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• W2097948608 abstract "Cbf1p is a basic-helix-loop-helix-zipper protein of Saccharomyces cerevisiae required for the function of centromeres and MET gene promoters, where it binds DNA via the consensus core motif CACRTG (R = A or G). At MET genes Cbf1p appears to function in both activator recruitment and chromatin-remodeling. Cbf1p has been implicated in the regulation of other genes, and CACRTG motifs are common in potential gene regulatory DNA. A recent genome-wide location analysis showed that the majority of intergenic CACGTG palindromes are bound by Cbf1p. Here we tested whether all potential Cbf1p binding motifs in the yeast genome are likely to be bound by Cbf1p using chromatin immunoprecipitation. We also tested which of the motifs are actually functional by assaying for Cbf1p-dependent chromatin remodeling. We show that Cbf1p binding and activity is restricted to palindromic CACGTG motifs in promoter-proximal regions. Cbf1p does not function through CACGTG motifs that occur in promoter-distal locations within coding regions nor where CACATG motifs occur alone except at centromeres. Cbf1p can be made to function at promoter-distal CACGTG motifs by overexpression, suggesting that the concentration of Cbf1p is normally limiting for binding and is biased to gene regulatory DNA by interactions with other factors. We conclude that Cbf1p is required for normal nucleosome positioning wherever the CACGTG motif occurs in gene regulatory DNA. Cbf1p has been shown to interact with the chromatin-remodeling ATPase Isw1p. Here we show that recruitment of Isw1p by Cbf1p is likely to be general but that Isw1p is only partially required for Cbf1p-dependent chromatin structures. Cbf1p is a basic-helix-loop-helix-zipper protein of Saccharomyces cerevisiae required for the function of centromeres and MET gene promoters, where it binds DNA via the consensus core motif CACRTG (R = A or G). At MET genes Cbf1p appears to function in both activator recruitment and chromatin-remodeling. Cbf1p has been implicated in the regulation of other genes, and CACRTG motifs are common in potential gene regulatory DNA. A recent genome-wide location analysis showed that the majority of intergenic CACGTG palindromes are bound by Cbf1p. Here we tested whether all potential Cbf1p binding motifs in the yeast genome are likely to be bound by Cbf1p using chromatin immunoprecipitation. We also tested which of the motifs are actually functional by assaying for Cbf1p-dependent chromatin remodeling. We show that Cbf1p binding and activity is restricted to palindromic CACGTG motifs in promoter-proximal regions. Cbf1p does not function through CACGTG motifs that occur in promoter-distal locations within coding regions nor where CACATG motifs occur alone except at centromeres. Cbf1p can be made to function at promoter-distal CACGTG motifs by overexpression, suggesting that the concentration of Cbf1p is normally limiting for binding and is biased to gene regulatory DNA by interactions with other factors. We conclude that Cbf1p is required for normal nucleosome positioning wherever the CACGTG motif occurs in gene regulatory DNA. Cbf1p has been shown to interact with the chromatin-remodeling ATPase Isw1p. Here we show that recruitment of Isw1p by Cbf1p is likely to be general but that Isw1p is only partially required for Cbf1p-dependent chromatin structures. Saccharomyces cerevisiae centromere binding factor 1, Cbf1p (also known as Cpf1, CBP1, and CP1), is a dimeric DNA-binding protein of the basic-helix-loop-helix-leucine zipper family. Present at an estimated 6890 molecules per cell during vegetative growth, Cbf1p is a relatively abundant DNA-binding protein (1Ghaemmaghami S. Huh W-K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Nature. 2003; 425: 737-741Crossref PubMed Scopus (2948) Google Scholar). The core binding motif for Cbf1p (2Niedenthal R. Stoll R. Hegemann J.-H. Mol. Cell. Biol. 1991; 11: 3545-3553Crossref PubMed Scopus (39) Google Scholar, 3Wilmen A. Pick H. Niedenthal R.K. Sen-Gupta M. Hegemann J-H. Nucleic Acids Res. 1994; 22: 2791-2800Crossref PubMed Scopus (15) Google Scholar) is the sequence CACRTG (R = A or G), which is typical of the CANNTG-type, or E-box, motifs bound by other members of the basic-helix-loop-helix and basic-helix-loop-helix-leucine zipper classes of proteins (4Robinson K.A. Lopes J.M. Nucleic Acids Res. 2000; 28: 1499-1505Crossref PubMed Google Scholar). Cbf1p was the first factor to be shown to bind to yeast centromere DNA (5Bram R.J. Kornberg R.D. Mol. Cell. Biol. 1987; 7: 403-409Crossref PubMed Scopus (113) Google Scholar, 6Baker R.E. Fitzgerald-Hayes M. O'Brien T.C. J. Biol. Chem. 1989; 264: 10843-10850Abstract Full Text PDF PubMed Google Scholar, 7Cai M. Davis R.W. Mol. Cell. Biol. 1989; 9: 2544-2550Crossref PubMed Scopus (59) Google Scholar, 8Jiang W. Philippsen P. Mol. Cell. Biol. 1989; 9: 5585-5593Crossref PubMed Scopus (39) Google Scholar) via the CACRTG motif, which is found within the consensus centromere DNA Element 1 (CDEI) 1The abbreviations used are: CDEI, centromere DNA element I; MNase, micrococcal nuclease; ChIP, chromatin immunoprecipitation.1The abbreviations used are: CDEI, centromere DNA element I; MNase, micrococcal nuclease; ChIP, chromatin immunoprecipitation. RTCACRTG. Deletion of the CBF1 gene or deletion of CDEI motifs from centromeric DNA led to similar defects in mitotic and meiotic centromere function, indicating that Cbf1p is an important component of the centromere-kinetochore complex (9Ortiz J. Lechner J. Protoplasma. 2000; 211: 12-19Crossref Scopus (5) Google Scholar, 10McAinsh A.D. Tytell J.D. Sorger P.K. Annu. Rev. Cell Dev. Biol. 2003; 19: 519-539Crossref PubMed Scopus (179) Google Scholar). Although the precise role of Cbf1p at the centromere is not yet understood, it is known to interact with other protein components of the kinetochore complex to form a compact nucleosome-based structure (11Bloom K.S. Carbon J. Cell. 1982; 29: 305-317Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 12Funk M. Hegemann J.H. Philippsen P. Mol. Gen. Genet. 1989; 219: 153-160Crossref PubMed Scopus (44) Google Scholar, 13Meluh P.B. Koshland D. Genes Dev. 1997; 11: 3401-3412Crossref PubMed Scopus (150) Google Scholar, 14Baker R.E. Harris K. Zhang K. Genetics. 1998; 149: 73-85Crossref PubMed Google Scholar, 15Hemmerich P. Stoyan T. Wieland G. Koch M. Lechner J. Diekmann S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12583-12588Crossref PubMed Scopus (34) Google Scholar). The CACRTG motif is not restricted to centromeric DNA but also occurs in one or several copies in the regulatory DNA of almost every gene encoding a protein involved in the methionine biosynthetic pathway (the MET genes). Loss of Cbf1p function leads to methionine auxotrophy, indicating that Cbf1p also functions as a transcription factor (16Thomas D. Cherest H. Surdin-Kerjan Y. Mol. Cell. Biol. 1989; 9: 3292-3298Crossref PubMed Scopus (95) Google Scholar, 17Baker R.E. Masison D.C. Mol. Cell. Biol. 1990; 10: 2458-2467Crossref PubMed Scopus (127) Google Scholar, 18Cai M. Davis R.W. Cell. 1990; 61: 437-446Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 19Mellor J. Jiang W. Funk M. Rathjen J Barnes C.A. Hinz T. Hegemann J.H. Philippsen P. EMBO J. 1990; 9: 4017-4026Crossref PubMed Scopus (147) Google Scholar, 20Kuras L. Thomas D. FEBS Lett. 1995; 367: 15-18Crossref PubMed Scopus (30) Google Scholar). The role of Cbf1p at MET gene promoters has turned out to be quite complicated and appears to involve two separate functionalities. First, Cbf1p acts in the recruitment of Met4p, the MET gene transcriptional activator. Met4p is a basic-zipper protein that is a target for regulation through S-adenosyl methionine concentration-dependent ubiquitinylation via the Cdc34p·SCFMet30p complex (21Patton E.E. Peyraud C. Rouillon A. Surdin-Kerjan Y. Tyers M. Thomas D. EMBO J. 2000; 19: 1613-1624Crossref PubMed Google Scholar, 22Kaiser P. Flick K. Wittenberg C. Reed S.I. Cell. 2000; 102: 303-314Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 23Kuras L. Rouillon A. Lee T. Barbey R. Tyers M. Thomas D. Mol. Cell. 2002; 10: 69-80Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). On its own Met4p will not bind DNA (24Kuras L. Barbey R. Thomas D. EMBO J. 1997; 16: 2441-2451Crossref PubMed Scopus (71) Google Scholar). At the MET16 promoter, Met4p is recruited as part of a co-operatively bound complex consisting of Met4p, the basic-helix-loop-helix factor Met28p, and Cbf1p, which binds to a single CACGTG motif (24Kuras L. Barbey R. Thomas D. EMBO J. 1997; 16: 2441-2451Crossref PubMed Scopus (71) Google Scholar, 25Kuras L. Cherest H. Surdin-Kerjan Y. Thomas D. EMBO J. 1996; 15: 2519-2529Crossref PubMed Scopus (96) Google Scholar). In the absence of Cbf1p the complex does not form at the promoter, and the MET16 gene cannot be activated. Interestingly, Cbf1p is not absolutely required for regulation of all MET genes (20Kuras L. Thomas D. FEBS Lett. 1995; 367: 15-18Crossref PubMed Scopus (30) Google Scholar). This apparent paradox has been resolved by experiments showing that recruitment of Met4p to different MET genes requires an overlapping set of context-dependent factors, which only sometimes include and/or require Cbf1p (26Blaiseau P.L. Isnard A.D. Surdin-Kerjan Y. Thomas D. Mol. Cell. Biol. 1997; 17: 3640-3648Crossref PubMed Scopus (98) Google Scholar, 27Blaiseau P.L. Thomas D. EMBO J. 1998; 17: 6327-6336Crossref PubMed Scopus (86) Google Scholar). The second role of Cbf1p at MET gene promoters appears to be in ensuring the correct position of promoter-proximal nucleosomes; in the absence of Cbf1p, changes in cleavage with micrococcal nuclease (MNase) are observed at MET gene promoters that are consistent with alteration in the translational position of two to three nucleosomes packaging MET gene regulatory DNA (28Kent N.A. Tsang J.S.H. Crowther D.J. Mellor J. Mol. Cell. Biol. 1994; 14: 5229-5241Crossref PubMed Scopus (35) Google Scholar, 29O'Connell K.F. Surdin-Kerjan Y. Baker R.E. Mol. Cell. Biol. 1995; 15: 1879-1888Crossref PubMed Scopus (45) Google Scholar). This function is likely to involve the recruitment of the chromatin remodeling ATPase Isw1p (30Moreau J.-L. Lee M. Mahachi N. Vary J. Mellor J. Tsukiyama T. Goding C.R. Mol. Cell. 2003; 11: 1609-1620Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Although the chromatin-remodeling function of Cbf1p is not absolutely required for MET gene activation, it appears to influence the kinetics of activation (20Kuras L. Thomas D. FEBS Lett. 1995; 367: 15-18Crossref PubMed Scopus (30) Google Scholar). Although the ubiquitous presence of CACRTG motifs at centromeres and MET gene promoters is striking, the motif is widespread throughout the yeast genome, occurring in both intergenic regions and within coding DNA. The haploid yeast genome contains 4820 potential Cbf1p binding CACRTG core motifs, of which 953 are CACGTG palindromes that show 10-fold higher binding affinity for Cbf1p than CACATG in vitro (3Wilmen A. Pick H. Niedenthal R.K. Sen-Gupta M. Hegemann J-H. Nucleic Acids Res. 1994; 22: 2791-2800Crossref PubMed Scopus (15) Google Scholar). Previous work has concentrated on potential Cbf1p binding motifs that occur in a gene regulatory context; a recent series of genome-wide protein localization experiments tested the binding of a variety of transcription factors to yeast promoter regions and suggested that 83% of intergenic CACGTG palindromes were likely to be bound by Cbf1p (31Lee T.I. Rinaldi N.J. Robert F. Odom D.T. Bar-Joseph Z. Gerber G.K. Hannett N.M. Harbison C.T. Thompson C.M. Simon I. Zeitlinger J. Jennings E.G. Murray H.L. Gordon D.B. Ren B. Wyrick J.J. Tagne J.B. Volkert T.L. Fraenkel E. Gifford D.K. Young R.A. Science. 2002; 298: 799-804Crossref PubMed Scopus (2352) Google Scholar). Mutation of the CBF1 gene or mutation of potential CACRTG binding motifs in the regulatory DNA of the GAL2, TRP1, CYT1, PGK, RPL45, QCR8, and GSH1 genes leads to perturbation of transcriptional regulation (19Mellor J. Jiang W. Funk M. Rathjen J Barnes C.A. Hinz T. Hegemann J.H. Philippsen P. EMBO J. 1990; 9: 4017-4026Crossref PubMed Scopus (147) Google Scholar, 32Mellor J. Rathjen J. Jiang W. Dowell S.J. Nucleic Acids Res. 1991; 19: 2961-2969Crossref PubMed Scopus (45) Google Scholar, 33Oechsner U. Bandlow W. Nucleic Acids Res. 1996; 24: 2395-2403Crossref PubMed Scopus (9) Google Scholar, 34Packham E.A. Graham I.R. Chambers A. Mol. Gen. Genet. 1996; 250: 348-356PubMed Google Scholar, 35Kraakman L.S. Mager W.H. Grootjans J.J. Planta R.J. Biochim. Biophys. Acta. 1991; 1090: 204-210Crossref PubMed Scopus (15) Google Scholar, 36DeWinde J.H. Grivell L. Eur. J. Biochem. 1995; 233: 200-208Crossref PubMed Scopus (12) Google Scholar, 37Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellor J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In addition, altered patterns of nuclease accessibility in chromatin consistent with changes in nucleosome position have been reported in association with Cbf1p binding at the TRP1 and QCR8 promoters (19Mellor J. Jiang W. Funk M. Rathjen J Barnes C.A. Hinz T. Hegemann J.H. Philippsen P. EMBO J. 1990; 9: 4017-4026Crossref PubMed Scopus (147) Google Scholar, 28Kent N.A. Tsang J.S.H. Crowther D.J. Mellor J. Mol. Cell. Biol. 1994; 14: 5229-5241Crossref PubMed Scopus (35) Google Scholar, 38De Winde J.H. Van Leeuwen H.C. Grivell L. Yeast. 1993; 9: 847-857Crossref PubMed Scopus (16) Google Scholar), suggesting that chromatin remodeling is a general function of DNA-bound Cbf1p. The experiments above suggest that Cbf1p could be associated with a large number of yeast loci. In this work we have attempted to investigate which CACRTG motifs in the yeast genome are bound by Cbf1p and, more importantly, which motifs support Cbf1p-dependent chromatin remodeling. In particular, we have sought to answer the question of whether Cbf1p is required for correct nucleosome positioning at every potential chromosomal binding site (which would suggest a structural function for the protein throughout the genome) or whether Cbf1p is biased to gene regulatory sequences (which would suggest a general function as a transcription factor). We, therefore, chose to examine a panel of yeast loci that contain CACRTG motifs in various combinations and contexts using chromatin immunoprecipitation and in vivo nuclease digestion methods. By choosing a mixture of loci, some with known association to Cbf1p and some at random from one arm of a yeast chromosome, we expect that our results should be representative of the yeast genome as whole. Yeast Strains and Microbiological Culture—Epitope tagging was performed by the gene replacement technology described in Longtine et al. (39Longtine M.S. McKenzie A.D. Demarini J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4082) Google Scholar); 13 Myc repeats were added in-frame to the C-terminal amino acid of either Cbf1p or Isw1p in yeast of the CEN.PK2 reference background (MAT α, leu2-3, leu2-112, his3Δ1, trp1-289, ura3–52). Isw1p-Myc, which is fully functional in chromatin remodeling, is described in more detail in Kent et al. (40Kent N.A. Karabetsou N. Politis P.K. Mellor J. Genes Dev. 2001; 15: 619-626Crossref PubMed Scopus (97) Google Scholar). Disruptions of the CBF1 locus were created by gene replacement as described (19Mellor J. Jiang W. Funk M. Rathjen J Barnes C.A. Hinz T. Hegemann J.H. Philippsen P. EMBO J. 1990; 9: 4017-4026Crossref PubMed Scopus (147) Google Scholar). For chromatin analysis in Figs. 2, 3, 4 the wild-type and mutant strains were of the DBY745 background (MATα, ade1-100, leu2-3, leu2-112, ura3-5). For the chromatin analysis in Fig. 5, the strains were of the CEN.PK2 background as above. Overexpression of Cbf1p was achieved using the plasmid pYGCBF1 (28Kent N.A. Tsang J.S.H. Crowther D.J. Mellor J. Mol. Cell. Biol. 1994; 14: 5229-5241Crossref PubMed Scopus (35) Google Scholar), which expresses the CBF1 gene under control of the GAL1-10 promoter. Chromatin immunoprecipitation (ChIP)1 experiments and chromatin analyses were performed with yeast grown in 100 ml of rich media (1% w/v Bacto-peptone, 1% w/v yeast extract, and 2% w/v d-glucose). Yeast were grown at 29 °C, and cells were harvested at densities of between 1.0 × 107 and 2.5 × 107 cells/ml (midlog/pre-diauxic shift). For Cbf1p overexpression experiments, yeast were grown overnight in 100 ml of synthetic complete medium supplemented with the appropriate amino acids and either 2% w/v d-glucose or 2% w/v d-galactose.Fig. 3A CACGTG pGAL::CBF1 palindrome deep within the FUN30-coding region does not show a Cbf1p-dependent chromatin structure unless Cbf1p is overexpressed.A, indirect end-label analysis of in vivo chromatin MNase accessibility at FUN30. Chromatin in wild-type (WT) and cbf1 yeast grown in glucose (GLU) was digested as described in Fig. 2. Chromatin in cbf1 yeast transformed with a plasmid pGAL::CBF1, which expresses the CBF1 gene under control of the GAL1-10 promoter, was digested with 75 and 150 units/ml MNase after growth overnight in either glucose (GLU) or galactose (GAL). Chromatin in cbf1 yeast not containing the plasmid was digested with 150 units/ml MNase after growth in galactose to control for any effect that carbon source might have on MNase accessibility. The blot is annotated as described in Fig. 2; note the alterations in MNase accessibility surrounding the CACGTG motif in the two galactose induced cbf1 + pGAL::CBF1 samples. B, electrophoretic mobility shift assay of Cbf1p binding to an excess of CACGTG-containing oligonucleotide probe. Equal amounts of total protein extracted from cells grown under the conditions used for chromatin analysis in A were added, and overexpression of Cbf1p is observed in the induced cbf1 + pGAL::CBF1 cells (based on the increased amount of Cbf1p·DNA complex formed).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Destabilizing Cbf1p binding to the CACGTG motif at MET16 by removal of Met4p affects its ability to modulate chromatin structure. Blots are annotated as described in Fig. 2, and Cbf1p-dependent changes in MNase cleavage are highlighted with black diamonds and gray rectangles. A, indirect end-label analysis of in vivo chromatin MNase accessibility at the MET16 promoter. Chromatin in isogenic wild-type (WT), cbf1 null, and met4 null yeast strains was digested with 75 and 150 units/ml MNase. The MNase cleavage pattern at MET16 in the absence of Met4p is similar to the cbf1 mutant pattern, although some faint wild-type bands are also present. B, indirect end-label analysis of in vivo chromatin MNase accessibility at the MET16 promoter under conditions of Cbf1p overexpression as described in Fig. 3, showing that overexpression of Cbf1p creates similar chromatin structure to wild-type. C, identical chromatin digests to panel A analyzed with a DRS2 end-label probe showing that loss of Met4p per se does not affect all Cbf1p-dependent chromatin remodeling.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Cbf1p functions to recruit Isw1p to DRS2, but the contribution of each factor to DRS2 chromatin structure (and DRS2 expression) is not the same.A, Isw1p recruitment to the DRS2 promoter and 5′-coding region is dependent on Cbf1p. ChIP recovery of DRS2 DNA from isogenic yeast strains expressing C-terminal Myc-tagged Isw1p (40Kent N.A. Karabetsou N. Politis P.K. Mellor J. Genes Dev. 2001; 15: 619-626Crossref PubMed Scopus (97) Google Scholar) in a CBF1 (WT) or cbf1 mutant background was assayed as described in Fig. 1. NoAb and IP lanes contain DNA amplified from Cbf1p-Myc ChIP without or with the addition of anti-Myc antibody, respectively. B, Cbf1p is not required for ISW1 expression. A Western blot of Isw1p-Myc levels in strains used above plus an isogenic untagged strain detected with an anti-Myc antibody and using α-tubulin levels as a loading control. C, indirect end-label analysis of in vivo chromatin MNase accessibility at the DRS2, as described in Fig. 2, comparing isogenic wild-type, cbf1, isw1, and cbf1 isw1 mutants. The relatively subtle change in the positions of MNase cleavage in isw1 yeast (40Kent N.A. Karabetsou N. Politis P.K. Mellor J. Genes Dev. 2001; 15: 619-626Crossref PubMed Scopus (97) Google Scholar) are marked between the blots with black boxes and white diamonds. D, Cbf1p is required to maintain the basal transcript level from DRS2, whereas Isw1p is not. Northern blot of total RNA extracted from the strains used in C and grown under the same conditions. The blot was probed for DRS2 and ACT1 transcripts. DRS2 is a low abundance transcript, and the blot was exposed to film for 20-times longer with the DRS2 probe than the ACT1 probe.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ChIP—ChIP was performed using the general method of Meluh and Broach (41Meluh P.B. Broach J.R. Methods Enzymol. 1999; 304: 414-430Crossref PubMed Scopus (47) Google Scholar) as described in Kent et al. (40Kent N.A. Karabetsou N. Politis P.K. Mellor J. Genes Dev. 2001; 15: 619-626Crossref PubMed Scopus (97) Google Scholar). Cultures of yeast strains were fixed in 1% formaldehyde for 2 h at room temperature. Chromatin from 3.0 × 108 cell equivalents was incubated for 15 h with (see Fig. 1; IP) or without (NoAb) anti-Myc monoclonal antibody from clone 9E10 (Sigma) at a 1:200 final dilution. After purification, IP and NoAb DNA samples were re-suspended in 150 μl for Cbf1p-Myc experiments and in 40 μl for Isw1p-Myc experiments. 5-μl aliquots of DNA were analyzed by semiquantitative PCR (20-μl reactions) using primer pairs that amplify ∼300-bp products. DNA from input chromatin (see Fig. 1; three TOT samples) represents 0.5% of the total input with 1:10 and 1:20 dilutions. PCR products were fractionated on 1.5% agarose gels containing ethidium bromide and photographed in negative. The samples used for the ChIP analysis in Fig. 1 were matched, i.e. they all came from the same immunoprecipitation. ChIP primer pairs amplified DNA from the following nucleotide positions relative to the start codon of the quoted coding region or CDEI for centromeres: CLB2 (-850 to -551); CEN6 (-90 to +210); CEN3 (-90 to +210); MET17 (-512 to -212); GAL2 (-600 to -300); DRS2 (-250 to +50); PSK1 (-737 to -437); GSH1 (-584 to -285); SHU1 (+7 to +307); GDH3 (+3 to +300); FUN30 (+1475 to +1775); ERV46 (+200 to +504); GAL3 (-400 to -100); MET16 (-300 to +1). Chromatin Indirect End-label Analysis—Chromatin was digested with MNase in permeabilized yeast cells according to the general methods (42Kent N.A. Bird L.E. Mellor J Nucleic Acids Res. 1993; 21: 4653-4654Crossref PubMed Scopus (40) Google Scholar, 43Wu L. Winston F. Nucleic Acids Res. 1997; 25: 4230-4234Crossref PubMed Scopus (53) Google Scholar). All chromatin samples contained DNA from 2.0 × 108 cells and were digested with 75, 150, and 300 units/ml of MNase for 3–5 min at 37 °C. Equivalent amounts of purified genomic DNA were digested with 10 units/ml MNase at room temperature for 30–50 s to provide “naked” DNA controls. Further samples of purified DNA were cleaved with restriction enzymes and pooled in various combinations to provide marker digests. MNase-treated samples and marker mixes were digested to completion with appropriate restriction enzymes and analyzed by indirect end-labeling (44Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (745) Google Scholar). DNAs were separated on 1.5% agarose gels and Southern-blotted to nylon membranes (MSI/Osmonics). Probes were derived from DNA fragments amplified by PCR from yeast genomic DNA. PCR products were typically designed to be 1.0–1.5 kilobases in size and were then digested with the appropriate restriction enzymes to generate the required end-label. End-label probes were gel-purified before radiolabeling by random priming (Stratagene). Hybridizations and washes were carried out in aqueous buffer at 64 °C as described (28Kent N.A. Tsang J.S.H. Crowther D.J. Mellor J. Mol. Cell. Biol. 1994; 14: 5229-5241Crossref PubMed Scopus (35) Google Scholar). Probe fragments were as follows with nucleotide positions relative to the quoted coding region: CLB2, EcoRV (+787) to a PCR primer at +304; GAL2, HaeIII (+440) to BamHI (+873); DRS2, XhoI (+460) to EcoRV (+810); PSK1, BamHI (+727) to PCR primer at +327; SHU1, SphI (+697) to NcoI (+940); FUN30, BclI (+1109) to ClaI (+1472); ERV46, ScaI (+940) to HindIII (+1401); GAL3, NheI (-655) to PstI (-867); MET16, MscI (+412) to EcoRI (+736). Northern Analysis—RNA was isolated from 2 × 108 cell aliquots of cultures grown under identical conditions to those used for chromatin analysis using the RNeasy midi kit (Qiagen) and processed according to the manufacturer's protocols. Probes were prepared and hybridized exactly as described for the chromatin analyses. The DRS2 probe was the 390-bp XhoI/EcoRV fragment used above as an end-label. ACT1 transcript was detected using a BamHI fragment that contains the entire ACT1 gene. Cbf1p Electrophoretic Mobility Assay—2.0 × 108 cell samples were treated with glass beads to extract total protein into buffer containing 420 mm NaCl as described (28Kent N.A. Tsang J.S.H. Crowther D.J. Mellor J. Mol. Cell. Biol. 1994; 14: 5229-5241Crossref PubMed Scopus (35) Google Scholar). Samples containing 20 μg of total protein were incubated with an end-labeled double-stranded 21-bp oligonucleotide containing a CACGTG palindrome and analyzed by electrophoretic mobility shift assay on a 4% polyacrylamide, 0.5% Tris borate EDTA gel as described (28Kent N.A. Tsang J.S.H. Crowther D.J. Mellor J. Mol. Cell. Biol. 1994; 14: 5229-5241Crossref PubMed Scopus (35) Google Scholar). Western Analysis—Protein samples were prepared as for electrophoretic mobility assay, and 20 μg of total protein were separated on a 7.5% SDS-polyacrylamide gel and electroblotted to a polyvinylidene difluoride membrane. Blots were incubated with the 9E10 anti-Myc monoclonal antibody at a 1:200 dilution (Sigma) or an anti-α-tubulin monoclonal antibody (a generous gift from K. Gull) at a 1:2000 dilution. Bioinformatics—Positions of CACRTG motifs were taken from sequence data available the Stanford Saccharomyces Genome Data Base (www.yeastgenome.org) and the Regulatory Sequence Analysis Tools website (www.ucmb.ulb.ac.be/bioinformatics/rsa-tools). The presence of motifs within the loci was confirmed by electrophoretic mobility assay of cloned DNA fragments with wild-type and cbf1 mutant protein extracts as described above and/or DNA sequencing. 2S. M. Eibert and N. A. Kent, unpublished observation. Maps of CACRTG motif distribution in budding yeast in upstream DNA or by chromosome are available at the Kent lab website (www2.bioch.ox.ac.uk/~nakent). To examine the extent of Cbf1p function as a chromatin modulator throughout the yeast genome, we assayed in vivo Cbf1p binding and MNase accessibility at a panel of loci with potential Cbf1p binding CACRTG motifs. (Table I; Fig. 1A). We examined both palindromic CACGTG motifs and non-palindromic CACATG motifs. Motifs within the GAL2, GSH1, and SHU1 loci were chosen based on reports of Cbf1p-dependent changes in transcription (5Bram R.J. Kornberg R.D. Mol. Cell. Biol. 1987; 7: 403-409Crossref PubMed Scopus (113) Google Scholar, 19Mellor J. Jiang W. Funk M. Rathjen J Barnes C.A. Hinz T. Hegemann J.H. Philippsen P. EMBO J. 1990; 9: 4017-4026Crossref PubMed Scopus (147) Google Scholar, 37Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellor J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 45Nutten P.R. Regulation of S. cerevisiae Filamentous Growth in Response to the Nature and Availability of the Extracellular Carbon Source. 2000; (Ph.D. thesis, University of Oxford)Google Scholar). The remaining loci, DRS2, GDH3, PSK1, FUN30 and ERV46 (46Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (414) Google Scholar, 47Avedano A. Deluna A. Olivera H. Valenzuela L. Gonzalez A. J. Bacteriol. 1997; 179: 5594-5597Crossref PubMed Google Scholar, 48Clark M.W. Zhong W.W. Keng T. Storms R.K. Barton A. Kaback D.B. Bussey H. Yeast. 1992; 8: 133-145Crossref PubMed Scopus (23) Google Scholar, 49Clark M.W. Zhong W.W. Keng T. Storms R.K. Ouellette B.F. Barton A. Kaback D.B. Bussey H. Yeast. 1993; 9: 543-549Crossref PubMed Scopus (3) Google Scholar, 50Otte S. Belden W.J. Heidtman M. Liu J. Jensen O.N. Barlowe S. J. Cell Biol. 2001; 152: 503-518Crossref PubMed Scopus (134) Google Scholar), were chosen at random from the left arm of chromosome I with the exception of GAL3 (51Yano K. Fukasawa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1721-1726Crossref PubMed Scopus (89) Google Scholar) on chromosome IV, which was chosen because it is associated with three CACATG motifs. The CACRTG motifs at GAL2, DRS2, PSK1, GSH1, and GAL3 are predominantly 5′ to the coding region in known or potential gene-regulatory/promoter sequences. The motifs at SHU1, GDH3, and ERV46 occur within the coding regions of the genes.Table ICACRTG motifs associated with loci analyzed in this studyLocusChromosomeMotifaMotif sequences plus two flanking bases are written in the 5′ to 3′ orientation relative to" @default.
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• W2097948608 title "Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast" @default.
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