Bok, Bcl-2-related Ovarian Killer, Is Cell Cycle-regulated and Sensitizes to Stress-induced Apoptosis
2006; Elsevier BV; Volume: 281; Issue: 32 Linguagem: Inglês
10.1074/jbc.m604705200
ISSN1083-351X
AutoresJosé María Ros Rodríguez, Michele A. Glozak, Yihong Ma, W. Douglas Cress,
Tópico(s)MicroRNA in disease regulation
ResumoBok/Mtd (Bcl-2-related ovarian killer/Matador) is considered a pro-apoptotic member of the Bcl-2 family. Although identified in 1997, little is known about its biological role. We have previously demonstrated that Bok mRNA is up-regulated following E2F1 overexpression. In the current work, we demonstrate that Bok RNA is low in quiescent cells and rises upon serum stimulation. To determine the mechanism underlying this regulation, we cloned and characterized the mouse Bok promoter. We find that the mouse promoter contains a conserved E2F binding site (−43 to −49) and that a Bok promoter-driven luciferase reporter is activated by serum stimulation dependent on this site. Chromatin immunoprecipitation assays demonstrate that endogenous E2F1 and E2F3 associate with the Bok promoter in vivo. Surprisingly, we find that H1299 cells can stably express high levels of exogenous Bok protein. However, these cells are highly sensitive to chemotherapeutic drug treatment. Taken together these results demonstrate that Bok represents a cell cycle-regulated pro-apoptotic member of the Bcl-2 family, which may predispose growing cells to chemotherapeutic treatment. Bok/Mtd (Bcl-2-related ovarian killer/Matador) is considered a pro-apoptotic member of the Bcl-2 family. Although identified in 1997, little is known about its biological role. We have previously demonstrated that Bok mRNA is up-regulated following E2F1 overexpression. In the current work, we demonstrate that Bok RNA is low in quiescent cells and rises upon serum stimulation. To determine the mechanism underlying this regulation, we cloned and characterized the mouse Bok promoter. We find that the mouse promoter contains a conserved E2F binding site (−43 to −49) and that a Bok promoter-driven luciferase reporter is activated by serum stimulation dependent on this site. Chromatin immunoprecipitation assays demonstrate that endogenous E2F1 and E2F3 associate with the Bok promoter in vivo. Surprisingly, we find that H1299 cells can stably express high levels of exogenous Bok protein. However, these cells are highly sensitive to chemotherapeutic drug treatment. Taken together these results demonstrate that Bok represents a cell cycle-regulated pro-apoptotic member of the Bcl-2 family, which may predispose growing cells to chemotherapeutic treatment. The E2F family of transcription factors has key roles in regulating the G1/S transition (1Johnson D.G. Schwarz J.K. Cress W.D. Nevins J.R. Nature. 1993; 365: 349-352Crossref PubMed Scopus (833) Google Scholar, 2Lukas J. Petersen B.O. Holm K. Bartek J. Helin K. Mol. Cell. Biol. 1996; 16: 1047-1057Crossref PubMed Scopus (265) Google Scholar, 3Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar, 4Mann D.J. Jones N.C. Curr. Biol. 1996; 6: 474-483Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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Following plaque purification, seven purified positive plaques were identified. Phage DNA was extracted from plate lysates using the Qiagen MIDI λ Kit according to manufacturer's specifications. NotI digestion of the phage DNA indicated that each clone had a different sized insert, each in the ∼15-20-kb range. Restriction digestion and Southern blotting showed that the clones were unique and covered the entire Bok genomic locus. Each of the phage DNAs was then digested with NotI to excise the entire insert for cloning into pBluescript (pBS). In addition, based on differential hybridization patterns, phage DNAs were also digested with XhoI or SstI to subclone smaller fragments into pBS. Bluescript clones containing inserts were sequenced with T3 and T7 promoter to confirm the ends of each clone against the published genomic sequence (NT_039173).Plasmids—Mouse Bok promoter reporters were generated by digestion of pBS-13S2 with SstI and ligated into pGL3 basic. Initial PCR primers were designed to amplify 331 bp (−244/+87) of our sequenced Bok promoter, which are numbered relative to the transcriptional start site. The forward (192 forward) and reverse (141 reverse) PCR primers for the Bok promoter were 5′-GGTACCAGAACTTGTGCTGGCCTTTCT-3′ and 5′-AAGCTTAGTTCTGGTTTCAGGACCCGC-3′, respectively. The forward primer added a KpnI site, and the reverse added a HindIII site to facilitate subcloning. The E2F binding site mutant of the Bok promoter was generated by site-directed mutagenesis with PCR. The initial reaction was done using 192 forward and 192 reverse (5′-TCCGCCGGTCTTCCATCGCGC-3′); a second reaction used primer 141 forward (5′-CGCGATGGAAGACCGGCGGA-3′) and 141 reverse. The PCR products from these reactions, 192 and 141 bp, respectively, were band purified, phenol/chloroform extracted, and ethanol precipitated. They were then resuspended in water, combined, and used as template in another PCR using the flanking primers 192 forward and 141 reverse. The resulting PCR product was inserted in pCRII-TOPO, followed by digestion with KpnI and HindIII (to excise PCR insert). Insert was then band purified and ligated to the pGL3 luciferase vector. The E2F1 mutant constructs, E2F1-(1-284) and E2F1-(Eco132) have been previously described (52Cress W.D. Nevins J.R. J. Virol. 1994; 68: 4213-4219Crossref PubMed Google Scholar, 53Cress W.D. Johnson D.G. Nevins J.R. Mol. Cell. Biol. 1993; 13: 6314-6325Crossref PubMed Scopus (109) Google Scholar).Cell Culture—Mouse NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 5% calf serum. The H1299 lung cancer cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. H1299 cells that constitutively express the FLAG-Bok fusion protein were obtained by transfecting with pcDNA3-FLAG-Bok (a gift from Gabriel Nunez, University of Michigan) and selecting for transformants in 400 μg/ml G418. G418-resistant lines were screened for expression of FLAG-Bok. Adenoviruses were described previously (54Ma Y. Croxton R. Moorer Jr., R.L. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (95) Google Scholar) and titered by plaque assay. Cell cycle parameters were measured by fixing cells with 70% ethanol-PBS, 2The abbreviations used are: PBS, phosphate-buffered saline; PARP, poly-(ADP)-ribose polymerase; PI, propidium iodide; FACS, fluorescence-activated cell sorter. staining with propidium iodide (PI), and analyzing by FACS, using ModFit.Biochemical Assays—Transfections were performed using Lipofectamine PLUS® Reagent from Invitrogen with test DNA totaling 2.85 μg of DNA per 60-mm dish. Transfections included 100 ng of expression plasmids (pcDNA3-based vectors), 2.5 μg of test construct firefly luciferase reporter plasmid (pGL3, Promega), and 250 ng of Renilla luciferase reporter plasmid (pRL-TK, Promega). Cells were harvested 48 h after transfection, and luciferase assays were performed using the Dual Luciferase Reporter Assay System following the manufacturer's protocol (Promega). Experiments were done in duplicate or triplicates, and the relative activities and standard deviation values were determined. To control for transfection efficiency, firefly luciferase values were normalized to the values for Renilla luciferase. Western blots were performed as previously described (39Ma Y. Cress W.D. Haura E.B. Mol. Cancer Ther. 2003; 2: 73-81PubMed Google Scholar) using monoclonal antibody against FLAG epitope (F3165, Sigma) or against poly(ADP)-ribose polymerase (PARP) antibody (Cell Signaling 9542). Western blots were stripped and re-probed with an antibody to actin (A5441, Sigma) to ensure equivalent loading.Reverse Transcriptase-PCR—Isolation of total RNA was done using the RNeasy mini kit (Qiagen 74104) as recommended by manufacturer. Total RNA was primed with random hexamers, and cDNA was created using the SuperScrip® First Strand Synthesis System for reverse transcriptase-PCR (Invitrogen 11904-018). PCR primers were designed to amplify 490 bp. The forward and reverse primers were 5′-CGCTCGCCCACAGACAAGGA-G-3′ and 5′-TCTGTGCTGACCACACACTTG-3′.Chromatin Immunoprecipitation—Chromatin immunoprecipitation assays were performed as previously described (39Ma Y. Cress W.D. Haura E.B. Mol. Cancer Ther. 2003; 2: 73-81PubMed Google Scholar, 55Wells J. Boyd K.E. Fry C.J. Bartley S.M. Farnham P.J. Mol. Cell. Biol. 2000; 20: 5797-5807Crossref PubMed Scopus (207) Google Scholar, 56Wells J. Graveel C.R. Bartley S.M. Madore S.J. Farnham P.J. Proc. Natl. Acad. Sci. U. S. 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Antibodies used included E2F1 (sc-193X), E2F3 (sc-878X), and IgG (sc-2027) (from Santa Cruz Biotechnology).RESULTSBok mRNA Is Induced by E2F1 Overexpression and by Serum Stimulation—In a previous microarray screen (54Ma Y. Croxton R. Moorer Jr., R.L. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (95) Google Scholar), we identified Bok as a potential E2F1 target gene. To confirm this observation, we tested if overexpression of E2F1 would correlate with increased expression of Bok mRNA. NIH 3T3 cells were brought to quiescence by a 48-h incubation in 0.5% calf serum. Cells were then stimulated with 10% fetal calf serum or were infected with 10 plaque-forming units of the indicated adenovirus per cell. Fig. 1A highlights the observation that Bok mRNA is very low in quiescent NIH 3T3 fibroblasts (lane 3), but is highly induced following infection with an E2F1-expressing adenovirus (lane 1). Lane 4 reveals that serum treatment, which stimulates quiescent cells to enter S phase, also elevated Bok message (lane 4), suggesting that Bok is E2F and cell cycle regulated. Data provided in Fig. 1B confirm the cell cycle status of treated cells (Fig. 1A).The Bok Promoter Contains a Conserved E2F Binding Sequence Central to Its Cell Cycle Regulation—To understand how Bok is regulated in an E2F/cell cycle-dependent manner, we compared the genomic sequences of human (AC110299) and mouse Bok (NT_039173). To obtain authentic Bok genomic sequence from mouse, we screened a λ phage library using a mixture of human cDNA probes and mouse untranslated region Bok probes. Fig. 2A shows a schematic of the various clones obtained. One of the subclones, 13S2, which contains the first two Bok exons and over 900 bp of upstream promoter region, was sequenced. Comparison of the mouse and human Bok 5′ regions (shown in Fig. 3) revealed significant sequence homology within the first exon (non-coding) and in a region-244 upstream of the putative transcriptional start site in mouse (60Maglott D. Ostell J. Pruitt K.D. Tatusova T. Nucleic Acids Res. 2005; 33: D54-D58Crossref PubMed Scopus (727) Google Scholar).FIGURE 2Overlapping subclones in pBS encompassing the entire Bok genomic locus. A, subclones were excised from the phage clones with SstI (S), XhoI (X), or NotI (N). NotI subclones represent the entire insert of the phage clones, whereas SstI and XhoI subclones contain only part of the original phage clone. Numbering is relative to the Mus musculus chromosome 1 genomic contig NT_039173.2, which contains the Bok locus. Solid boxes indicate exons. Exon 1 is noncoding. The ATG start codon is located at position 8083483 in exon 2. The stop codon is located at position 8092198 in exon 5. B, the pBS-13S2 was further subcloned into pGL3 luciferase vector using SstI, SmaI, or XbaI. These subclones contain the Bok promoter region and the four putative E2F binding sites marked by black circles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Conserved E2F binding site. An alignment between the mouse (NT_039173) and human (AC110299) Bok gene sequences using MegAlign (DNASTAR, Inc.) showed a conserved putative E2F binding site that extends from position −42 to −49 relative to the putative transcriptional start site in the mouse sequence. Shaded blocks indicate sequence identity of at least five base pairs. Boxed areas indicate putative transcription factor binding sites identified by MatInspector (Genomatix). The highlighted G at +1 in the mouse sequence indicates the putative transcription start site based on NCBI annotations (60Maglott D. Ostell J. Pruitt K.D. Tatusova T. Nucleic Acids Res. 2005; 33: D54-D58Crossref PubMed Scopus (727) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Crude deletion analysis localized the promoter to −244/+87 (not shown). Potentially important motifs within this region include numerous SP1 binding sites and, most importantly, a conserved E2F1 consensus-binding site. We used PCR to generate a luciferase reporter vector using the mouse genomic sequence from −244/+87. To examine the role of the conserved E2F1 site spanning from position −43 to −49, we also generated a mutated version of the −244/+87 construct in which the E2F1 site was rendered nonfunctional. Fig. 4A shows a schematic representation of the constructs generated. They differ in that the consensus E2F binding site CGCGCGGGAAGACCGGCGGA (wild type) is changed to CGCGATGGAAGACCGGCGGA (mutant).FIGURE 4The Bok promoter is activated by addition of serum dependent upon a conserved E2F binding site. A, schematic representation of the Bok promoter containing the wild type (closed circle) or mutated (×) E2F binding site. These fragments were then cloned into pGL3. B, NIH 3T3 cells were transfected with the WT or MUT −244/+87 Bok promoter luciferase construct and then brought to quiescent by 48 h incubation with 0.5% calf serum. Following starvation cells were stimulated with 10% fetal calf serum and harvested every 6 h and assayed for luciferase activity. C, cell cycle progression of NIH 3T3 cells after treatment as in B. Cells were fixed with 70% ethanol/PBS, stained with PI, and analyzed by FACS. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To characterize the activity of the cloned Bok promoter throughout the cell cycle, NIH 3T3 cells were transfected with Bok −244/+87 WT or MUT promoter/reporter. Cells were brought to quiescence by incubation with 0.5% calf serum for 48 h and were then serum stimulated with 10% fetal calf serum and harvested every 6 h. In parallel, cells were fixed with 70% ethanol/PBS, stained with PI, and analyzed by FACS to determine cell cycle status. Fig. 4B shows that the activity of the WT Bok promoter is maximal at 6 and 12 h after addition of serum corresponding to the mid to late G1 phase of the
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