ownloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press Kuo et al with TBS. Cells were lysed for 2 h(Vibrax-VXR 2000)with glass Author contributions: D K, K.T., T.R., and T.I. designed the beads and sonicated for four cycles of 20 sec(+100-sec rest) at study. D K, K.L., S B, R C, J.C., and C L. performed the experi power setting 2 (Misonex Sonicator 3000) on ice. Lysate was in- mental work. D K and K.T. analyzed the data D K and T.I. wrote cubated with Dynabeads M-280 conjugated with anti-TAP anti- the manuscript. K.T. and T I supervised the work. body(Open Biosystems CAB1001)overnight. Cross-link reversal was performed overnight at 65 C with antibody-enriched and References (Invitrogen) with Cy5/Cy3 dyes. Sc and C samples were hybrid Altschul SE, Gish w. 1996. Local alignment statistics Methods Enzymol 266: ized to commercial or custom(see below) Agilent tiling arrays and washed as recommended by Agilent(Agilent Technologies). Amberg dC, Burke D], Strathern JN. 2005. Metl ' ress, Cold spring Harbor Ny. mamual Cold Spring Harbor laboratory Cg tiling microarray design and validation Berger ME, Badis G, Gehrke AR, Talukder S, Philippakis AA, Pena-Castillo L, Alleyne TM, Mnaimneh S, Botvinnik OB, Chan ET, et al. 2008. Variatio resolution with -44,000 60-mer probes designed teat-250-bp We designed a custom microarray tiling the Cg genom in homeodomain DNA binding revealed by high-resolution analysis of Borneman AR, Gianoulis TA, Zhang ZD, Yu H, Rozowsky J, dimerization and variability in melting temperatures (Mfold; Zuker 2003), low-complexity and repetitive sequences(RepeatMasker; http://repeatmasker.org),andcross-hybridization(wUblasT2:Brachman Altschul and Gish 1996). Default settings were used for each pre seful set of strains and plasmids for PCR-mediated gene disruption and (Agilent Technologies). ChIP results were validated by qPCR of five Bradley Rk,Li x-Y, Trapnell C, Davidson S, Pachter L, Chu HC, Tonkin LA, rgets compared against CyACTI(Supplemental Fig. 5) Biggin MD, Eisen MB. 2010. Binding site turnover produces pervasive closely related Drosophila species. PLos Biol 8: e1000343. doi: 10.1371/ journal. pbo 100034 Microarray data processing Brem RB, Kruglyak L. 2005. The landscape of genetic complexity across Intensities were background subtracted and normalized by LOESS 5,700 gene expression traits in yeast. Proc Natl Acad Sci 102: 1572- (Smyth 2005). Expression microarrays were analyzed using the Bullard JH, Mostovoy Y, Dudoit S, Brem RB. 2010. Polygenic and directional linmma package(Smyth 2005)with default parameters. ChIP tiling evolution across pathways in Saccharomyces. Proc Natl Acad Sci array errors were estimated by the Rosetta error model (Weng et a 107:5058-5063 2006)with resulting P-values of binding for each promoter calcu- Cliften P, Sudarsanam P, Desikan A n L Fu B, Majors J, Waterston R, Cohen BA, Johnston M. 2003. Finding lated by combining P-values of adjacent probes as previously de- Saccharomyces genomes by phylogenetic footprinting Science 301: 71- cribed (Tan et al. 2008). EN, Crane-Robinson C privalov 2004a. Thermodynam of GCN4-bzIP binding to DNA Phylogenetic trees, orthologs, and evolutionary trace analysis ruminating between the AP-1 and AtF/ CREB sites. Mol Biol 343: 865-878. agan Al, Liu Y, Makeyeva EN, Privalov PL 2004b DNA-binding domain of with BEAST(Drummond and Rambaut 2007) using default set. DNA. Nndeicesciens ges3 2: 5192tsrs e ATF/CREB and AP-1 binding sites of tings Sequences, species trees, and orthologs were obtained from the Fungal Orthogroups Repository(Wapinskiet al 2007). Multiple- sequence alignment was preformed using MUSCLE (Edgar 2004) nd high throughput. Nucleic Acids Res 32: 1792-179 with default parameters. Evolutionary trace analysis was performed Emerson J, Hsieh LC, Sung HM, Wang TY, Huang C), Lu HH, Lu MY, wu SH, using Trace Suite II (Innis et al. 2000)with default setting H. 2010. Natural selection on cis and trans enome res20:826-836 Motif finding charormyces cerevisiae with distinct biological Cell bio17:69826993 Maclsaac et al. 2006; Tan et al. 2008)using the default settings for is coupled to evolution of DNA-encoded nucleosome organization. Nat et41: STAMP (Mahony et al. 2005 ). Promoters were scanned with the Fujii Y, Shimizu T, Toda T, Yanagida M, Hakoshima T2000.Structural basis default settings for Patser (Hertz and Stormo 1999) for motif en- richment by the hyper netric test with multiple test correction Struct biol 7: 889-893 (Storey and Tibshirani 2003). A motif was considered"present"in Gasch AP, Moses AM, Chiang DY, Fraser HB, Berardini M, Eisen MB. 2004 a promoter for(fraction of maximal information content)20.7 transcription factors cause natural variation in yeast. Science Acknowledgments Hannett NM, Tagne JB, Reynolds DB, You Paul Russell for pFA6a-kanMX6, Nevan Krogan for pFA6a-natMX6, Hertegula stror mode d ggg rdenit ge statistically significant alignments of multiple sequences. Bioinformatics Wapinski, Lorraine Pillus, and members of the T I laboratory for 15:563-5 helpful discussions was supported by the National Science Hittinger CT, Carroll SB. 2007. Gene duplication and the adaptive evolution and Engineering Research Council of Canada. K.T. and T.I. were Hogues H Lavoie H, sellam A, mangos m. Roemer t supported by the David and Lucille Packard Foundation and NIh Whiteway M. 2008. Transcription factor substitution during the grant no. RO1 ESO14811 to T.I. evolution of fungal ribosome regulation. Mol Cell 29: 552-562.with TBS. Cells were lysed for 2 h (Vibrax-VXR 2000) with glass beads and sonicated for four cycles of 20 sec (+100-sec rest) at power setting 2 (Misonex Sonicator 3000) on ice. Lysate was in￾cubated with Dynabeads M-280 conjugated with anti-TAP anti￾body (Open Biosystems CAB1001) overnight. Cross-link reversal was performed overnight at 65° C with antibody-enriched and unenriched DNA, and amplified (Sigma-Aldrich) and labeled (Invitrogen) with Cy5/Cy3 dyes. Sc and Cg samples were hybrid￾ized to commercial or custom (see below) Agilent tiling arrays and washed as recommended by Agilent (Agilent Technologies). Cg tiling microarray design and validation We designed a custom microarray tiling the Cg genome at ;250-bp resolution with ;44,000 60-mer probes designed to avoid self￾dimerization and variability in melting temperatures (Mfold; Zuker 2003), low-complexity and repetitive sequences (RepeatMasker; http://repeatmasker.org), and cross-hybridization (WUBLAST2; Altschul and Gish 1996). Default settings were used for each pro￾gram. Microarrays were manufactured using Agilent technology (Agilent Technologies). ChIP results were validated by qPCR of five targets compared against CgACT1 (Supplemental Fig. 5). Microarray data processing Intensities were background subtracted and normalized by LOESS (Smyth 2005). Expression microarrays were analyzed using the limma package (Smyth 2005) with default parameters. ChIP tiling array errors were estimated by the Rosetta error model (Weng et al. 2006) with resulting P-values of binding for each promoter calcu￾lated by combining P-values of adjacent probes as previously de￾scribed (Tan et al. 2008). Phylogenetic trees, orthologs, and evolutionary trace analysis The yAP-1 DNA-binding domain phylogenetic tree was created with BEAST (Drummond and Rambaut 2007) using default set￾tings. Sequences, species trees, and orthologs were obtained from the Fungal Orthogroups Repository (Wapinski et al. 2007). Multiple￾sequence alignment was preformed using MUSCLE (Edgar 2004) with default parameters. Evolutionary trace analysis was performed using TraceSuite II (Innis et al. 2000) with default settings. Motif finding De novo motifs were identified by SOMBRERO (Mahony et al. 2005) using default parameters and compared with literature (MacIsaac et al. 2006; Tan et al. 2008) using the default settings for STAMP (Mahony et al. 2005). Promoters were scanned with the default settings for Patser (Hertz and Stormo 1999) for motif en￾richment by the hypergeometric test with multiple test correction (Storey and Tibshirani 2003). A motif was considered ‘‘present’’ in a promoter for (fraction of maximal information content) $0.7. Acknowledgments We thank JonathanWeissman for providing pFA6a-TAP-HIS3MX6, Paul Russell for pFA6a-kanMX6, Nevan Krogan for pFA6a-natMX6, and Richard Kolodner for pRS303 and pRS306. We also thank Ilan Wapinski, Lorraine Pillus, and members of the T.I. laboratory for helpful discussions. D.K. was supported by the National Science and Engineering Research Council of Canada. K.T. and T.I. were supported by the David and Lucille Packard Foundation and NIH grant no. R01 ES014811 to T.I. Author contributions: D.K., K.T., T.R., and T.I. designed the study. D.K., K.L., S.B., R.C., J.C., and C.L. performed the experi￾mental work. D.K. and K.T. analyzed the data. D.K. and T.I. wrote the manuscript. 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