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• W2015278341 abstract "Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis. Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis. Two genes, cell death specification protein 1 (ces-1) and ces-2, control the decisions of neuro-secretory motor sister cells in Caenorhabditis elegans to undergo apoptosis. A genetic approach showed that these factors regulate the genes required for apoptosis and that a gain of ces-1 function or a reduction of ces-2 function prevents these cells from dying (1Ellis R.E. Horvitz H.R. Development (Camb.). 1991; 112: 591-603Crossref PubMed Google Scholar). The pro-apoptotic CES-2 protein negatively regulates CES-1, which prevents the death of the neuro-secretory motor sister cells by transcriptional silencing of Egl-1, a BH3-only protein required for apoptosis in C. elegans (2Thellmann M. Hatzold J. Conradt B. Development (Camb.). 2003; 130: 4057-4071Crossref PubMed Scopus (89) Google Scholar, 3Metzstein M.M. Horvitz H.R. Mol. Cell. 1999; 4: 309-319Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). CES-2 is similar to members of the proline- and acid-rich (PAR) 4The abbreviations used are: PAR, proline- and acid-rich subfamily; bZIP, basic region leucine zipper transcription factors; TEF, thyrotroph embryonic factor; DBP, albumin D-site-binding protein; tDBP, truncated DBP; HLF, hepatic leukemia factor; EMSA, electrophoretic mobility shift assay; RT, reverse transcription; GFP, green fluorescent protein; siRNA, small interfering RNA. subfamily of basic region leucine zipper (bZIP) transcription factors, and both share the DNA binding specificity (4Metzstein M.M. Hengartner M.O. Tsung N. Ellis R.E. Horvitz H.R. Nature. 1996; 382: 545-547Crossref PubMed Scopus (140) Google Scholar). Mammalian homologs of CES-2 include thyrotroph embryonic factor (TEF), albumin D-site-binding protein (DBP), and hepatic leukemia factor (HLF) (5Hunger S.P. Ohyashiki K. Toyama K. Cleary M.L. Genes Dev. 1992; 6: 1608-1620Crossref PubMed Scopus (185) Google Scholar, 7Khatib Z.A. Inaba T. Valentine M. Look A.T. Genomics. 1994; 23: 344-351Crossref PubMed Scopus (26) Google Scholar). These PAR bZIP proteins have recently been shown to be involved in amino acid and neurotransmitter metabolism through transcriptome profiling analyses in both liver and brain (8Gachon F. Fonjallaz P. Damiola F. Gos P. Kodama T. Zakany J. Duboule D. Petit B. Tafti M. Schibler U. Genes Dev. 2004; 18: 1397-1412Crossref PubMed Scopus (194) Google Scholar). Thus, despite the pro-apoptotic activity described for CES-2 promoting the induction of Egl-1 through an indirect pathway, none of its human homologs have been associated with the transcriptional regulation of BH3-only genes or other executors of apoptosis. BH3-only proteins are a pro-apoptotic subgroup of the Bcl-2 family of apoptosis regulators, which share only the short BH3 region with the rest of the family (9Bouillet P. Strasser A. J. Cell Sci. 2002; 115: 1567-1574Crossref PubMed Google Scholar). Genetic experiments have shown that these proteins are essential initiators of programmed cell death in species as distantly related as mice and C. elegans. They are regulated transcriptionally and by post-translational modifications such as phosphorylation, ubiquitination, and proteolytic cleavage (10Yu J. Zhang L. Biochem. Biophys. Res. Commun. 2005; 331: 851-858Crossref PubMed Scopus (366) Google Scholar, 13del Gonzalez-Garcia M. Peso L. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1986) Google Scholar). BH3-only proteins include Bid, Bad, Bim, Puma, Noxa, Hrk, Bik, Bmf, Bnip3, and Bnip3L (14Gelinas C. White E. Genes Dev. 2005; 19: 1263-1268Crossref PubMed Scopus (98) Google Scholar, 15Strasser A. Nat. Rev. Immunol. 2005; 5: 189-200Crossref PubMed Scopus (516) Google Scholar). Another member, Bcl-gS, the short splice variant of the bcl-g gene, which is uniquely expressed in testis, has recently joined this list (16Guo B. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 2780-2785Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). BH3-only proteins serve as upstream sentinels that selectively respond to apoptotic stimuli inducing activation of other members, mainly bax and bak, required for execution of cell death (17Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar). Blockade of expression or activation of different BH3-only proteins has been associated with cancer cell survival. To this end, Hrk is methylated in colorectal and gastric cancer cell lines, which correlates with loss of expression of this proapoptotic gene. Restoration of Hrk promotes apoptosis and enhances adriamycin-induced cell death (18Obata T. Toyota M. Satoh A. Sasaki Y. Ogi K. Akino K. Suzuki H. Murai M. Kikuchi T. Mita H. Itoh F. Issa J.P. Tokino T. Imai K. Clin. Cancer Res. 2003; 9: 6410-6418PubMed Google Scholar). We previously described that Hrk expression is silenced in hematopoietic progenitors by a growth factor-dependent transcriptional repressor mechanism that avoids inappropriate apoptosis in these cells (19Sanz C. Mellstrom B. Link W. Naranjo J. Fernandez-Luna J. EMBO J. 2001; 20: 2286-2292Crossref PubMed Scopus (86) Google Scholar). Blockade of other BH3-only genes such as Noxa or Bim also promotes resistance to chemotherapeutic agents in different tumor cell models (20Tan T.T. Degenhardt K. Nelson D.A. Beaudoin B. Nieves-Neira W. Bouillet P. Villunger A. Adams J.M. White E. Cancer Cell. 2005; 7: 227-238Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 21Qin J.Z. Ziffra J. Stennett L. Bodner B. Bonish B.K. Chaturvedi V. Bennett F. Pollock P.M. Trent J.M. Hendrix M.J. Rizzo P. Miele L. Nickoloff B.J. Cancer Res. 2005; 65: 6282-6293Crossref PubMed Scopus (264) Google Scholar). Therefore, inactivation or transcriptional repression of BH3-only proteins may represent an advantage for tumor cells to escape chemotherapy-induced apoptosis, and consequently, deciphering the regulation of these genes is crucial to understand the mechanism by which they contribute to cell fate. We report here a novel transcriptional pathway, mediated by PAR bZIP proteins, mainly TEF, that promotes the expression of a BH3-only gene. TEF binds to a consensus site in the promoter region of bcl-gS and induces its expression. bcl-gS is upregulated by transfection of tumor cells with TEF or drastically reduced by NFIL3, a putative antagonist of PAR bZIP proteins, or by a dominant negative isoform of DBP. Additionally, the TEF-bcl-gS transcriptional pathway can be triggered by chemotherapeutic agents and mediates in part the apoptotic response. This is the first description of PAR bZIP transcription factors that activate the expression of a BH3-only gene. Cells—Human embryonal carcinoma cell line NTERA-2 and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Flow Laboratories, Irvine, CA). Colorectal carcinoma cells HCT116 and their p53 (–/–) derivatives (22Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2538) Google Scholar), a gift from B. Vogelstein, were grown in McCoy's 5A medium (Invitrogen) supplemented with 10% fetal calf serum. In some experiments, cells were treated with the indicated concentrations of etoposide or cisplatin (Sigma). Antibody Production—TEF cDNA was cloned into NdeI and XhoI sites of the pET30b expression vector (Novagen, Madison, WI), and this construct was used for transformation of BL21pLysS bacteria (Stratagene, La Jolla, CA). Expression of TEF was induced by treating cells with 1 mm isopropyl-β-d-thiogalactopyranosid, and the recombinant protein was purified by using the nickel-nitrilotriacetic acid spin kit (Qiagen, Hilden, Germany). C57BL/6 mice were immunized subcutaneously with 50 μg of the purified protein emulsified 1:1 in complete Freund's adjuvant. After 21 days, the mice were boosted with the same amount of protein, and by day 35, blood was collected, and the serum was tested for the presence of TEF-specific antibodies. Analysis of Apoptosis—Apoptosis was assessed by an enzyme-immunoassay method that quantifies the histone-associated DNA fragments present in the cytosol (Roche Applied Science, Mannheim, Germany) as described previously (23Real P.J. Benito A. Cuevas J. Berciano M.T. de Juan A. Coffer P. Gomez-Roman J. Lafarga M. Lopez-Vega J.M. Fernandez-Luna J.L. Cancer Res. 2005; 65: 8151-8157Crossref PubMed Scopus (61) Google Scholar). Values were represented as the percentage of apoptosis with respect to a positive internal control. Apoptosis was also confirmed by using a flow cytometry analysis with an active caspase 9 staining kit (MBL Int., Woburn, MA). Electrophoretic Mobility Shift Assay (EMSA)—Cells were lysed, and nuclear fractions were resuspended in 20 mm HEPES, pH 7.9, 420 mm NaCl, 1 mm EDTA, 1 mm EGTA, and 20% glycerol. Nuclear extracts (5–10 μg of total protein) were incubated with a 32P-labeled double-stranded DNA probe from the promoter region of bcl-gS (5′-CAGATACATAAGCA-3′). Samples were run on a 5% non-denaturing polyacrylamide gel in 200 mm Tris borate, 2 mm EDTA. Gels were dried and visualized by autoradiography. Supershifts were performed using monoclonal antibodies specific for FLAG (Sigma) and Myc (BD Biosciences) tags. Analyses of RNA—Total RNA was prepared using TRIzol reagent (Invitrogen). To assess mRNA expression, semiquantitative RT-PCR and quantitative real-time PCR were performed as described previously (24Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nunez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-41705Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). The generated cDNA was amplified by using primers for human bid, bim, bik, bnip3L, gapdh (23Real P.J. Benito A. Cuevas J. Berciano M.T. de Juan A. Coffer P. Gomez-Roman J. Lafarga M. Lopez-Vega J.M. Fernandez-Luna J.L. Cancer Res. 2005; 65: 8151-8157Crossref PubMed Scopus (61) Google Scholar), bcl-gS (5′-AAGGCCACGTGCCTGTAGC-3′ and 5′-CCCTGGATGCTGGTGTCAAC-3′), puma (5′-ACGACCTCAACGCACAGTACG-3′ and 5′-TGGGTAAGGGCAGGAGTCC-3′), tef (5′-TGGTCCTGAAGAAGCTGATGG-3′ and 5′-TCCAGGTCCATGTACTCCAGG-3′), and mouse dbp (5′-GCGCGGCCTGTGAGCGACAGGA-3′ and 5′-TCACAGGGCCCCGTGCTGGGC-3′). Western Blot Analysis—Protein expression was determined by Western blotting as described previously (25Benito A. Silva M. Grillot D. Nunez G. Fernandez-Luna J. Blood. 1996; 87: 3837-3843Crossref PubMed Google Scholar). Proteins (30–60 μg) were separated on a 12% polyacrylamide gel and transferred to nitrocellulose. Blots were blocked with 3% bovine serum albumin and incubated with rabbit anti-Bcl-g antibodies, which recognize both long and short isoforms (Abgent, San Diego, CA), or mouse anti-FLAG and anti-α-tubulin (both from Sigma) antibodies and then incubated with goat anti-rabbit or anti-mouse antibodies conjugated to alkaline phosphatase (Sigma). Bound antibody was detected by a chemiluminescence system (Applied Biosystems, Foster City, CA). Gene Silencing—Cells were transfected with a 100 nm solution of a pool of four SMARTselection™-designed siRNA duplexes specific for TEF, for all splicing variants of the bcl-g gene, or with a similar pool of irrelevant siRNA duplexes (Dharmacon, Chicago, IL) by using Lipofectamine 2000 (Invitrogen). After 24 h of transfection, cells were incubated in the presence or in the absence of 80 μm etoposide, and 24 h later, transfected cells were analyzed for both bcl-gS mRNA expression and apoptosis. Transfections and Gene Reporter Assays—A genomic PCR fragment from the promoter region of bcl-gS (413 bp) was cloned into KpnI and HindIII sites of the pGL2-basic luciferase reporter vector (Promega Corp., Madison, WI). The authenticity of the construct was confirmed by sequencing. HEK293T cells were cotransfected with 1 μg of the promoter-containing pGL2 construct, 1 μgof TEF, DBP, HLF, or NFIL3 cDNAs cloned into FLAG-containing pcDNA3 expression vector, and 20 ng of pRSV-β-gal by lipofection using Superfect (Qiagen). When indicated, cells were cotransfected with pGL2-bcl-gS promoter and a combination of both TEF and a truncated isoform of DBP (tDBP). 24 h after transfection, cell extracts were prepared and analyzed for the relative luciferase activity by a dual-light reporter gene assay system (Applied Biosystems). Results were normalized for transfection efficiency with values obtained with pRSV-β-gal. PCR site-directed mutagenesis of the PAR bZIP consensus site at position –13 in the bcl-gS promoter was carried out by an overlap extension method (26Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). The following primers were used to generate two DNA fragments having overlapping ends (changed nucleotides are underlined): sense, 5′-TCTGCCCATGTGGCTGGGGAGGTATGCAATG-3′, antisense, 5′-CCAGCCACATGGGCAGAGAAGACTGAGTAACG-3′. These fragments were then combined, allowing the 3′ overlap of each strand to serve as a primer for the 3′ extension of the complementary strand. The bcl-gS promoter DNA inserts were sequenced to verify the mutations. Transfection of cells with the pcDNA3 vector containing FLAG-tagged TEF or tDBP or with the vector alone (2 μg each) was performed using Lipofectamine 2000. In some experiments, NTERA2 cells were cotransfected with 1 μgof TEF or NFIL3 cDNAs and 0.2 μg of a green fluorescent protein (GFP)-containing vector (Genscript, Piscataway, NJ) by using Lipofectamine 2000. After 24 h of transfection, GFP-positive cells were selected by a cell sorter (BD Biosciences, Heidelberg, Germany) and cultured for another 24-h period. These cells were then subjected to gene expression analyses. PAR bZIP Proteins Promote the Expression of a BH3-only Gene—To assess whether PAR bZIP transcription factors were able to regulate the expression of BH3-only genes, we first looked for consensus sequences within the promoter regions of these genes and found that bcl-gS contains a putative binding sequence located 13 bases upstream from the transcription start site (Fig. 1A). To study the functionality of this sequence, HEK293T cells were cotransfected with a promoter fragment from the bcl-gS gene cloned into a luciferase reporter vector and each of the PAR bZIP genes inserted into a FLAG-containing expression vector (pcDNA3-FLAG). As shown in Fig. 1B, TEF increased luciferase activity more than 35-fold. DBP also activated the bcl-gS promoter (about 10-fold), whereas HLF had no transcriptional activity. Interestingly, the transcriptional repressor NFIL3, which shares the DNA binding site with PAR bZIP proteins, down-regulated the basal activity of the bcl-gS promoter (Fig. 1B). Furthermore, activation of the bcl-gS promoter was abolished when a mutagenesis strategy changed four bases within the PAR bZIP site. After transfection with this construct, the TEF-mediated luciferase activity reached the same value as that obtained with the promoterless vector (Fig. 1C). To directly prove the binding of PAR bZIP proteins to the bcl-gS promoter, HEK293T cells were transfected with FLAG-tagged HLF, DBP, TEF, and NFIL3 (Fig. 2A), and then nuclear extracts were incubated with a radiolabeled probe from the bcl-gS promoter and subjected to EMSA. In all cases, we detected a protein-DNA binding complex that was supershifted in the presence of anti-FLAG antibodies (Fig. 2B). Next, we studied the expression of bcl-gS in a number of tumor cell lines and found the highest mRNA and protein levels in NTERA2 and 2102Ep embryonal cancer cells (Fig. 3, A, and B). Additionally, tef mRNA was detected in these cells as well as in RT112 and HCT116 (data not shown). To confirm the TEF-dependent transcription of bcl-gS, NTERA2 cells were cotransfected with TEF and a GFP-containing vector, and then the GFP-positive cell population was sorted and analyzed for expression of BH3-only genes by real-time RT-PCR. As shown in Fig. 3C, TEF promoted a 5-fold induction of bcl-gS mRNA levels when compared with empty vector-transfected cells, whereas other BH3-only genes did not show significant variations in response to TEF. Consistent with our previous data, NFIL3 down-regulated bcl-gS. Recently, it has been described that p53 induces bcl-g (no variant specified) in Saos cells (27Miled C. Pontoglio M. Garbay S. Yaniv M. Weitzman J.B. Cancer Res. 2005; 65: 5096-5104Crossref PubMed Scopus (69) Google Scholar). To assess whether p53 was able to modify the TEF-mediated expression of bcl-gS, we transfected p53-deficient and wild type HCT116 cells with pcDNA3-FLAG-TEF and then analyzed the expression of bcl-gS by RT-PCR. Western blot analyses revealed the levels of the transfected protein (Fig. 3D). As shown in Fig. 3D, HCT116 express no or very little constitutive levels of bcl-gS, which are significantly increased in response to TEF regardless of the presence of p53. Thus, TEF was able to up-regulate bcl-gS in a p53-independent manner, although no significant apoptotic response was observed (data not shown). tDBP Functions as a Competitive Inhibitor of TEF—Gene expression analyses by RT-PCR in mouse tissues revealed the presence of a shorter (truncated) form of DBP (tDBP) in the liver (Fig. 4B) that retains the PAR bZIP region but lacks the transactivation domain (Fig. 4, A and C). A similar study in more than 30 human cell lines failed to detect a tDBP-like isoform but identified two frameshift deletions that created premature stop codons, which are predicted to result in the production of truncated proteins lacking the PAR bZIP region and all or part of the transactivation domain (Fig. 4A). Fig. 4C shows that tDBP gives rise to a shorter protein of the expected size. Consistently, EMSA experiments demonstrated that tDBP, as well as DBP and TEF, binds specifically to a probe from the bcl-gS promoter containing the PAR bZIP binding site (Fig. 4D). The bZIP sequence contains the DNA binding and protein dimerization domains. Thus, tDBP could act as a non-functional competitor of TEF that is able to bind DNA and to dimerize with itself and with other PAR bZIP proteins. To test this hypothesis, we cotransfected HEK293T cells with pcDNA3-FLAG-TEF and increasing amounts of pcDNA3-Myc-tDBP. As shown in Fig. 5A, tDBP dimerizes with TEF to form heterodimers that bind to a consensus sequence from the bcl-gS promoter. Both anti-FLAG and anti-Myc antibodies confirmed the specificity of the protein-DNA binding complexes. A similar approach was used to analyze the transcriptional activity of TEF-tDBP heterodimers. Cells were cotransfected with TEF, tDBP, and a vector containing the luciferase gene driven by the bcl-gS promoter. The luciferase activity was progressively down-regulated in the presence of increasing amounts of tDBP (Fig. 5B). To further confirm the blocking activity of tDBP, we determined the endogenous expression of bcl-gS in HCT116 cells cotransfected with TEF and tDBP. As shown in Fig. 5C, both proteins were coexpressed in these cells following transfection. Consistent with our previous results, TEF up-regulated the mRNA levels of bcl-gS, which were significantly reduced (about 3-fold) in the presence of tDBP (Fig. 5C and data not shown). These data indicate that tDBP is a convenient molecular tool to study the TEF-bcl-gS transcriptional pathway.FIGURE 5tDBP down-regulates the expression of bcl-gS. A, radiolabeled probe containing the PAR bZIP site of the bcl-gS promoter was incubated with nuclear lysates from HEK293T cells transfected with FLAG-tagged TEF, Myc-tagged tDBP, or both, and the formation of binding complexes was analyzed by EMSA. Anti-FLAG or anti-Myc antibodies were used to show the specific binding of overexpressed proteins. B, HEK293T cells were transfected with a luciferase reporter vector containing the bcl-gS promoter in the presence of TEF, tDBP, or both. Following 24 h of transfection, cell extracts were prepared and analyzed for the relative luciferase activity. Results were normalized for transfection efficiency with values obtained with pRSV-β-gal. C, HCT116 cells were transfected with TEF or TEF plus tDBP (both tagged with the FLAG epitope), and the expression of the overexpressed proteins was determined by Western blot with anti-FLAG. Transfected cells were analyzed for the expression of bcl-gS mRNA by semiquantitative RT-PCR. gapdh mRNA was included as an amplification control. All histograms represent the means ± S.D. of triplicate analyses.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chemotherapy Promotes Activation of the TEF-bcl-gS Transcriptional Pathway—BH3-only proteins may be up-regulated after treatment with chemotherapy, contributing to the apoptotic response of the cell. Thus, we asked whether the TEF-bcl-gS transcriptional pathway was activated by chemotherapeutic agents. HCT116 cells were treated with increasing concentrations of etoposide or cisplatin, and the endogenous expression of bcl-gS was determined by real-time quantitative PCR (Fig. 6A). Both treatments provoked at least a 4-fold induction of bcl-gS mRNA levels when compared with untreated cells. The highest inductions were obtained with 40 μm etoposide (12-fold) and with 5 μm cisplatin (about 10-fold). However, no variation in the levels of tef mRNA was observed (data not shown). To confirm that chemotherapeutic agents activated TEF, we analyzed the binding of this transcription factor to the consensus site in the bcl-gS promoter by EMSA. As shown in Fig. 6B, both etoposide and cisplatin increased the formation of a protein-DNA complex in HCT116 cells. To detect the presence of endogenous TEF in this binding complex, we generated a polyclonal anti-TEF antibody that detected a single protein of the right size by Western blot analysis (Fig. 6C). This antibody partly supershifted the protein-DNA complex, confirming that TEF binds to the bcl-gS promoter in response to chemotherapy. A more direct way to demonstrate that TEF mediates the chemotherapy-induced transactivation of the bcl-gS gene is to block the transcriptional activity of TEF. HCT116 cells were transfected with either an empty vector or a FLAG-tagged tDBP (Fig. 6D) and then cultured in the presence or in the absence of etoposide. After 24 h of treatment, the etoposide-dependent induction of bcl-gS mRNA was virtually blocked in tDBP-expressing cells (Fig. 6E). Quantification of the histone-associated DNA fragments present in the cytosol, a feature of apoptosis, demonstrated that the level of apoptosis following treatment with etoposide decreased from 23% in control cells to 13% in tDBP-expressing cells (Fig. 6E). To further confirm the relevance of TEF in the chemotherapy-induced expression of bcl-gS, we used an RNA interference approach to deplete cells of endogenous TEF. Specific siRNA significantly reduced the TEF mRNA levels and impaired the up-regulation of bcl-gS (Fig. 6F). Furthermore, this interference strategy promoted a decrease in apoptotic cell death as assessed by quantification of the histone-associated DNA fragments (Fig. 6F) and annexin V binding (Fig. 6G). Blockade of bcl-gS expression gave similar results. A representative experiment is shown in Fig. 6H. Specific siRNA impaired the etoposide-induced expression of bcl-gS mRNA and gave rise to reduced levels of apoptotic cell death (from 28% to 14%) after treatment with etoposide. This result shows the relevance of bcl-gS in chemotherapy-induced apoptosis. BH3-only proteins are essential for the appropriate execution of apoptotic cell death. Thus, understanding the regulation of BH3-only genes is necessary to gain insight into the mechanisms of response to apoptotic stimuli. We have identified a DNA binding site for PAR bZIP proteins in the promoter region of bcl-gS, a BH3-only gene. We also found that TEF promoted the strongest induction. TEF has been shown to induce the expression of thyroid-stimulating hormone β (6Drolet D.W. Scully K.M. Simmons D.M. Wegner M. Chu K.T. Swanson L.W. Rosenfeld M.G. Genes Dev. 1991; 5: 1739-1753Crossref PubMed Scopus (207) Google Scholar), pyridoxal kinase, an enzyme involved in the activation of vitamin B6 (8Gachon F. Fonjallaz P. Damiola F. Gos P. Kodama T. Zakany J. Duboule D. Petit B. Tafti M. Schibler U. Genes Dev. 2004; 18: 1397-1412Crossref PubMed Scopus (194) Google Scholar), telokin, a smooth muscle-restricted protein (28Zhou J. Hoggatt A.M. Herring B.P. J. Biol. Chem. 2004; 279: 15929-15937Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), and the LMO2 transcription factor (29Crable S.C. Anderson K.P. Blood. 2003; 101: 4757-4764Crossref PubMed Scopus (25) Google Scholar). More recently, TEF has been associated with down-regulation of the common β chain of cytokine receptors, but no transcriptional mechanism was defined (30Inukai T. Inaba T. Dang J. Kuribara R. Ozawa K. Miyajima A. Wu W. Look A.T. Arinobu Y. Iwasaki H. Akashi K. Kagami K. Goi K. Sugita K. Nakazawa S. Blood. 2005; 105: 4437-4444Crossref PubMed Scopus (24) Google Scholar). Although this transcription factor is expressed in the pituitary gland during embryonic development, it appears in several tissues in the mature organism, including brain, lung, liver, spleen, and kidney (7Khatib Z.A. Inaba T. Valentine M. Look A.T. Genomics. 1994; 23: 344-351Crossref PubMed Scopus (26) Google Scholar). We have determined the expression of TEF mRNA in NTERA2, 2102Ep, HCT116, and RT112 cells. However, we were unable to clearly detect the endogenous protein with our polyclonal anti-TEF antibody, most likely because of the low affinity of this primary antibody, as it readily revealed the overexpressed protein in transfected cells. We determined that the highest expression levels of bcl-gS were found in NTERA2 and 2102Ep, which are testicular embryonal carcinoma cells. Consistently, bcl-gS mRNA has been found only in testis (16Guo B. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 2780-2785Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Overexpression of Bcl-gS in COS-7 cells induces apoptosis, which is suppressed by coexpression of anti-apoptotic Bcl-xL protein (16Guo B. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 2780-2785Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We have observed that transfection of HCT116 and NTERA2 cells with TEF promoted the expression of bcl-gS but had no effect on cell survival. One possible explanation for these apparently contradictory results may be in the endogenous levels of Bcl-gS, which are presumably higher in cells transfected with this gene than in cells overexpressing its transcription factor. Alternatively, the levels of Bcl-2 or Bcl-xL in the different cell lines used in both experiments may determine the cell fate in response to Bcl-gS overexpression, as both anti-apoptotic proteins counteract the action of BH3-only family members (12Puthalakath H. Strasser A. Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (631) Google Scholar, 31Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (807) Google Scholar, 32Zhu Y. Swanson B.J. Wang M. Hildeman D.A. Schaefer B.C. Liu X. Suzuki H. Mihara K. Kappler J. Marrack P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7681-7686Crossref PubMed Scopus (112) Google Scholar). We also identified a splicing variant of DBP (tDBP) in mouse liver that retains the DNA binding capacity but has lost the transactivation domain. TEF and DBP bind the same DNA sequence (33Fonjallaz P. Ossipow V. Wanner G. Schibler U. EMBO J. 1996; 15: 351-362Crossref PubMed Scopus (124) Google Scholar) and form heterodimers (6Drolet D.W. Scully K.M. Simmons D.M. Wegner M. Chu K.T. Swanson L.W. Rosenfeld M.G. Genes Dev. 1991; 5: 1739-1753Crossref PubMed Scopus (207) Google Scholar). Consistently, we showed that tDBP competes with TEF for binding to the bcl-gS promoter and counteracts the TEF-mediated induction of endogenous bcl-gS. Although we use tDBP here as a convenient molecular tool for the study of TEF-bcl-gS transcriptional pathway, it may be that this or a similar truncated protein in human normal or tumor cells is acting as a regulator of TEF and other PAR bZIP proteins. Splice variants of transcription factors involved in the expression of apoptosis regulators have been previously described. To this end, a naturally occurring repressor isoform of Stat3 has been shown to be coexpressed with the activator Stat3 in a variety of cell types including leukemia cells (34Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A. Lammers J.W. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). This variant inhibits the transactivation potential of Stat3 and may interfere with the anti-apoptotic activity of this transcription factor. In addition, p73 gene generates a truncated isoform that lacks the transactivation domain, frequently overexpressed in human cancers (35Zaika A.I. Slade N. Erster S.H. Sansome C. Joseph T.W. Pearl M. Chalas E. Moll U.M. J. Exp. Med. 2002; 196: 765-780Crossref PubMed Scopus (305) Google Scholar). This variant acts as a potent dominant inhibitor of wild type p53 and transactivation-competent p73, blocking their apoptotic response. Chemotherapeutic agents have been shown to promote apoptosis in numerous cell systems, including cancer cells, and BH3-only proteins play an essential role in mediating this process. In line with this, paclitaxel has been recently described to induce apoptosis in vitro and in tumors in vivo through a Bim-dependent mechanism (20Tan T.T. Degenhardt K. Nelson D.A. Beaudoin B. Nieves-Neira W. Bouillet P. Villunger A. Adams J.M. White E. Cancer Cell. 2005; 7: 227-238Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In this model, inactivation of Bim conferred resistance of cancer cells to paclitaxel. Other BH3-only proteins such as Noxa, Puma, Bik, Bid, and Hrk have also been associated with sensitivity to chemotherapy-induced apoptosis (36Daniel P.T. Pun K.T. Ritschel S. Sturm I. Holler J. Dorken B. Brown R. Blood. 1999; 94: 1100-1107Crossref PubMed Google Scholar, 38Iyer N.G. Chin S.F. Ozdag H. Daigo Y. Hu D.E. Cariati M. Brindle K. Aparicio S. Caldas C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7386-7391Crossref PubMed Scopus (123) Google Scholar). Consistent with this, we found that etoposide and cisplatin, two commonly used chemotherapeutic agents, activated the TEF-bcl-gS transcriptional pathway. Furthermore, tDBP or siRNA duplexes specific for all splicing variants of the bcl-g gene efficiently reduced the levels of bcl-gS in response to chemotherapy and partly blocked apoptosis, indicating a significant participation of this pathway in chemotherapy-induced apoptosis. Based on these data, TEF-bcl-gS may participate in a regulatory network that controls the apoptotic response to chemotherapeutic agents. Together, this study shows a novel role for PAR bZIP proteins in the transcriptional regulation of BH3-only genes, mainly bcl-gS, and supports that this pathway can be activated by chemotherapeutic agents in cancer cells, thus contributing to the apoptotic response to chemotherapy. Further studies will need to deepen the understanding of the physiological relevance of this transcriptional pathway in normal and cancer cells. It will also be of interest to find other targets of PAR bZIP proteins that may be involved in apoptosis in different cell systems. We are grateful to Valentina Riancho for valuable technical assistance." @default.
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• W2015278341 title "A Novel Role for Proline- and Acid-rich Basic Region Leucine Zipper (PAR bZIP) Proteins in the Transcriptional Regulation of a BH3-only Proapoptotic Gene" @default.
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