamily proteins in Cctq gene transcription and possible common effects of the Ras/MAPK pathway on the growth-dependent expression of different CCT subunit genes. Chemicals and Antibodies—MEK1/2 inhibitor PD98059 was purchased from Calbiochem. Antibodies against human Elk-1 (I-20), Sap-1a (C-20), Net (C-20), SRF (G-20), and phosphorylated Elk-1 (B-4) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Reporter and Effector Constructs—The reporter construct containing the 5′-flanking region and first intron of the mouse Cctq gene in the firefly luciferase expression vector pGL3-basic (pCQL) was described previously (15Kubota H. Yokota S. Yanagi H. Yura T. Eur. J. Biochem. 1999; 262: 492-500Crossref PubMed Scopus (51) Google Scholar). The first intron of pCQL was removed by a PCR-mediated subcloning method using an intronless sequence as a primer. The resulting construct (pCQL-pro) was deleted unidirectionally from 5′ upstream of the Cctq promoter by a modified method of Henikoff (39Henikoff S. Gene (Amst.). 1984; 3: 351-359Crossref Scopus (2828) Google Scholar) using mung bean nuclease instead of S1 nuclease (Fig. 1A). Alternatively, some deletion constructs were produced by cloning PCR-amplified DNA fragments. Nucleotide substitutions, deletions, and insertions were also introduced into the reporter constructs by PCR. Tandem repeats of cis-acting DNA elements were produced by cloning self-ligated synthetic oligonucleotides. A firefly luciferase reporter gene driven by the herpes simplex virus thymidine kinase promoter (–80 to +38) was constructed by cloning a PCR-amplified DNA fragment into the pGL3-basic vector (named ptk-80Luc). Human Elk-1 (40Rao V.N. Huebner K. Isobe M. ar-Rushdi A. Croce C.M. Reddy E.S. Science. 1989; 244: 66-70Crossref PubMed Scopus (196) Google Scholar), Sap-1a (27Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (532) Google Scholar), and Net (Sap-2) (33Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar) cDNAs were amplified from human cDNA libraries (BD Biosciences Clontech, Palo Alto, CA) by PCR using previously published sequences as primers and cloned into the pCAGGS mammalian expression vector (41Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4524) Google Scholar). cDNA fragments encoding the DNA binding domains of Elk-1-(1–95), Sap-1a-(1–95), and Net-(1–95) were also subcloned into pCAGGS using PCR-based methods. Nucleotide sequences of the reporter and effector constructs were confirmed by sequencing. Ras expression vectors pCMV-Ras, pCMV-RasV12, and pCMV-RasN17 (Clontech) and the internal control sea pansy expression vector pRL-SV40 (Promega, Madison, WI) were purchased from the sources shown. Supercoiled plasmid DNA prepared by ultracentrifugation in CsCl solution was used for transfection. Cell Culture and Reporter Gene Assay—HeLa cells were maintained in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum (Invitrogen). NIH 3T3 cells were cultured in Dulbecco's modified essential medium containing 10% calf serum (Colorado Serum Company, Denver, CO). Cells (3.0 × 105) were plated in 35-mm dishes, cultured for 24 h, and then transfected with a total of 1.2 μg of reporter, effector, and internal control plasmid DNA using 10 μl of LipofectAMINE reagent (Invitrogen), according to the manufacturer's instructions. After 5 h of exposure to the DNA-LipofectAMINE complex, cells were cultured in medium containing 10% serum for 19 h. Luciferase activities of transfected cells were determined using the dual luciferase assay system (Promega, Madison, WI) according to the manufacturer's instructions, and the activity of firefly luciferase was normalized against that of the sea pansy enzyme. Purification of Glutathione S-Transferase (GST) Fusion Proteins— The cDNA fragments encoding DNA binding domains of human Elk-1, Sap-1a, or Net were subcloned into the GST fusion protein expression vector pGEX-6P (Amersham Biosciences). Escherichia coli carrying each plasmid were cultured overnight at 20 °C. Bacteria were sonicated in phosphate-buffered saline containing 0.25 mm (p-amidinophenyl-)methanesulfonyl fluoride hydrochloride, and the GST fusion protein was purified using glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. The purified proteins were stored in phosphate-buffered saline at 4 °C. Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extract was prepared according to Sadowski and Gilman (42Sadowski H.B. Gilman M.Z. Nature. 1993; 362: 79-83Crossref PubMed Scopus (234) Google Scholar). Radiolabeled probes were prepared by labeling double-stranded synthetic oligonucleotides with [32P]dCTP using the Klenow fragment. HeLa cell nuclear extract (5 μg) or purified GST fusion proteins (20 ng) were preincubated for 10 min on ice in the presence of 2 μg of poly(dI-dC) (Amersham Biosciences) in 20 μl of reaction buffer containing 10 mm Tris-HCl (pH 7.5), 1 mm MgCl2, 0.5 mm dithiothreitol, and 10% glycerol, and labeled probes (0.1 ng) were added and incubated for 30 min at room temperature. Complexes of probe DNA and binding proteins were separated by electrophoresis on 4% polyacrylamide gels (29:1) in 0.25× TBE buffer at 150 V and 4 °C for 2.25 h and visualized using a STORM 820 image analyzer (Amersham Biosciences). Cis-acting Elements Up-regulating Cctq Gene Transcription—Previously, we reported a firefly luciferase reporter gene construct driven by the mouse Cctq gene promoter/enhancer (pCQL) (15Kubota H. Yokota S. Yanagi H. Yura T. Eur. J. Biochem. 1999; 262: 492-500Crossref PubMed Scopus (51) Google Scholar). This contains a DNA fragment from 2027 bp upstream of the transcription start point to the second exon of Cctq and showed strong transcriptional activity: 1.6-fold a combination of the SV40 promoter and enhancer in HeLa cells. By removing the first intron, the reporter construct containing the 2027-bp promoter of Cctq (pCQL-pro) (Fig. 1A, top) was produced. To search for cis-acting elements in the Cctq promoter, the 2027-bp promoter region of pCQL-pro was unidirectionally deleted from the 5′-end (Fig. 1A). HeLa cells were transiently transfected with these constructs, and luciferase activities of cell extracts were determined. In the segment extending from –2027 to –176, activities down-regulating Cctq transcription were detected. Trans-acting factors repressing Cctq promoter activity may bind this region. No significant effect on Cctq transcription was observed for the region between –176 and –85. Deletion from –85 to –40 slightly reduced transcriptional activity, suggesting a contribution of weakly positive cis-acting elements, including a possible Sp1-binding site found in this region (Fig. 1C). Deletion from –40 to –19 remarkably reduced the activity to 30%, indicating the existence of strong cis-acting elements in this region. Since transcriptional activity was almost completely abolished by deleting the construct from –19 to –3, this region is probably important for the initiation of transcription. To analyze the strong cis-acting activity immediately downstream of –40 bp in more detail, double point mutations were introduced into the reporter gene construct containing the Cctq promoter up to –40 bp, and the luciferase activities of these constructs were analyzed using HeLa cells (Fig. 1B). Mutations on the residues from –36 to –29 bp reduced the activity by 40–60% relative to wild type, whereas mutations on residues from –40 to –37 bp or –28 to –25 bp exhibited little effect. These results indicate that the eight residues from –36 to –29 bp make a significant contribution to Cctq transcription and suggest that a trans-acting factor recognizes the 8-bp-long element CCGGAAGT. We named this element CQE1 (for Cctq gene transcription-activating element 1). Results very similar to Fig. 1, A and B, were obtained using a distinct cell line, mouse fibroblast NIH 3T3 (data not shown), implying that this element is recognized by transcription factors common to many cell types. Computational Search for CQE1-binding Proteins—We searched for possible trans-acting factors recognizing the CQE1 sequences by computer programs, and the results indicated that the CQE1 sequence is highly homologous to the binding consensus sequence of the human Ets domain transcription factor Elk-1, a member of TCF subfamily (Fig. 2B). Of the 11-bp Elk-1 binding consensus determined by PCR-mediated affinity selection of binding DNAs (30Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar), 10 residues match the CQE1 sequence, and only one substitution (A to G) occurs at the 5′-end of the consensus. This 5′-end substitution (A to G) was also found in the affinity selection (30Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar), suggesting that contact at the 5′-end residue may be weaker than that of other residues in the consensus. Consistent with this view, mutation at this site had no significant effect in our reporter gene assay at this site (Fig. 1B). Since another TCF subfamily transcription factor, Sap-1a (27Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (532) Google Scholar), shows characteristics very similar to Elk-1 (33Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar, 43Janknecht R. Ernst W.H. Nordheim A. Oncogene. 1995; 10: 1209-1216PubMed Google Scholar) (Fig. 2A), we also compared its binding consensus sequence (30Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar) with CQE1. The 9-bp Sap-1a binding consensus exhibited perfect match with CQE1. In addition, a third member of TCF subfamily, Net (44Giovane A. Pintzas A. Maira S.M. Sobieszczuk P. Wasylyk B. Genes Dev. 1994; 8: 1502-1513Crossref PubMed Scopus (202) Google Scholar), has been identified. Net differs from Elk-1 and Sap-1a in its transcriptional suppressor activity (35Maira S.M. Wurtz J.M. Wasylyk B. EMBO J. 1996; 15: 5849-5865Crossref PubMed Scopus (77) Google Scholar, 36Criqui-Filipe P. Ducret C. Maira S.M. Wasylyk B. EMBO J. 1999; 18: 3392-3403Crossref PubMed Scopus (135) Google Scholar) (Fig. 2A). Although the binding of all three TCF subfamily proteins to the known mammalian target sequence in the c-fos promoter is SRF-dependent (33Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar), a Drosophila gene, E74, is known to contain a DNA element with an Ets-binding motif (45Urness L.D. Thummel C.S. Cell. 1990; 63: 47-61Abstract Full Text PDF PubMed Scopus (143) Google Scholar) (E74-EBS in Fig. 2B) specifically recognized by human Elk-1 and/or Sap-1a in the absence of SRF both in vitro (37Mo Y. Vaessen B. Johnston K. Marmorstein R. Mol. Cell. 1998; 2: 201-212Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 38Rao V.N. Reddy E.S.P. Oncogene. 1992; 7: 65-70PubMed Google Scholar, 46Shore P. Bisset L. Lakey J. Waltho J.P. Virden R. Sharrocks A.D. J. Biol. Chem. 1995; 270: 5805-5811Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and in vivo (32Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (504) Google Scholar, 34Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar, 43Janknecht R. Ernst W.H. Nordheim A. Oncogene. 1995; 10: 1209-1216PubMed Google Scholar). We therefore compared the sequence of E74-EBS with CQE1 and found that these sequences are nearly identical at the Elk-1/Sap-1a binding region (Fig. 2B). These observations suggest that CQE1 is a sequence recognized by the TCF subfamily proteins in the absence of SRF. Sequence-specific Recognition of CQE1 by HeLa Cell Nuclear Factors—To characterize the DNA binding activities of transcription factors acting on CQE1 in HeLa cells, HeLa cell nuclear extract was analyzed by EMSA using synthetic oligonucleotide probes (Fig. 3). The CQE1 probe gave two bands: a broad band with a strong signal (band I) and a thin band with a much weaker signal (band II). Although the binding of labeled CQE1 for the two shift bands was strongly competed by excess unlabeled CQE1 with wild-type sequence, a mutant CQE1 carrying nucleotide substitutions in the GGAA motif, QDM4 (see Fig. 1B), did not compete even at a 300-fold excess, consistent with the results of the reporter assay (Fig. 1B). A mutation in the region 3′ to the GGAA motif, QDM6, also significantly weakened the ability of competition. The slightly weaker competition of QDM4 than QDM6 is also consistent with the reduced activity of reporter in vivo. The recognition by CQE1-binding factor is likely to be more strict for the GGAA motif than for surrounding sequences, in agreement with common characteristics of Ets family transcription factors. These results indicate that the factors giving bands I and II specifically recognize the CQE1 sequence and that these factors may correspond to the same trans-acting factors postulated by the results of reporter gene assay. Since CQE1 shows a strong resemblance to the SRF-independent Elk-1 binding sequence found in the Drosophila E74 gene, E74-EBS (see Fig. 2B), we tested whether the CQE1-binding factors recognize E74-EBS by competition experiments. E74-EBS strongly competed with the CQE1 probe for binding (Fig. 3B), indicating that the factors giving bands I and II recognize both CQE1 and E74-EBS. Results similar to Fig. 3B were obtained using E74-EBS as a labeled probe, and the mobility of bands I and II was the same as that of CQE1 probe (Fig. 3C). Excess unlabeled CQE1 competed strongly with E74-EBS for band I but relatively weakly for band II, suggesting that binding of the protein giving band II is slightly stronger for E74-EBS than for CQE1. These results indicate that binding factors are common between E74-EBS and CQE1 for bands I and II and support the idea that the factors recognizing CQE1 belong to the TCF subfamily because the Drosophila E74-EBS sequence was previously shown to be recognized by Elk-1 and Sap-1a in mammalian cells (32Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (504) Google Scholar, 34Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar, 43Janknecht R. Ernst W.H. Nordheim A. Oncogene. 1995; 10: 1209-1216PubMed Google Scholar). The Major HeLa Cell Nuclear Factors Recognizing CQE1 Are Elk-1 and Sap-1a—To determine whether the CQE1-binding factors identified in EMSA are TCF subfamily proteins, we performed supershift analysis using antibodies against Elk-1, Sap-1a, and Net. With the antibodies against Elk-1 or Sap-1a, CQE1-bound proteins of band I were supershifted to more slowly migrating bands. The shift pattern indicated that the broad band I was doublet of Elk-1 and Sap-1a; Elk-1 migrated slightly more slowly than Sap-1a (Fig. 4A). The slightly slower mobility of Elk-1 relative to Sap-1a in EMSA is consistent with previous observations using E74 probe (34Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar). Using a mixture of anti-Elk-1 and anti-Sap-1a antibodies, almost all proteins consisting of band I were supershifted, confirming that band I is a doublet of Elk-1 and Sap-1a. Since Elk-1 is known to be regulated by the phosphorylation of its transactivation domain, we examined whether CQE1-bound Elk-1 was phosphorylated, using a monoclonal antibody recognizing phosphorylated Elk-1 transactivation domain. All proteins in band I were supershifted with this antibody. Since the amino acid sequence of the phosphorylated Elk-1 synthetic polypeptide used for antibody production shows significant identity to that of Sap-1a, the antibody against phosphorylated Elk-1 probably recognized phospho
Referência(s)