Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

Abstract

The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1β. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPβ carboxylterminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity. The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1β. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPβ carboxylterminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity. The ETS family of transcription factors serves a variety of roles in development and differentiation (1Sementchenko V.I. Watson D.K. Oncogene. 2000; 19: 6533-6548Crossref PubMed Scopus (314) Google Scholar). A highly conserved DNA-binding domain (DBD) 3The abbreviations used are: DBDDNA-binding domainIL1Bgene encoding the interleukin-1β proteinbZIPbasic leucine zipper domainTADtransactivation domainUISupstream induction sequence for the interleukin-1β gene.3The abbreviations used are: DBDDNA-binding domainIL1Bgene encoding the interleukin-1β proteinbZIPbasic leucine zipper domainTADtransactivation domainUISupstream induction sequence for the interleukin-1β gene. defines the family. Spi-1/PU.1 (Spi-1), one of the most diverse members, is critical for myeloid and B cell differentiation and function (2Friedman A.D. Oncogene. 2002; 21: 3377-3390Crossref PubMed Scopus (269) Google Scholar, 3Shin M.K. Koshland M.E. Genes Dev. 1993; 7: 2006-2015Crossref PubMed Scopus (122) Google Scholar, 4Oikawa T. Yamada T. Kihara-Negishi F. Yamamoto H. Kondoh N. Hitomi Y. Hashimoto Y. Cell Death Differ. 1999; 6: 599-608Crossref PubMed Scopus (76) Google Scholar, 5Fisher R.C. Scott E.W. Stem Cells. 1998; 16: 25-37Crossref PubMed Scopus (134) Google Scholar, 6Auron P.E. Lotze M.T. Thomson A.W. Measuring Immunity-Basic Science and Clinical Practice. 2005: 91-109Google Scholar). It also regulates the expression of many effector genes, including IL1B, coding for the IL-1β protein (7Kominato Y. Galson D. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1995; 15: 59-68Crossref Google Scholar). The Spi-1 molecule possesses a mutipartite transactivation domain (TAD) consisting of the amino half of the molecule that binds TBP, RB (8Hagemeier C. Bannister A.J. Cook A. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1580-1584Crossref PubMed Scopus (273) Google Scholar), and CBP/p300 (9Yamamoto H. Kihara-Negishi F. Yamada T. Hashimoto Y. Oikawa T. Oncogene. 1999; 18: 1495-1501Crossref PubMed Scopus (95) Google Scholar) (Fig. 1a). The carboxyl end of the TAD contains a phosphorylatable PEST sequence that aids in the formation of a protein-protein interaction with transcription factors IRF-4 and -8 (10Escalante C.R. Brass A.L. Pongubala J.M. Shatova E. Shen L. Singh H. Aggarwal A.K. Mol Cell. 2002; 10: 1097-1105Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Marecki S. Riendeau C.J. Liang M.D. Fenton M.J. J. Immunol. 2001; 166: 6829-6838Crossref PubMed Scopus (97) Google Scholar). Located at the COOH-terminal end of the protein is the ETS DBD. Besides DNA binding, this domain is also a tethering site for multiple transactivators, some of which occur at composite DNA-binding sites (reviewed in Ref. 12Li R. Pei H. Watson D.K. Oncogene. 2000; 19: 6514-6523Crossref PubMed Scopus (189) Google Scholar), and contains the previously coarsely mapped nuclear localization signal (NLS) (13Boulukos K.E. Pognonec P. Rabault B. Begue A. Ghysdael J. Mol. Cell. Biol. 1989; 9: 5718-5721Crossref PubMed Scopus (92) Google Scholar). The structure of the Spi-1 DBD, like other ETS factors, consists of 85 conserved residues folded into a winged-helix-turn-helix (wHTH) conformation (14Kodandapani R. Pio F. Ni C.Z. Piccialli G. Klemsz M. McKercher S. Maki R.A. Ely K.R. Nature. 1996; 380: 456-460Crossref PubMed Scopus (267) Google Scholar) (Fig. 1b). Two arginines, which are solvent-exposed in helix α3, are conserved in all ETS family members and form direct and indirect hydrogen bonds to a core DNA sequence of A/GGAA in Spi-1 (1Sementchenko V.I. Watson D.K. Oncogene. 2000; 19: 6533-6548Crossref PubMed Scopus (314) Google Scholar, 7Kominato Y. Galson D. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1995; 15: 59-68Crossref Google Scholar, 15Pio F. Kodandapani R. Ni C.Z. Shepard W. Klemsz M. McKercher S.R. Maki R.A. Ely K.R. J. Biol. Chem. 1996; 271: 23329-23337Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The Spi-1 DBD is 98% conserved at the amino acid level between mouse and human, suggesting stringent structural and functional requirements. DNA-binding domain gene encoding the interleukin-1β protein basic leucine zipper domain transactivation domain upstream induction sequence for the interleukin-1β gene. DNA-binding domain gene encoding the interleukin-1β protein basic leucine zipper domain transactivation domain upstream induction sequence for the interleukin-1β gene. Spi-1 plays a necessary role in the regulation of the IL1B promoter by cooperating functionally with C/EBPβ, a basic leucine zipper (bZIP) family member (16Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1204) Google Scholar, 17Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (438) Google Scholar). This cooperativity may be derived, in part, by a physical interaction between the DBD of both molecules and the two composite DNA-binding sites in the IL1B promoter (Fig. 2a) (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The downstream TATA-proximal site binds relatively well to Spi-1 but weakly to C/EBPβ (7Kominato Y. Galson D. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1995; 15: 59-68Crossref Google Scholar). C/EBPβ DNA binding at this site appears to be supported by a protein interaction with the adjacent DNA-binding domain of Spi-1 (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Relative binding of these two factors at the upstream site is reversed relative to the downstream site and may play an especially critical role in serving to tether Spi-1 to C/EBPβ bound at a site (located between -2768 and -2761) in the IL1B upstream induction sequence (UIS) enhancer (19Tsukada J. Saito K. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1994; 14: 7285-7297Crossref PubMed Google Scholar), based upon a greater loss of general enhancer function when the upstream site is deleted from the promoter (7Kominato Y. Galson D. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1995; 15: 59-68Crossref Google Scholar). Analogous phenomena have been described between ETS and other transcription factors (reviewed in (20Verger A. Duterque-Coquillaud M. BioEssays. 2002; 24: 362-370Crossref PubMed Scopus (119) Google Scholar)). For example, the cooperative function observed for Spi-1 and IRF-4 on the IgL λ enhancer is explained, in part, by the enhanced DNA binding of IRF4 mediated by sidechain contacts with Arg222 and Lys223 on the adjacent bound Spi-1 (10Escalante C.R. Brass A.L. Pongubala J.M. Shatova E. Shen L. Singh H. Aggarwal A.K. Mol Cell. 2002; 10: 1097-1105Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). C-Myb, another HTH protein, was shown to interact with the bZIP domain of C/EBPβ while each was bound to a remote DNA site, demonstrating a model of tethering between an enhancer and promoter (21Tahirov T.H. Sato K. Ichikawa-Iwata E. Sasaki M. Inoue-Bungo T. Shiina M. Kimura K. Takata S. Fujikawa A. Morii H. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2002; 108: 57-70Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In contrast, Spi-1 interactions with several proteins including C/EBPβ and human cytomegalovirus (HCMV) IE2 reveal clear cooperative function but no obvious DNA binding cooperativity (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 22Behre G. Whitmarsh A.J. Coghlan M.P. Hoang T. Carpenter C.L. Zhang D-E. Davis R.J. Tenen D.G. J. Biol. Chem. 1999; 274: 4939-4946Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Wara-aswapati N. Yang Z. Waterman W.R. Koyama Y. Tetradis S. Choy B.K. Webb A.C. Auron P.E. Mol. Cell. Biol. 1999; 19: 6803-6814Crossref PubMed Google Scholar). This has led to a hypothesis of protein-tethered transactivation, whereby a direct DNA-binding protein tethers another protein, resulting in either activation or inhibition of function (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 23Wara-aswapati N. Yang Z. Waterman W.R. Koyama Y. Tetradis S. Choy B.K. Webb A.C. Auron P.E. Mol. Cell. Biol. 1999; 19: 6803-6814Crossref PubMed Google Scholar). Therefore, understanding the molecular basis for these kinds of interactions continues to be intensely studied. While investigating the nature of the Spi-1-C/EBPβ interaction, we uncovered new functions for the highly conserved arginines of helix 3 (Arg232 and Arg235) within the DNA recognition site. These functions expand upon the classic role these residues play in DNA binding and suggest new mechanisms of functional cooperativity and protein-DNA interaction for transcription factors. Plasmids—The pGL3B HT reporter plasmid was constructed as described previously (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The 3MEHT and 3MEHTΔIL6 expression plasmids contain the IL1B promoter sequence -131 to +12, and the IL1B promoter sequence -131 to +12 with deletion of the C/EBPβ-binding site at -95 to -80, respectively, ligated to pA10CAT3ME (24Shirakawa F. Saito K. Bonagura C.A. Galson D.L. Fenton M.J. Webb A.C. Auron P.E. Mol. Cell. Biol. 1993; 13: 1332-1344Crossref PubMed Google Scholar). The HCMV expression vector pEQ326 (IE2), gift from Adam Geballe (Fred Hutchinson Cancer Research Center, Seattle, WA), contains the genomic HCMV IE2 DNA inserted into pGEM1 vector. The C/EBPβ pCDNA3.1 expression vector contains the full-length cDNA for C/EBPβ (19Tsukada J. Saito K. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1994; 14: 7285-7297Crossref PubMed Google Scholar). The bZIP portion of C/EBPβ from amino acids 269-345 was previously cloned into pCDNA3.1 (Invitrogen) and pGEX 2T (Amersham Biosciences) by PCR (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The Spi-1 pRC/CMV expression vector contains the murine Spi-1 cDNA as reported previously (25Galson D.L. Hensold J.O. Bishop T.R. Schalling M. D'Andrea A.D. Jones C. Auron P.E. Mol. Cell. Biol. 1993; 13: PubMed Google Scholar). constructed PCR as described previously N. Yang Z. Waterman W.R. Koyama Y. Tetradis S. Choy B.K. Webb A.C. Auron P.E. Mol. Cell. Biol. 1999; 19: 6803-6814Crossref PubMed Google Scholar) or by to the DBD either within plasmids or cloned by PCR into either pRC/CMV for in or 2T (Amersham Biosciences) for in as described previously N. Yang Z. Waterman W.R. Koyama Y. Tetradis S. Choy B.K. Webb A.C. Auron P.E. Mol. Cell. Biol. 1999; 19: 6803-6814Crossref PubMed Google Scholar). by DNA The vector was used in for of and proteins from previously described N. Yang Z. Waterman W.R. Koyama Y. Tetradis S. Choy B.K. Webb A.C. Auron P.E. Mol. Cell. Biol. 1999; 19: 6803-6814Crossref PubMed Google Scholar). with for in with and one on and in and with proteins at with and with the in protein of for at with and by with (Invitrogen) for protein and for binding protein in and with (Amersham Biosciences) to the and by to in well with and in and DNA was in of of and of at was to the and an was to each of for with with and by an to as at the of with For all the DNA The and as described previously N. Yang Z. Waterman W.R. Koyama Y. Tetradis S. Choy B.K. Webb A.C. Auron P.E. Mol. Cell. Biol. 1999; 19: 6803-6814Crossref PubMed Google Scholar). and from a from on (Invitrogen) and to with and was by enhanced to IL1B promoter -131 to was and DNA in the of in and with Relative protein based on of of in and to to binding and DNA binding of and with in a of The binding at for and to on a and The was and by of Spi-1 DBD to C/EBPβ and transactivation of the IL1B promoter by Spi-1 and C/EBPβ (Fig. and a from this by in the of the upstream enhancer (Fig. A collection of of the Spi-1 DBD for as with of the C/EBPβ bZIP domain and at in and in C/EBPβ bZIP and IE2 binding, respectively, while at a on binding to C/EBPβ bZIP and or no on binding with IE2 (Fig. the in a in C/EBPβ bZIP binding. in residues previously shown to with interaction between a bZIP and an ETS factor with to also A. E. S. R. A. J. D. Duterque-Coquillaud M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). These at and in Spi-1 no on bZIP binding at and residues that Spi-1 DBD interaction with (10Escalante C.R. Brass A.L. Pongubala J.M. Shatova E. Shen L. Singh H. Aggarwal A.K. Mol Cell. 2002; 10: 1097-1105Abstract Full Text Full Text PDF PubMed Scopus (123) Google also interaction with bZIP or IE2 of Spi-1 DBD on was on and nuclear to the expression of Spi-1 or DBD expression and the by and with the of the proteins to DNA by was a in Spi-1 to the from to either or at and respectively, in and the in we DNA binding for these proteins (Fig. However, the no on nuclear while the with in to in nuclear localization (Fig. and respectively, and the in no binding was observed for the was DNA binding for the (Fig. at and in to of the protein in the and no DNA binding respectively, in and the in and with the and all or greater nuclear but was DNA binding observed for and and DNA binding for (Fig. respectively, and the in and that DNA binding in nuclear The protein was in the nuclear and in the (Fig. in DNA (Fig. These are with a loss of function for this (Fig. resulting from to the structural core of the The that a of Spi-1 to the with protein proteins, both and nuclear localization for the full-length molecule but nuclear localization for the DBD These suggest that some upstream portion of the Spi-1 molecule with or regulates Spi-1 nuclear these we also the of the Spi-1 DBD by and (Fig. The a in localization to the nuclear for the DBD to of the protein (Fig. of Spi-1 DBD for Spi-1 described in used in an IL1B promoter reporter to protein expression with function (Fig. expression from cell was and the nuclear localization of each protein be for in the of the in For example, when was at was Spi-1 at a This was with the that this protein possesses DNA binding and nuclear of expression the the other at or both and in reporter at expression and at expression the function at expression and function when expression was This well with the nuclear localization and but DNA binding at Arg222 and Lys223 have been by reporter but of protein and function to the Spi-1 at the COOH-terminal of C/EBPβ to the Spi-1 ETS and Spi-1 on the IL1B C/EBPβ molecule contains a transactivation domain at and a basic leucine zipper structure at (Fig. C/EBPβ, like other bZIP proteins via the leucine which the basic of each at two the DNA a DNA The structure of the zipper from the DNA helix at into the (21Tahirov T.H. Sato K. Ichikawa-Iwata E. Sasaki M. Inoue-Bungo T. Shiina M. Kimura K. Takata S. Fujikawa A. Morii H. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2002; 108: 57-70Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, W. P. J. Mol. Biol. 1995; PubMed Scopus Google Scholar, S.C. Nature. 1995; PubMed Scopus Google Scholar, A.C. PubMed Scopus Google Scholar). The bZIP of C/EBPβ also interact with transcription factors, including other ETS family members and (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, B. Mol. Cell. Biol. 1993; PubMed Google Scholar, Y. Chen C. Auron P.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (134) Google Scholar, J. M. Y. R. S. M. S. Y. Auron P.E. S. 90: PubMed Google Scholar). However, the of the bZIP molecule these contacts was the that residues of the COOH-terminal end of the C/EBPβ bZIP molecule are required for interaction with while bound to a remote DNA site (21Tahirov T.H. Sato K. Ichikawa-Iwata E. Sasaki M. Inoue-Bungo T. Shiina M. Kimura K. Takata S. Fujikawa A. Morii H. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2002; 108: 57-70Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). that a of interaction occur between C/EBPβ and this we a bZIP residues at the COOH-terminal end Using protein we that bZIP loss of binding to a protein with the interaction between the bZIP and (Fig. previously reported that a promoter the composite site in 2a) cooperativity when the site was by that C/EBPβ binding (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Using a we the role of tethering at the upstream composite site. of the observed multiple functions of in the Spi-1 DBD, we an to the functional role of we that the of the reporter an upstream C/EBPβ site deletion be by the of Spi-1 expression vector (Fig. This led to a hypothesis that the of the bZIP domain Spi-1 This was by determining the of the IL1B reporter to with either bZIP or bZIP when with Spi-1 and C/EBPβ (Fig. The that cooperative transactivation of the IL1B promoter was enhanced by the bZIP domain but when the COOH-terminal The loss of function by bZIP be by the loss of DNA binding by this (Fig. In to sites on the Spi-1 DBD, we have uncovered may be novel functions for conserved arginines in the DBD of all ETS these residues are well for role in DNA binding, other functions to nuclear localization and protein The for ETS factors was previously to the DBD (13Boulukos K.E. Pognonec P. Rabault B. Begue A. Ghysdael J. Mol. Cell. Biol. 1989; 9: 5718-5721Crossref PubMed Scopus (92) Google Scholar). However, reveal a signal for this family of reveal that nuclear localization is mediated by a helix which has and into the The that deletion of the Spi-1 TAD to a greater of protein in the suggests that a of the to helix This may to either with or nuclear While many DNA-binding proteins have to DBD 1995; PubMed Scopus Google Scholar), we are of a that have function and DNA binding to the These the bZIP family members and 2001; PubMed Scopus Google Scholar, G. Mol. 1991; 5: PubMed Scopus Google Scholar, S.C. Johnson P.F. 6: Google Scholar). was that at and binding to C/EBPβ with a on binding to this to the of the interaction between these which was to interaction at a surface other that previously reported that Spi-1 tether C/EBPβ to the promoter the downstream composite site binds to Spi-1 but C/EBPβ and that required the of the C/EBPβ TAD (18Yang Z. Wara-Aswapati N. Chen C. Tsukada J. Auron P.E. J. Biol. Chem. 2000; 275: 21272-21277Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Using a we the role of tethering at the upstream composite site. This was we previously reported that the downstream Spi-1 site and the upstream site each required for However, deletion of the upstream C/EBPβ site that was important but (7Kominato Y. Galson D. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1995; 15: 59-68Crossref Google Scholar). that the of the reporter the upstream C/EBPβ site deletion be by the of Spi-1 expression vector. These suggest that the function of this site the of the C/EBPβ TAD but a cooperativity as a protein-protein The observed cooperative of a C/EBPβ bZIP on Spi-1 and transactivation of the IL1B promoter upstream composite site is to the hypothesis that the bZIP domain Spi-1 binding to this site. This is in to the function for the TAD in other of bZIP cooperativity by deletion of COOH-terminal residues to a that the tethering function with Spi-1 DBD. The loss of cooperative function by the deletion be to loss of or DNA binding as we observed DNA binding by bZIP C/EBPβ and IE2 interact at a site on but with some that the in the of binding to C/EBPβ and IE2 when and are direct structural be to the the role of protein tethering at a critical for DNA binding. of the Spi-1 DBD structure (14Kodandapani R. Pio F. Ni C.Z. Piccialli G. Klemsz M. McKercher S. Maki R.A. Ely K.R. Nature. 1996; 380: 456-460Crossref PubMed Scopus (267) Google Scholar) that helix 3 in the with at the of the helix to (Fig. However, that this residue be for interaction with the of C/EBPβ bound to an adjacent DNA binding site. However, C/EBPβ in or with a remote DNA site to this site. This model is to that shown for tethering of a remote C/EBPβ enhancer site to on the the molecular interactions (21Tahirov T.H. Sato K. Ichikawa-Iwata E. Sasaki M. Inoue-Bungo T. Shiina M. Kimura K. Takata S. Fujikawa A. Morii H. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2002; 108: 57-70Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The C/EBPβ leucine zipper with a surface of that while with the DNA of the (21Tahirov T.H. Sato K. Ichikawa-Iwata E. Sasaki M. Inoue-Bungo T. Shiina M. Kimura K. Takata S. Fujikawa A. Morii H. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2002; 108: 57-70Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In contrast, the of Spi-1 within the suggests a novel of the by the of the leucine zipper (Fig. This is the basic the C/EBPβ leucine zipper as with that of (Fig. This a model the nature of DNA at sites of are greater hydrogen and of Scholar). These to the bZIP leucine zipper within the and the for interaction with at one of the two amino of Spi-1 at the of helix α3, the interaction (Fig. and helix interact with tethering of This the of two functions that on and of the protein and DNA the protein interaction may to Spi-1 with proteins at composite DNA-binding sites (Fig. interactions cooperative binding on the This model be for composite promoter sites the for C/EBPβ binding to DNA is and Spi-1 is as is the for the upstream IL1B composite site (Fig. The is for the downstream TATA-proximal composite site (Fig. Therefore, C/EBPβ and Spi-1 either to the IL1B DNA via a interaction or C/EBPβ site and as a for In either the interaction of one factor with DNA the of the other factor in the of DNA-binding site, the interaction via protein-protein This occur for other composite Spi-1 sites that DNA binding cooperativity with transcription factors, including the interaction Spi-1 possesses a greater for site A.L. Singh H. EMBO J. 1999; 18: PubMed Scopus Google Scholar). which is a tether between the of C/EBPβ bound at the upstream composite site and Spi-1 bound at the composite site (Fig. tethering between C/EBPβ bound at the IL1B enhancer contains a functional and Spi-1 bound at a downstream composite site be the structural basis for functional cooperativity. In we have that the conserved ETS domain arginines have multiple functions in to DNA binding. These nuclear localization and protein binding with other The of this to both functions upon the and on ETS that the model the cooperativity observed between molecules on the IL1B also that the leucine zipper of C/EBPβ may function in classic bZIP but also as a DNA interaction an for long DNA the of and G. Tenen for and the of this L. Galson and we R. for critical of the