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• W2029294217 abstract "Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199–291) and the Ets-1 regulatory III domain (amino acids 190–257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2–80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2–45) and region 2 (R2, amino acids 46–80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD. Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199–291) and the Ets-1 regulatory III domain (amino acids 190–257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2–80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2–45) and region 2 (R2, amino acids 46–80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD. Pit-1/GHF-1 is a POU homeodomain transcription factor specifically expressed in the anterior pituitary which is responsible for determining the somatotroph, lactotroph, and thyrotroph lineages, as well as the regulated expression of their respective hormones: growth hormone, prolactin, and thyroid-stimulating hormone. Combinatorial interactions between Pit-1 and other trans-acting factors at composite or adjacent response elements have been shown to allow Pit-1 to serve as a cell-specific integrator of signaling pathways. As such, Pit-1 has been shown to physically interact with itself, ER, 1The abbreviations used are: ER, estrogen receptor; BPV, bovine papilloma virus; CBP, cAMP-responsive element-binding protein-binding protein; CMV, cytomegalovirus; DBD, DNA binding domain; EBS, Ets binding site; FPIV, footprint IV; GST, glutathione S-transferase; In1, In2, inhibitory domains 1 and 2, respectively; Luc, luciferase; m, mutant; MAP, mitogen-activated protein; P1, P2, pointed domains 1 and 2, respectively; PBS, phosphate-buffered saline; PRL, prolactin; r, rat; R1 and R2, amino acids 1–45 functioning as a modulatory region 1 and amino acids 46–80 functioning as an effector region 2, respectively; RI, RII, RIII, regulatory domains I, II, and III, respectively; RLU, relative light units; TAD, transcriptional activation domain; TK, thymidine kinase; WT, wild-type. thyroid hormone receptor, c-Jun, Oct-1, GATA-2, P-Lim, Ptx-1, and Ets-1 (1Jacobson E. Li P. Leon-del-Rio A. Rosenfeld M. Aggarwal A. Genes Dev. 1997; 11: 198-212Crossref PubMed Scopus (177) Google Scholar, 2Ingraham H.A. Flynn S.E. Voss J.W. Albert V.R. Kapiloff M.S. Wilson L. Rosenfeld M.G. Cell. 1990; 61: 1021-1033Abstract Full Text PDF PubMed Scopus (320) Google Scholar, 3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar, 4Farrow K. Manning N. Schaufele F. Gutierrez-Hartmann A. J. Biol. Chem. 1996; 271: 17139-17146Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 5Nowakowski B. Maurer R. Mol. Endocrinol. 1994; 8: 1742-1749Crossref PubMed Google Scholar, 6Gordon D. Lewis S. Haugen B. James R. McDermott M. Wood W. Ridgway E. J. Biol. Chem. 1997; 272: 24339-24347Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Szeto D. Ryan A. O'Connell S. Rosenfeld M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (247) Google Scholar, 8Voss J.W. Wilson L. Rosenfeld M.G. Genes Dev. 1991; 5: 1309-1320Crossref PubMed Scopus (172) Google Scholar, 9Bach I. Rhodes S.J. Pearse II, R.V. Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (293) Google Scholar). In precisely this manner, an Ets/Pit-1 combination, acting via the Ets binding site (EBS)/footprint IV (FPIV) composite element spanning positions –217 to –190, is critical for both basal lactotroph-specific expression of the rPRL gene and for the targeting of the Ras-MAP kinase pathway to the prolactin promoter (3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar, 10Bradford A.P. Conrad K.E. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1995; 15: 2849-2857Crossref PubMed Scopus (112) Google Scholar). Several lines of evidence indicate that both Ets-1 and Pit-1 factors are required for the synergistic activation and Ras responsiveness. For example, transient transfection of pituitary-derived GH4T2 somatolactotrope cells with a dominant-negative Ets factor encoding only the Ets DNA binding domain (DBD), or with Pit-1β (an alternatively spliced isoform that functions as a dominant-negative effector in pituitary cells), results in diminished activity of the rPRL promoter in an effector-dependent manner (3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar). Similarly, transient transfection of rPRL promoter constructs containing site-specific mutations of either the EBS or FPIV sites within the composite element results in a loss of Ras responsiveness (10Bradford A.P. Conrad K.E. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1995; 15: 2849-2857Crossref PubMed Scopus (112) Google Scholar, 11Bradford A. Conrad K. Tran P. Ostrowski M. Gutierrez-Hartmann A. J. Biol. Chem. 1996; 271: 24639-24648Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In non-pituitary HeLa cells, it has been shown that although individual Ets-1 or Pit-1 effectors strongly activate the rPRL promoter, the combination results in a response that is 6-fold greater than the sum of the individual responses (i.e. 6-fold synergy) (3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar). In addition, Ets-1 and Pit-1 have been shown to bind physically to each other in dilute solution and in the absence of DNA by utilizing the Ets-1 regulatory III (RIII) transcriptional activation domain (TAD) and Pit-1 homeodomain (3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar, 12Augustijn K.D. Duval D.L. Wechselberger R. Kaptein R. Gutierrez-Hartmann A. Van der Vliet P.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12657-12662Crossref PubMed Scopus (26) Google Scholar, 13Bradford A.P. Brodsky K.S. Diamond S.E. Kuhn L.C. Liu Y. Gutierrez-Hartmann A. J. Biol. Chem. 2000; 275: 3100-3106Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Finally, Thr-82 of chicken p68 c-Ets-1 was identified as a MAP kinase phosphorylation site, and site-specific mutation of Thr-82 to alanine resulted in loss of the Ets-1-mediated enhancement of the Ras response (14Wasylyk C. Bradford A. Gutierrez-Hartmann A. Wasylyk B. Oncogene. 1997; 14: 899-913Crossref PubMed Scopus (93) Google Scholar). Taken together, these results support a two-step model of Ets-1/Pit-1 activation of the rPRL promoter in which binding of Ets-1/Pit-1 to the composite element and to each other regulates basal promoter activity, and Ras activation occurs in part as a response to phosphorylation of Ets-1 at Thr-82. Structure-function analysis of p68 Ets-1 has highlighted a number of features. Specifically, the TAD is composed of three regulatory domains, RI–RIII. RI and RIII function as positive TADs, whereas RII functions as a domain that negatively regulates the function of RI and positively regulates RIII. Overlapping the RII TAD is the pointed domain which contains the MAP kinase phosphorylation site, Thr-82. The latter is highly conserved in the Ets subfamily, comprising Ets-1, Ets-2, and pointed P1 and P2 (15Graves B. Petersen J. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar). In the Ets subfamily, RI and RIII are unique to either Ets-1 or Ets-2. Amino acids 180–300 within RIII have only 6% similarity between Ets-1 and Ets-2 compared with ∼90% similarity for the DBD and 66% conservation for RII. Finally, the ETS DBD, located near the carboxyl terminus of Ets-1, is flanked by two domains that inhibit DNA binding, In1 and In2. The DBD is a highly conserved 84–90-amino acid domain referred to as the ETS domain (16Donaldson L. Petersen J. Graves B. McIntosh L. EMBO J. 1996; 15: 125-134Crossref PubMed Scopus (135) Google Scholar, 17Donaldson L. Petersen J. Graves B. McIntosh L. Biochemistry. 1994; 33: 13509-13516Crossref PubMed Scopus (69) Google Scholar). The ETS domain, which serves to define the family, utilizes a winged helix-turn-helix structural motif to bind to Ets binding sites. The core sequence of the Ets binding sites consists of the sequence 5′-GGA(A/T)-3′. In addition, flanking DNA sequences on either side of this core binding element may contribute to specific Ets factor binding, such that the actual binding site may consist of ∼9 bp (15Graves B. Petersen J. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar). The composite EBS/FPIV binding site located at –217 to –190 consists of a core GGAA site on the antisense strand separated by 8 bp from the core binding site for Pit-1 (see Fig. 1). Typically, monomeric Ets factors bind to DNA weakly and require a vicinally bound factor to stabilize Ets factor-DNA interactions (18Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar). Consequently, Ets factors and their partners are often recruited to bipartite binding sites on DNA, forming ternary complexes and resulting in synergistic transcriptional responses. The spacing and orientation of these bipartite binding sites do not appear to be critical determinants for the binding of these ternary complexes. For example, ternary complexes of SRF and the Ets factors Elk-1 and SAP-1 can occur even when the respective binding sites are inverted with respect to each other or moved apart over two helical turns of DNA (19Treisman R. Marais R. Wynne J. EMBO J. 1992; 11: 4631-4640Crossref PubMed Scopus (137) Google Scholar). Rather, DNA binding appears to be modulated by the flanking autoinhibitory sequences, In1 and In2, which can be regulated by specific protein partnerships and further modulated by protein phosphorylation (15Graves B. Petersen J. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 18Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar, 20Hill C. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar, 21Wasylyk B. Hagman J. Gutierrez-Hartmann A. Trends Biochem. Sci. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 22Dittmer J. Nordheim A. Biochim. Biophys. Acta. 1998; 1377: F1-F11PubMed Google Scholar, 23Wasylyk B. Nordheim A. Papavassiliou A. Transcription Factors in Eukaryotes. Landes Bioscience Publishing, Georgetown, TX1997: 251-284Google Scholar). Indeed, the most important mechanism for achieving target gene specificity is cofactor-induced alterations of Ets protein interactions. Usually, Ets proteins interact with partners through the EtsDBD (15Graves B. Petersen J. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 18Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar, 22Dittmer J. Nordheim A. Biochim. Biophys. Acta. 1998; 1377: F1-F11PubMed Google Scholar, 23Wasylyk B. Nordheim A. Papavassiliou A. Transcription Factors in Eukaryotes. Landes Bioscience Publishing, Georgetown, TX1997: 251-284Google Scholar). However, core-binding factor and Pit-1 interact with Ets-1 via domains distinct from the DBD, with core-binding factor binding to the In1 autoinhibitory domain and Pit-1 binding to the RIII TAD (3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar, 24Kim W.-Y. Sieweke M. Ogawa E. Wee H.-J. Englmeier U. Graf T. Ito Y. EMBO J. 1999; 18: 1609-1620Crossref PubMed Scopus (197) Google Scholar). Further analysis has refined the Pit-1 interaction face of Ets-1 to a 67-amino acid subdomain (amino acids 190–257) within this Ets-1 isoform-specific region (3Bradford A. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1997; 17: 1065-1074Crossref PubMed Scopus (102) Google Scholar, 12Augustijn K.D. Duval D.L. Wechselberger R. Kaptein R. Gutierrez-Hartmann A. Van der Vliet P.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12657-12662Crossref PubMed Scopus (26) Google Scholar). Conversely, POU homeodomain transcription factors utilize a bipartite DBD that can partially encircle the DNA. The DNA element thus dictates the configurations of subdomains and subsequent recruitment of specific coregulators to control transcription (25Phillips K. Luisi B. J. Mol. Biol. 2000; 302: 1023-1039Crossref PubMed Scopus (200) Google Scholar). Specifically, the ability of Pit-1 to bind to DNA as either a monomer or a dimer dictates the domains that it uses to synergize with heterologous transcription factors (26Holloway J. Szeto D. Scully K. Glass C. Rosenfeld M. Genes Dev. 1995; 9: 1992-2006Crossref PubMed Scopus (88) Google Scholar). Similarly, the spacing of the contact points for the POU-specific domain and POU homeodomain is sufficient to transform Pit-1 from a trans-activating factor to a repressor (27Scully K.M. Jacobson E. Jepsen K. Lunyak V. Viadiu H. Carriere C. Rose D.W. Hooshmand F. Aggarwal A.K. Rosenfeld M.G. Science. 2000; 290: 1127-1131Crossref PubMed Scopus (219) Google Scholar). In addition to its role in DNA binding, the homeodomain of Pit-1 is necessary and sufficient for physical interaction with Ets-1. Although it is unclear what precise role the physical interaction of Ets-1/Pit-1 plays in the synergistic activation of the rPRL promoter, it is intriguing to note that the β-domain of the Pit-1β isoform also interacts with this Ets-1-specific region (13Bradford A.P. Brodsky K.S. Diamond S.E. Kuhn L.C. Liu Y. Gutierrez-Hartmann A. J. Biol. Chem. 2000; 275: 3100-3106Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In this study we examined the role played by spacing and composition of the combinatorial EBS/FPIV element or the physical interaction of Ets and Pit-1 in Ras responsiveness and synergistic activation of the rPRL promoter. We show that the Ets-1/Pit-1 composite element represents a highly flexible site through which the assembly of select transcriptional activation domains stimulates synergistic activation and Ras responsiveness of the rPRL promoter. Moreover, we show that the Ets-1/Pit-1 synergism and Ras responses require different subdomains of the Pit-1 TAD, indicating that these two responses are mediated by distinct transcription control mechanisms. Taken together, these results provide novel insights into molecular mechanisms that specify transcription synergy and oncogenic Ras transcription responses. pA3–425PRL-Luc containing the proximal 425 bp of 5′-flanking sequence from the rPRL promoter in the pA3Luc reporter has been described previously (28Conrad K.E. Gutierrez-Hartmann A. Oncogene. 1992; 7: 1279-1286PubMed Google Scholar). Mutations mEBS and mFPIV have been previously described (11Bradford A. Conrad K. Tran P. Ostrowski M. Gutierrez-Hartmann A. J. Biol. Chem. 1996; 271: 24639-24648Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Mutations of pA3–425PRL-Luc including monomer, dimer, 12 bp, 16 bp, and 24 bp were generated by overlap extension PCR amplification from –425PRLpGEM7 using mutant internal primers and SP6 and T7 primers with dual rounds of amplification. The 12-bp and 16-bp insertions incorporate an SalI site into the space between the EBS and FPIV Pit-1 binding site. The 24-bp insertion incorporates SalI, SpeI, and NheI sites. The mutant primers are as follows: monomer S, GCA TTA AAA AAT GCA TAT CCT TCC; monomer AS, GAT ATG CAT TTT TTA ATG CAA AAG G; dimer S, CTT TTG ATG TAT ATA CAT AAA ATC C; dimer AS, GAT TTT ATG TAT ATA CAT CAA AAG G; 12-bp S, TCC TTT TGT CGA CTG TAA TTA ATC AAA ATC C; 12-bp AS, ACA GTC GAC AAA AGG AAA TGA GAG A; 16-bp S, TGT CGA CGT CCT GTA ATT AAT CAA AAT CC; 16-bp AS, TAC AGG ACG TCG ACA AAA GGA AAT GAG AGA; 24-bp S, TCG ACA CTA GTG CTA GCT GTA ATT AAT CAA AAT CC; 24-bp AS, CTA GCA CTA GTG TCG ACA AAA GGA AAT GAG AGA. Mutant promoter 28 bp was generated by digesting the 24-bp insertion construct with SpeI, filling in the overhangs by Klenow treatment, and religating the vector. Mutant promoter 32 bp was generated by digesting 28 bp with SalI, filling in the overhangs by Klenow treatment, and religating the vector. Sequences were verified by sequencing. Plasmid pSG5c-Ets-1 and Ets-2 encode the p68 chicken Ets-1 and chicken Ets-2, respectively, under control of the SV40 early promoter. These constructs as well as the amino-terminal deletions of Ets-1, pSG5-Ets-1Δ5–4 and Δ5–6, n70 Ets-1DBD (EtsDBD), VP16TAD-n70Ets-1DBD (VP16EtsDBD), VP16Ets280–440, and VP16Ets330–440 (29Schneikert J. Lutz Y. Wasylyk B. Oncogene. 1992; 7: 249-256PubMed Google Scholar) were generously provided by Dr. Bohdan Wasylyk (Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch Cedex, France). The pSG5 Ets-1 BPV-1, BPV-2, and BPV-3 are mutant constructs in which overlapping regions of p68 chicken Ets-1 amino acids 190–257 are replaced by 26 amino acids from the bovine papilloma virus L-1 capsid (amino acids 415–440 including the AU1 epitope). These mutations were made utilizing overlap extension PCR to amplify mutated fragments of Ets-1 from nucleotides 401–1054. The fragments were cloned into PCR 2.1 and subcloned into pSG5 Ets-1 by digesting at internal BglII and AflIII sites. PCR primers utilized are: BPV-1 S, CTA AAT GTG CCA GCA ATG TGA TTC CTG CTA AAG AAG ACC CTT ATG CCC CCT CTG AGT TCT CTG; BPV-1 AS, TTG CTG GCA CAT TTA GTT GCA GGA GAC TCT ATA TAG CGA TAG GTG TCC ACT CCG TTT GCT GGG; BPV-2 S, CTA AAT GTG CCA GCA ATG TGA TTC CTG CTA AAG AAG ACC CTT ATG CCT CGG; BPV-2 AS, TTG CTG GCA CAT TTA GTT GCA GGA GAC TCT ATA TAG CGA TAG GTG TCG TGC TCG ATG CCA TAA; BPV-3 S, CTA AAT GTG CCA GCA ATG TGA TTC CTG CTA AAG AAG ACC CTT ATG CCG TCC AGA CGG ACT CCC; BPV-3 AS, TTG CTG GCA CAT TTA GTT GCA GGA GAC TCT ATA TAG CGA TAG GTG TCG GTC TGG TAG GAC TCT; p68 Ets-1 401 S, TGA AGG GAG TGG ATT TCC; p68 Ets-1 1054 AS, CAT AGT CCT TGA AGG TGC. Gal4ADΔ5–4 and Δ5–6 were constructed by subcloning the Gal4AD from pACT2 into pBluescript utilizing HindIII and XhoI restriction sites. The Gal4AD was then isolated from pBluescript by a partial digest with EcoRI and complete digest with XhoI and ligated into either pSG5 Δ5–4 or Δ5–6 that had also been partially digested with EcoRI and completely digested with XhoI. The mutant Ets-1/2 chimera was generated using overlap extension PCR to replace amino acids 185–257 of Ets-1 with amino acids 178–251 of c-Ets-2. The resulting PCR product was digested at internal BglII and AflIII restriction sites and ligated into a pSG5 Ets-1 vector in which the BglII site in the multiple cloning site was destroyed. This vector was partially digested with AflIII and completely digested with BglII. The primers used for PCR were as follows: the previously listed p68 Ets-1 401 S and p68 Ets-1 1054 AS; Ets1–2A, TAT TGA TCT GGG TAT GGT TTT GCC T; Ets1–2B, CCA TAC CCA GAT CAA TAT GTG GAG AG; Ets1–2C, GTC TGG ACT TTA GGG AAC AAT GAA AAG; Ets1–2D, TCC CTA AAG TCC AGA CGG ACT CC. Plasmid pRSV-GHF-1 encoding rat Pit-1 was provided by M. Karin (University of California, San Diego). The plasmids pCGN2-Pit-1, pCGN2-Pit-1Δ2–80, and pCGN2-Pit-1Δ2–45 express amino-terminally hemagglutinin-tagged Pit-1 and amino-terminal TAD deletion mutants under the control of the CMV promoter (13Bradford A.P. Brodsky K.S. Diamond S.E. Kuhn L.C. Liu Y. Gutierrez-Hartmann A. J. Biol. Chem. 2000; 275: 3100-3106Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 30Gordon D.F. Woodmansee W.W. Black J.N. Dowding J. Bendrick-Peart J. Wood W.M. Ridgway E.C. Mol. Cell. Endocrinol. 2002; 196: 53-66Crossref PubMed Scopus (32) Google Scholar) and were generously provided by Dr. David Gordon, University of Colorado Health Sciences Center. The wild-type (WT) pGex Pit-1 (199–291) expression vector (13Bradford A.P. Brodsky K.S. Diamond S.E. Kuhn L.C. Liu Y. Gutierrez-Hartmann A. J. Biol. Chem. 2000; 275: 3100-3106Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) contains the homeodomain of rat Pit-1 fused in-frame to glutathione S-transferase in a modified pGex 2TK vector, pGexDFGK, incorporating the multiple cloning site derived from pCGN2, constructed and provided by Dr. David Gordon, University of Colorado Health Sciences Center. Plasmid pSV-Ras contains the T24 bladder carcinoma Harvey Ras valine 12 mutant oncogene (V-12 Ras) (10Bradford A.P. Conrad K.E. Wasylyk C. Wasylyk B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1995; 15: 2849-2857Crossref PubMed Scopus (112) Google Scholar). GST Fusion Protein Preparation—Recombinant fusion proteins were prepared from bacterial extracts. Overnight cultures of Escherichia coli BL-21 (DE3)pLysS (Stratagene), transformed with pGex plasmids, were diluted 1:10 in fresh Luria broth supplemented with 100 μg/ml ampicillin and grown at 30 °C to an absorbance of 0.5 at 600 nm. The cultures were induced by addition of isopropyl-β-d-thiogalactopyrano-side to a final concentration of 1 mm. Cultures were grown for an additional 2 h at 30 °C. The bacteria were harvested by centrifugation at 5,000 × g for 10 min at 4 °C and stored at –80 °C until preparation. The bacterial pellets were resuspended in 5 ml of Bugbuster Reagent (Novagen)/g of bacterial pellet supplemented with a 1× concentration of Complete protease inhibitor mixture (Roche Applied Science) and 25 units of benzonase/ml of reagent and incubated at room temperature with rocking for 20 min to allow for lysis. Alternatively, some preparations were resuspended in a 1/10 volume of phosphate-buffered saline (PBS) supplemented with a 1× concentration of Complete protease inhibitor mixture and sonicated for three 10-s bursts at 4 °C. Following sonication, Triton X-100 was added to a final concentration of 1%, and the bacterial pellets were incubated with rocking at room temperature for 30 min. If necessary, bacterial DNA was sheared with three additional bursts of sonication. Following lysis, cellular debris was removed by centrifugation at 16,000 × g for 20 min at 4 °C. The supernatant was transferred to a clean tube and bound to glutathione-Sepharose CL-4B (Amersham Biosciences) for 1 h at room temperature. The Sepharose was washed extensively with 1× PBS supplemented with Complete protease inhibitors and 1 mm dithiothreitol. Ets-1 fusions were supplemented with 10 mm dithiothreitol. Protein concentration was measured by the Bio-Rad assay (Bio-Rad). Bound protein was analyzed for intactness and mass by SDS-PAGE in parallel with a known amount of bovine serum albumin and Gelcode Blue or Coomassie Blue staining. In Vitro Binding Assays— 35S-Labeled proteins were synthesized and labeled with [35S]methionine (PerkinElmer Life Sciences) using the TNT-coupled transcription-translation reticulocyte lysate system with T7 polymerase and supercoiled plasmids pSG5-Ets-1, BPV-1, BPV-2, BPV-3, Ets-2, Ets-1/2, VP16EtsDBD, and Gal4ADΔ5–6 according to the manufacturer's protocol (Promega, Madison, WI). Approximately equal amounts (5 μg) of GST fusion proteins bound to 20 μl of glutathione-Sepharose beads were suspended in a total volume of 0.5 ml of binding buffer (40 mm HEPES, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 0.1 mm EDTA, 0.05% Nonidet P-40, 1 mm dithiothreitol, and 1× Complete protease inhibitors). Ethidium bromide was added to a final concentration of 50 μg/ml to eliminate binding of any contaminating DNA (31Lai J. Herr W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6958-6962Crossref PubMed Scopus (397) Google Scholar) and incubated for 30 min at 4 °C on a rotator. 35S-Labeled proteins (5 μl of a 50-μl synthesis reaction) were added to the GST fusion samples and incubated on a rotator at room temperature for 1 h. The Sepharose beads were pelleted by centrifugation at 2,500 × g for 5 min. Aliquots of the supernatant were reserved to assess input, and the beads were washed four times with 1 ml of binding buffer containing 0.1% Triton X-100. The 35S-labeled proteins were eluted from the beads by boiling in SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. Bands were quantified using a Molecular Dynamics PhosphorImager with ImageQuant software. HeLa and GH4T2 cells were maintained in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 15% horse serum and 2.5% fetal calf serum (Invitrogen). Cells were grown at 37 °C in 5% CO2. The medium was changed 4–16 h prior to transfection. Electroporation—Cells were harvested with 1× PBS with 3 mm EDTA and resuspended in culture medium. Aliquots of ∼2–4 × 106 cells in 200 μl of medium were added to plasmid DNA as described in the figure legends and transfected by electroporation at 220 V and 500 microfarads using a Bio-Rad Gene Pulser with 4-mm gap cuvettes. Following transfection, cells were plated on 60-mm tissue culture plates in culture medium and incubated for 24 h. All electroporations included 0.3 μg of CMV-β-galactosidase (Clontech) as an internal control for transfection efficiency. Total DNA was kept constant, and nonspecific effects of viral promoters were controlled for by transfecting appropriate control vectors. Approximately 24 h following the electroporation, the cells were harvested, and luciferase and β-galactosidase activities were measured. The transfected cells were harvested in PBS containing 3 mm EDTA, pelleted by centrifugation, and lysed by three freeze-thaw cycles in 100 μl of 100 mm potassium phosphate, pH 7.8 with 1 mm dithiothreitol. Cell debris was pelleted by centrifugation at 10,000 × g for 10 min at 4 °C, and aliquots of the supernatant were used in subsequent assays. Samples were assayed in duplicate for luciferase activity as described previously (28Conrad K.E. Gutierrez-Hartmann A. Oncogene. 1992; 7: 1279-1286PubMed Google Scholar) using a Monolight 3010 luminometer (Analytical Luminescence Laboratories). β-Galactosidase activity was determined spectrophotometrically using the chromogenic substrate o-nitrophenyl-β-d-galactopyranoside as described (28Conrad K.E. Gutierrez-Hartmann A. Oncogene. 1992; 7: 1279-1286PubMed Google Scholar). Lipid-mediated Transfection—HeLa cells were" @default.
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