Poly(ADP-ribose) Polymerase Is a B-MYB Coactivator

B-MYB is implicated in cell growth control, differentiation, and cancer and belongs to the MYB family of nuclear transcription factors. Evidence exists that cellular proteins bind directly to B-MYB, and it has been hypothesized that B-MYB transcriptional activity may be modulated by specific cofactors. In an attempt to isolate proteins that interact with the B-MYB DNA-binding domain, a modular domain that has the potential to mediate protein-protein interaction, we performed pull-down experiments with a glutathione S-transferase-B-MYB protein and mammalian protein extracts. We isolated a 110-kDa protein associated endogenously with B-MYB in the nuclei of HL60 cells. Microsequence analysis and immunoprecipitation experiments determined that the bound protein was poly(ADP-ribose) polymerase (PARP). Transient transfection assays showed that PARP enhanced B-MYB transactivation and that PARP enzymatic activity is not required for B-MYB-dependent transactivation. These results suggest that PARP, as a transcriptional cofactor of a potentially oncogenic protein, may play a role in growth control and cancer. B-MYB is implicated in cell growth control, differentiation, and cancer and belongs to the MYB family of nuclear transcription factors. Evidence exists that cellular proteins bind directly to B-MYB, and it has been hypothesized that B-MYB transcriptional activity may be modulated by specific cofactors. In an attempt to isolate proteins that interact with the B-MYB DNA-binding domain, a modular domain that has the potential to mediate protein-protein interaction, we performed pull-down experiments with a glutathione S-transferase-B-MYB protein and mammalian protein extracts. We isolated a 110-kDa protein associated endogenously with B-MYB in the nuclei of HL60 cells. Microsequence analysis and immunoprecipitation experiments determined that the bound protein was poly(ADP-ribose) polymerase (PARP). Transient transfection assays showed that PARP enhanced B-MYB transactivation and that PARP enzymatic activity is not required for B-MYB-dependent transactivation. These results suggest that PARP, as a transcriptional cofactor of a potentially oncogenic protein, may play a role in growth control and cancer. poly(ADP-ribose) polymerase glutathione S-transferase wild type DNA-binding domain B-MYB is a nuclear transcription factor belonging to theMYB family, which is expressed ubiquitously and is involved in cell growth control, differentiation, and cancer (1.Sala A. Watson R. J. Cell. Physiol. 1999; 179: 245-250Crossref PubMed Scopus (63) Google Scholar, 2.Nomura N. Takahashi M. Matsui M. Ishii S. Date T. Sasamoto S. Ishizaki R. Nucleic Acids Res. 1988; 16: 11075-11089Crossref PubMed Scopus (200) Google Scholar). Virtually all proliferating cell lines transcribe the B-MYB gene in the G1/S phase of the cell cycle, although lower but detectable levels of B-MYB protein are also observed during the G0 and G2/M phases of the murine fibroblast cell cycle (3.Reiss K. Travali S. Calabretta B. Baserga R. J. Cell. Physiol. 1991; 148: 338-343Crossref PubMed Scopus (65) Google Scholar, 4.Golay J. Capucci A. Arsura M. Castellano M. Rizzo V. Introna M. Blood. 1991; 77: 149-158Crossref PubMed Google Scholar, 5.Lam E.W. Robinson C. Watson R.J. Oncogene. 1992; 7: 1885-1890PubMed Google Scholar, 6.Lam E.W. Bennett J.D. Watson R.J. Gene (Amst.). 1995; 160: 277-281Crossref PubMed Scopus (75) Google Scholar, 7.Robinson C. Light Y. Groves R. Mann D. Marias R. Watson R. Oncogene. 1996; 12: 1855-1864PubMed Google Scholar). B-MYB, similarly to other members of the family, binds to DNA through the consensus sequence (C/T)AACNG, resulting in transactivation of consensus site-bearing promoters. In practice, however, it has been quite difficult to detect B-MYB-dependent transactivation because of variable response in different cell types (8.Tashiro S. Takemoto Y. Handa H. Ishii S. Oncogene. 1995; 10: 1699-1707PubMed Google Scholar). The leading hypothesis describes B-MYB as a constitutively repressed molecule that requires post-translational modifications to disclose its activity. In this regard, several groups (9.Saville M.K. Watson R.J. Oncogene. 1998; 17: 2679-2689Crossref PubMed Scopus (72) Google Scholar, 10.Sala A. Kundu M. Casella I. Engelhard A. Calabretta B. Grasso L. Paggi M.G. Giordano A. Watson R.J. Khalili K. Peschle C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 532-536Crossref PubMed Scopus (91) Google Scholar) have shown that phosphorylation of the B-MYB protein induced by the CDK2/cyclin A kinase results in activation of the B-MYB transactivating function. Activation is also achieved by truncation of the carboxyl terminus of the B-MYB molecule (11.Lane S. Farlie P. Watson R. Oncogene. 1997; 14: 2445-2453Crossref PubMed Scopus (69) Google Scholar, 12.Ziebold U. Bartsch O. Marais R. Ferrari S. Klempnauer K.H. Curr. Biol. 1997; 7: 253-260Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Although cyclin-induced phosphorylation relieves inhibition exerted by the carboxyl terminus, a B-MYB mutant deleted at the carboxyl terminus is activated by enforced expression of CDK2/cyclin A (11.Lane S. Farlie P. Watson R. Oncogene. 1997; 14: 2445-2453Crossref PubMed Scopus (69) Google Scholar). This suggests that, in addition to relieving intramolecular repression, phosphorylation may enhance B-MYB cross-talk with putative co-activators or may inhibit binding of co-repressors. This hypothesis is corroborated by evidence suggesting that the B-MYB transactivating function depends on the cellular context and that it correlates with the binding of cellular proteins to specific B-MYB domains (8.Tashiro S. Takemoto Y. Handa H. Ishii S. Oncogene. 1995; 10: 1699-1707PubMed Google Scholar). Numerous reports have established a fundamental requirement for B-MYB expression during the growth and survival of normal and tumorigenic cell lines, whereas ectopic expression of a B-MYB transgene results in a perturbed cell cycle and differentiative pattern (13.Sala A. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10415-10419Crossref PubMed Scopus (102) Google Scholar, 14.Lin D. Fiscella M. O'Connor P.M. Jackman J. Chen M. Luo L.L. Sala A. Travali S. Appella E. Mercer W.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10079-10083Crossref PubMed Scopus (116) Google Scholar, 15.Raschell G. Negroni A. Sala A. Pucci S. Romeo A. Calabretta B. J. Biol. Chem. 1995; 270: 8540-8545Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16.Sala A. Casella I. Bellon T. Calabretta B. Watson R.J. Peschle C. J. Biol. Chem. 1996; 271: 9363-9367Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17.Bies J. Hoffman B. Amanullah A. Giese T. Wolff L. Oncogene. 1996; 12: 355-363PubMed Google Scholar, 18.Arsura M. Introna M. Passerini F. Mantovani A. Golay J. Blood. 1992; 79: 2708-27016Crossref PubMed Google Scholar). These results correlate well with circumstantial evidence suggesting that abnormal B-MYBexpression may contribute to tumorigenesis. For example, B-MYB is overexpressed during the progression of human small cell lung carcinoma, and high B-MYB expression is predictive, independently from other markers such as N-MYC, of poor survival of child bearing neuroblastoma tumors (19.Hibi K. Liu Q. Beaudry G.A. Madden S.L. Westra W.H. Wehage S.L. Yang S.C. Heitmiller R.F. Bertelsen A.H. Sidransky D. Jen J. Cancer Res. 1998; 58: 5690-5694PubMed Google Scholar, 20.Raschella‘ G. Cesi V. Amendola R. Negroni A. Tanno B. Altavista P. Tonini G.P. De Bernardi B. Calabretta B. Cancer Res. 1999; 59: 3365-3368PubMed Google Scholar). A current hypothesis is that B-MYB may activate sets of genes involved in the regulation of the cell cycle and/or DNA synthesis. Klempnauer and co-workers (21.Mink S. Kerber U. Klempnauer K.H. Mol. Cell. Biol. 1996; 16: 1316-1325Crossref PubMed Scopus (86) Google Scholar) have shown that the v-MYB DNA-binding domain, which consists of three imperfect reiterations shared among all MYB proteins, interacts with the mammalian transactivator C/EBPβ. C/v-MYB and MYB-like proteins from mammalian to yeast also interact with regulatory proteins through the conserved tryptophan repeat structure (22.Pfitzner E. Kirfel J. Becker P. Rolke A. Schule R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5539-5544Crossref PubMed Scopus (28) Google Scholar, 23.Ganter B. Fu S.L. Lipsick J.S. EMBO J. 1998; 17: 255-268Crossref PubMed Scopus (82) Google Scholar, 24.Inoue K. Sherr C.J. Mol. Cell. Biol. 1998; 18: 1590-1600Crossref PubMed Scopus (143) Google Scholar, 25.Berger S.L. Pina B. Silverman N. Marcus G.A. Agapite J. Regier J.L. Triezenberg S.J. Guarente L. Cell. 1992; 70: 251-265Abstract Full Text PDF PubMed Scopus (357) Google Scholar). As a basis of the present study, we hypothesized that proteins binding to the B-MYB DNA-binding domain may function as regulatory molecules, contributing to B-MYB transcriptional activity. To address this issue, a hybrid protein consisting of GST fused to the B-MYB DNA-binding domain was used as bait and mixed with HL60 cell lysate to isolate binding proteins. A nuclear protein of 110 kDa, which was identified as poly(ADP-ribose) polymerase (PARP),1 bound to the GST-B-MYB fusion protein. PARP is associated with chromatin and catalyzes the transfer of poly(ADP)-ribose units to acceptor proteins. Interestingly, PARP and B-MYB share several characteristics. Both proteins are nuclear, and transcription of their genes is down-regulated during mammalian cell differentiation, and they are both able to suppress in vitro induced differentiation when expressed ectopically in mammalian cells (15.Raschell G. Negroni A. Sala A. Pucci S. Romeo A. Calabretta B. J. Biol. Chem. 1995; 270: 8540-8545Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17.Bies J. Hoffman B. Amanullah A. Giese T. Wolff L. Oncogene. 1996; 12: 355-363PubMed Google Scholar, 26.Bhatia M. Kirkland J.B. Meckling-Gill K.A. Cell Growth Differ. 1996; 7: 91-100PubMed Google Scholar, 27.Bhatia M. Kirkland J.B. Meckling-Gill K.A. Biochem. J. 1995; 308: 131-137Crossref PubMed Scopus (61) Google Scholar). Among other functions attributed to PARP is apoptosis regulation and DNA repair (28.Jeggo P.A. Curr. Biol. 1998; 8: 49-51Abstract Full Text Full Text PDF PubMed Google Scholar, 29.Pieper A.A. Verma A. Zhang J. Snyder S.H. Trends Pharmacol. Sci. 1999; 20: 171-181Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Recent studies have implicated PARP in the regulation of gene transcription through association with specific transcription factors. PARP can behave both as an inhibitor or stimulator of the bound transcription factors, suggesting that its biological role may depend on the availability of specific partners, the cell type, and the differentiative and/or proliferative status of the cell. For example, PARP binds to and inactivates the DNA-binding function of p53, and it is a suppressor of RAR/RXR signaling (30.Kumari S.R. Mendoza-Alvarez H. Alvarez-Gonzalez R. Cancer Res. 1998; 58: 5075-5078PubMed Google Scholar, 31.Miyamoto T. Kakizawa T. Hashizume K. Mol. Cell. Biol. 1999; 19: 2644-2649Crossref PubMed Scopus (85) Google Scholar). On the other hand, PARP increases the on-rate binding of nuclear proteins to thePAX-6 gene enhancer, resulting in increased gene transcription, and it is a co-activator of the AP2 transcription factor (32.Plaza S. Aumercier M. Bailly M. Dozier C. Saule S. Oncogene. 1999; 18: 1041-1051Crossref PubMed Scopus (37) Google Scholar, 33.Kannan P., Yu, Y. Wankhade S. Tainsky M.A. Nucleic Acids Res. 1999; 27: 866-874Crossref PubMed Scopus (124) Google Scholar). Furthermore, PARP associates with OCT-1, enhancing its recruitment onto DNA (34.Nie J. Sakamoto S. Song D. Qu Z. Ota K. Taniguchi T. FEBS Lett. 1998; 424: 27-32Crossref PubMed Scopus (84) Google Scholar). Here we show that endogenous B-MYB and PARP co-immunoprecipitate from nuclear extracts of HL60 cells. Overexpression of PARP and B-MYB results in synergistic activation of a MYB-responsive promoter, suggesting that PARP is a B-MYB-specific co-activator. The human osteosarcoma tumor cell line SAOS2 was obtained from ATCC and was cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum. The human hematopoietic HL60 cell line was cultured in RPMI 1640 medium with 10% fetal calf serum. An EcoRI-BamHI fragment (900 base pairs) containing the DNA-binding domain of human B-MYB, was cloned into pGEX2T (Amersham Pharmacia Biotech) and cut withEcoRI-BamHI to generate pGST-B-MYB-DBD. This construct encodes the glutathioneS-transferase protein (GST) in-frame with the B-MYB DNA-binding domain. The construction was verified by sequencing. Bacteria strain BL21 was used for producing the GST-B-MYB-DBD fusion protein. After induction for 2 h with 0.1 mmisopropyl-β-d-thiogalactopyranoside, the bacteria were harvested and resuspended in phosphate-buffered saline. The cells were treated by several cycles of sonication on ice and centrifuged. The supernatant, containing soluble recombinant protein, was incubated with glutathione-agarose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C, washed three times with phosphate-buffered saline, and used directly for protein binding assays. 50 × 10−6 exponentially growing HL60 cells were cultured for 3 h in methionine- and cysteine-free RPMI medium supplemented with 10% dialyzed fetal bovine serum (Life Technologies, Inc.) containing pro-Mix 35Smethionine and -cysteine cell labeling mix (NEN Life Science Products). Labeled cells were lysed in 1 ml of Buffer 1 (20 mm KOH-Hepes, pH 8.0, 25 mm KCl, 5 mm MgCl2, 0.5% Nonidet P-40, 5 mmdithiothreitol, protease inhibitors) and centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant, containing the cytoplasmic fraction, was stored for further analysis, whereas the nuclear pellet was incubated in 1 ml of Buffer 2 (20 mm KOH-Hepes, pH 8.0, 0.1 mm EDTA, 5 mm MgCl2, 0.5m NaCl, 20% glycerol, protease inhibitors) for 10 min at 4 °C and cleared by centrifugation. Both cytoplasmic and nuclear extracts were pre-cleared by mixing 100 μl of GST-Sepharose beads (50% slurry, Santa Cruz Biotechnology) in binding buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.03% Nonidet P-40, 2 mmphenylmethylsulfonyl fluoride, 1 mm ZnSO4, 20 mm β-mercaptoethanol). After rocking for 1 h at 4 °C, the beads were discarded and the supernatants recovered. Aliquots of 0.5 ml of pre-cleared lysates were incubated with 20 μl of glutathione-Sepharose beads loaded with 4 μg of GST-B-MYB-DBD with or without 3 μg of a synthetic double-stranded oligonucleotide containing the MYB-binding site (tcgacacatt ataacgg ttttttagc). As further controls, we mixed lysates with GST-A-MYB-DBD (a kind gift of Martino Introna) and GST. The beads were washed five times with 1 ml of binding buffer, and the bound proteins were eluted with SDS sample buffer and analyzed on a 10% SDS-polyacrylamide gel. PARP protein was translatedin vitro from the pSPARP plasmid (a kind gift of Dr. Perry Kannan) with the TNT kit according to manufacturer's instructions (Promega). A total of 1 × 109 HL60 cells were lysed. After pre-clearing with GST-coated beads, nuclear extracts were adsorbed with GST-B-MYB-DBD beads. After washings, the bound proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining. was by analysis performed by the at the of HL60 cell lysates were pre-cleared with 50 μl of protein and with PARP or Cruz or B-MYB A. Kundu M. Casella I. Engelhard A. Calabretta B. Grasso L. Paggi M.G. Giordano A. Watson R.J. Khalili K. Peschle C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 532-536Crossref PubMed Scopus (91) Google or control Santa Cruz Biotechnology) for 1 h at 4 °C in a buffer containing 10 mm Tris-HCl, pH mm NaCl, Nonidet μl of protein was and the were for an at 4 °C. After five were on an SDS-polyacrylamide and to was performed with B-MYB or PARP in in phosphate-buffered Nonidet After and with to the was a according to manufacturer's instructions Pharmacia SAOS2 cells were in according to the as A. Kundu M. Casella I. Engelhard A. Calabretta B. Grasso L. Paggi M.G. Giordano A. Watson R.J. Khalili K. Peschle C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 532-536Crossref PubMed Scopus (91) Google Scholar). D. Fiscella M. O'Connor P.M. Jackman J. Chen M. Luo L.L. Sala A. Travali S. Appella E. Mercer W.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10079-10083Crossref PubMed Scopus (116) Google A. B. De P. Casella I. Watson R. Peschle C. Oncogene. 1999; 18: PubMed Scopus Google or T. Kakizawa T. Hashizume K. Mol. Cell. Biol. 1999; 19: 2644-2649Crossref PubMed Scopus (85) Google Scholar) were at a μg B-MYB through MYB and A. B. De P. Casella I. Watson R. Peschle C. Oncogene. 1999; 18: PubMed Scopus Google we the of used for transfection For this was performed in and experiments were at three was with a kit according to manufacturer's instructions (Promega). Light was with the of a and expressed as In to proteins with the terminus of the B-MYB protein, HL60 cells were with and nuclear or cytoplasmic protein lysates were lysates were incubated with GST-B-MYB fusion protein, and cellular proteins binding to GST-B-MYB were analyzed by SDS-PAGE and cytoplasmic and nuclear proteins bound to the and a nuclear protein of 110 was eluted in The was bound the DNA-binding domain, which is to the B-MYB domain, with lower and it not bind to GST cytoplasmic proteins bound the fusion proteins, and a protein was with both and B-MYB The of this cytoplasmic protein was not A was loaded with the from GST-B-MYB protein mixed with nuclear lysates from 109 HL60 cells. determined that the 110-kDa protein was To PARP as the protein, the eluted was loaded onto an SDS-PAGE and to analysis with a from but not from GST PARP protein that was by the PARP To the hypothesis that PARP and B-MYB are able to a we mixed PARP with GST-B-MYB fusion protein. In vitro PARP bound to B-MYB, but not to as by pull-down experiments 2 The is in the analysis showed that the PARP but not the control was able to co-immunoprecipitate B-MYB protein from HL60 nuclear extracts with this a B-MYB-specific was able to co-immunoprecipitate PARP protein from HL60 cells 3 endogenous is in nuclear extracts of HL60 cells. analysis was performed as and of HL60 nuclear lysates were with the as the and to analysis with a B-MYB or a PARP PARP is binding to the B-MYB DNA-binding domain, it is that the may B-MYB transactivating function. To address this we a containing with expression and of transcription, to its activity on polymerase S.L. J. M. M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In of PARP and B-MYB in synergistic activation of the MYB-responsive To the enzymatic activity of PARP is required in the we of and an at the domain V. M. G. 1997; PubMed Scopus Google Scholar). Although at different both PARP were able to with B-MYB suggesting that activity is not required for the function of PARP evidence suggests that transcription are by association with co-activators and with protein The DNA-binding domain of MYB proteins, the tryptophan repeat has the potential of with both DNA and proteins. In this we show that PARP binds to the domain and its transactivating activity. Interestingly, of the MYB family, binds PARP with suggesting that PARP may B-MYB, but not activity Although the and proteins show a in the DNA-binding domain and B-MYB is the different for PARP by the proteins N. Takahashi M. Matsui M. Ishii S. Date T. Sasamoto S. Ishizaki R. Nucleic Acids Res. 1988; 16: 11075-11089Crossref PubMed Scopus (200) Google Scholar). reports have that PARP can behave as an inhibitor or stimulator of transcription in D. S. I. G. Biochem. J. 1999; PubMed Google Scholar). has been shown that is required for both inhibition and of activated transcription and that PARP enzymatic activity is enhanced by DNA (30.Kumari S.R. Mendoza-Alvarez H. Alvarez-Gonzalez R. Cancer Res. 1998; 58: 5075-5078PubMed Google Scholar, 31.Miyamoto T. Kakizawa T. Hashizume K. Mol. Cell. Biol. 1999; 19: 2644-2649Crossref PubMed Scopus (85) Google Scholar, D. S. I. G. Biochem. J. 1999; PubMed Google Scholar). PARP to through association with the DNA and PARP is required for DNA polymerase activity F. J.L. G. J. Nucleic Acids Res. 1998; PubMed Scopus Google Scholar). In the present we evidence that PARP B-MYB and that activity is not required for this PARP proteins of enzymatic activity the of with B-MYB suggest a in in the of DNA PARP is activated and to repair and gene M. M. B. Mol. Cell. Biochem. 1999; PubMed Google Scholar, F. V. C. C. E. De G. J. V. J. G. 1999; PubMed Scopus Google Scholar). On the other hand, during the normal cell cycle, PARP may be by transactivator proteins, such as B-MYB, to gene PARP enzymatic activity not a it is that the PARP and the B-MYB DNA-binding domain is for the synergistic of the carboxyl terminus of B-MYB B-MYB transactivating suggesting that B-MYB is a constitutively repressed molecule (1.Sala A. Watson R. J. Cell. Physiol. 1999; 179: 245-250Crossref PubMed Scopus (63) Google Scholar). B-MYB transactivating function also can be enhanced by induced phosphorylation (1.Sala A. Watson R. J. Cell. Physiol. 1999; 179: 245-250Crossref PubMed Scopus (63) Google Scholar). bound to different intramolecular may be by phosphorylation of B-MYB protein and/or by with cofactor such as PARP, resulting in a and increased transactivating cofactor may the cross-talk B-MYB and the transcription In experiments we have been able to show that the is increased in the of an oligonucleotide containing an MYB-binding site not This suggests that a containing PARP and B-MYB may bind to the MYB-binding site at resulting in synergistic gene The B-MYB and PARP are of further to the role of PARP in activated transcription have that abnormal expression of the may be implicated in the progression of human through its to transcription of genes to cell cycle progression and/or apoptosis (1.Sala A. Watson R. J. Cell. Physiol. 1999; 179: 245-250Crossref PubMed Scopus (63) Google Scholar, K. Liu Q. Beaudry G.A. Madden S.L. Westra W.H. Wehage S.L. Yang S.C. Heitmiller R.F. Bertelsen A.H. Sidransky D. Jen J. Cancer Res. 1998; 58: 5690-5694PubMed Google Scholar, 20.Raschella‘ G. Cesi V. Amendola R. Negroni A. Tanno B. Altavista P. Tonini G.P. De Bernardi B. Calabretta B. Cancer Res. 1999; 59: 3365-3368PubMed Google Scholar). be of to B-MYB on cells with a PARP gene to further the regulation of gene transcription with cellular functions such as differentiation, and We Dr. De and for and We also Dr. and Dr. Perry for the and pSPARP expression and Dr. Martino Introna for the Dr. G. De and T. are for the and