NaDeoxycholate, with protease and phosphatase inhibitors as described above). After centrifugation, protein concentration was determined with the Bio-Rad protein assay kit, and 1 mg of soluble extract was incubated with 5–10 ml anti-Adr1 antibody [10] for 3 hours at 4uC. After centrifugation, the supernatant was transferred to a tube with 60 ml of a 1:1 suspension of protein A sepharose : PBS (GE Healthcare) and incubated on a rocking platform for 1 hour, 4uC. After 3 washes with lysis buffer, Adr1 was eluted with 60 ml 46 LDS sample buffer (Invitrogen) at 70uC for 10 minutes and analyzed by Western blot with as described above. Expression of Adr1-dependent genes and Adr1 DNA binding RNA expression and ADH2 promoter - lacZ reporter activity was measured as described in Tachibana et al. [8]. ChIP assays were performed as described in Young et al. [4]. The sequences of oligonucleotides used for cloning and diagnostics are available on request. Electrophoretic Mobility Shift Assays (EMSA) EMSA was conducted as in Thukral et al. [17], using a double- stranded probe containing the UAS1 sequence (underlined) from References 1. Carlson M (1999) Glucose repression in yeast. Curr Opin Microbiol 2: 202–207. 2. Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62: 334–361. 3. Schuller HJ (2003) Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae. Curr Genet 43: 139–160. 4. Young ET, Dombek KM, Tachibana C, Ideker T (2003) Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8. J Biol Chem 278: 26146–26158. 5. Dombek KM, Young ET (1997) Cyclic AMP-dependent protein kinase inhibits ADH2 expression in part by decreasing expression of the transcription factor gene ADR1. Mol Cell Biol 17: 1450–1458. 6. Sloan JS, Dombek KM, Young ET (1999) Post-translational regulation of Adr1 activity is mediated by its DNA binding domain. J Biol Chem 274: 37575–37582. 7. Young ET, Kacherovsky N, Van Riper K (2002) Snf1 protein kinase regulates Adr1 binding to chromatin but not transcription activation. J Biol Chem 277: 38095–38103. 8. Tachibana C, Biddick R, Law GL, Young ET (2007) A poised initiation complex is activated by SNF1. J Biol Chem 282: 37308–37315. 9. Voronkova V, Kacherovsky N, Tachibana C, Yu D, Young ET (2006) Snf1- dependent and Snf1-independent pathways of constitutive ADH2 expression in Saccharomyces cerevisiae. Genetics 172: 2123–2138. 10. Dombek KM, Camier S, Young ET (1993) ADH2 expression is repressed by REG1 independently of mutations that alter the phosphorylation of the yeast transcription factor ADR1. Mol Cell Biol 13: 4391–4399. 11. Denis CL, Kemp BE, Zoller MJ (1991) Substrate specificities for yeast and mammalian cAMP-dependent protein kinases are similar but not identical. J Biol Chem 266: 17932–17935. 12. Cherry JR, Johnson TR, Dollard C, Shuster JR, Denis CL (1989) Cyclic AMP- dependent protein kinase phosphorylates and inactivates the yeast transcrip- tional activator ADR1. Cell 56: 409–419. 13. Denis CL, Fontaine SC, Chase D, Kemp BE, Bemis LT (1992) ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230. Mol Cell Biol 12: 1507–1514. 14. Blumberg H, Eisen A, Sledziewski A, Bader D, Young ET (1987) Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function. Nature 328: 443–445. 15. Young ET, Kacherovsky N, Cheng C (2000) An accessory DNA binding motif in the zinc finger protein Adr1 assists stable binding to DNA and can be replaced by a third finger. Biochemistry 39: 567–574. 16. Bowers PM, Schaufler LE, Klevit RE (1999) A folding transition and novel zinc finger accessory domain in the transcription factor ADR1. Nat Struct Biol 6: 478–485. 17. Thukral SK, Eisen A, Young ET (1991) Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression. Mol Cell Biol 11: 1566–1577. 18. Camier S, Kacherovsky N, Young ET (1992) A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger. Mol Cell Biol 12: 5758–5767. Phosphorylation of Adr1 the ADH2 promoter (sequence of top strand: 59 GCA TTG ACT AAG TTC TCC AAC TTA TAA GTT GGA GAT GAA TCA GTT ACG 39) labeled with an infrared dye and quantitated with an Odyssey Infrared Imaging System (Li-Cor Biosciences). Signals from all shifted bands in a lane were divided by the combined signal for all bands in the lane to determine % bound. Acknowledgments Thanks to Ian Carter-O’Connell and the O’Shea lab for purified Pho80/ 85 and Pho4, Carly Cox and Rachel Chang for technical assistance, Lynn Law for critical reading of the manuscript and the members of the Young lab for helpful discussions. Author Contributions Conceived and designed the experiments: NK CT ETY. Performed the experiments: NK CT EA DFI ETY. Analyzed the data: NK CT EA DFI ETY. Contributed reagents/materials/analysis tools: ETY. Wrote the paper: CT ETY. 19. Schaufler LE, Klevit RE (2003) Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR. J Mol Biol 329: 931–939. 20. Cook WJ, Mosley SP, Audino DC, Mullaney DL, Rovelli A, et al. (1994) Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation. J Biol Chem 269: 9374–9379. 21. Tachibana C, Yoo JY, Tagne JB, Kacherovsky N, Lee TI, et al. (2005) Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8. Mol Cell Biol 25: 2138–2146. 22. Denis CL (1987) The effects of ADR1 and CCR1 gene dosage on the regulation of the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevi- siae. Mol Gen Genet 208: 101–106. 23. Irani M, Taylor WE, Young ET (1987) Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids. Mol Cell Biol 7: 1233–1241. 24. Nishizawa M, Katou Y, Shirahige K, Toh-e A (2004) Yeast Pho85 kinase is required for proper gene expression during the diauxic shift. Yeast 21: 903–918. 25. Zhou Z, Elledge SJ (1993) DUN1 encodes a protein kinase that controls the DNA damage response in yeast. Cell 75: 1119–1127. 26. Yuzyuk T, Foehr M, Amberg DC (2002) The MEK kinase Ssk2p promotes actin cytoskeleton recovery after osmotic stress. Mol Biol Cell 13: 2869–2880. 27. Huang D, Moffat J, Wilson WA, Moore L, Cheng C, et al. (1998) Cyclin partners determine Pho85 protein kinase substrate specificity in vitro and in vivo: control of glycogen biosynthesis by Pcl8 and Pcl10. Mol Cell Biol 18: 3289–3299. 28. Williamson VM, Bennetzen J, Young ET, Nasmyth K, Hall BD (1980) Isolation of the structural gene for alcohol dehydrogenase by genetic complementation in yeast. Nature 283: 214–216. 29. Jeffery DA, Springer M, King DS, O’Shea EK (2001) Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference. J Mol Biol 306: 997–1010. 30. Thukral SK, Tavianini MA, Blumberg H, Young ET (1989) Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1. Mol Cell Biol 9: 2360–2369. 31. Biddick RK, Law GL, Young ET (2008) Adr1 and Cat8 mediate coactivator recruitment and chromatin remodeling at glucose-regulated genes. PLoS ONE 3: e1436. 32. Young ET, Tachibana C, Chang HW, Dombek KM, Arms EM, et al. (2008) Artificial recruitment of mediator by the DNA-binding domain of Adr1 overcomes glucose repression of ADH2 expression. Mol Cell Biol 28: 2509–2516. 33. O’Neill EM, Kaffman A, Jolly ER, O’Shea EK (1996) Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex. Science 271: 209–212. 34. Huang D, Farkas I, Roach PJ (1996) Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae. Mol Cell Biol 16: 4357–4365. 35. Timblin BK, Tatchell K, Bergman LW (1996) Deletion of the gene encoding the cyclin-dependent protein kinase Pho85 alters glycogen metabolism in Saccha- romyces cerevisiae. Genetics 143: 57–66. 36. Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, et al. (1999) Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15: 963–972. PLoS ONE | www.plosone.org 8 September 2008 | Volume 3 | Issue 9 | e3213