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Originally published In Press as doi:10.1074/jbc.M403818200 on April 24, 2004

J. Biol. Chem., Vol. 279, Issue 26, 27116-27123, June 25, 2004
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Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast*

Nicholas A. Kent{ddagger}§, Sybille M. Eibert§||, and Jane Mellor§

From the {ddagger}Genetics Unit and §Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

Received for publication, April 6, 2004 , and in revised form, April 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1). The core binding motif for Cbf1p (2, 3) 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 (4). Cbf1p was the first factor to be shown to bind to yeast centromere DNA (5–8) via the CACRTG motif, which is found within the consensus centromere DNA Element 1 (CDEI)1 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 (9, 10). 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 (11–15).

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 (16–20). 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 (21–23). On its own Met4p will not bind DNA (24). 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 (24, 25). 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 (20). 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 (26, 27).

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 (28, 29). This function is likely to involve the recruitment of the chromatin remodeling ATPase Isw1p (30). Although the chromatin-remodeling function of Cbf1p is not absolutely required for MET gene activation, it appears to influence the kinetics of activation (20).

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 (3). 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 (31). 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 (19, 32–37). 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 (19, 28, 38), 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Microbiological Culture—Epitope tagging was performed by the gene replacement technology described in Longtine et al. (39); 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 {alpha}, leu2-3, leu2-112, his3{Delta}1, trp1-289, ura3–52). Isw1p-Myc, which is fully functional in chromatin remodeling, is described in more detail in Kent et al. (40). Disruptions of the CBF1 locus were created by gene replacement as described (19). For chromatin analysis in Figs. 2, 3, 4 the wild-type and mutant strains were of the DBY745 background (MAT{alpha}, 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 (28), 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 x 107 and 2.5 x 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.



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  		FIG. 2.
Cbf1p-dependent chromatin structures are associated with CACGTG motifs in promoter proximal DNA. Cbf1p-dependent changes in MNase accessibility at loci selected from those shown in Fig. 1 were mapped in yeast chromatin by indirect end-label analysis. All chromatin digestions were performed with MNase at concentrations of 75, 150, and 300 units/ml in permeabilized cells from isogenic wild-type (WT) and cbf1 null yeast. Naked/de-proteinized DNA controls are marked DNA. Marker restriction digests are shown in relation to schematic maps of each locus. Potential Cbf1p binding motifs are indicated, with black boxes for CACGTG and gray boxes for CACATG. Restriction enzyme cleavage sites are marked relative to the coding region closest to the CACRTG motifs. Indirect end-label probes are marked with a bar at the bottom right of the locus map. Positions of Cbf1p-dependent changes in MNase cleavage, where present, are highlighted next to the blots with black diamonds and gray rectangles. A, the CLB2 promoter, which does not contain any potential Cbf1p binding motif, does not show any Cbf1p-dependent change in MNase accessibility. B, the GAL2 5' region, which contains a single CACGTG palindrome known to bind Cbf1p in vivo (19), shows a Cbf1p-dependent change in MNase accessibility. C, the DRS2 promoter and 5'-coding region, which contain three potential Cbf1p-binding motifs, show a Cbf1p-dependent change in MNase accessibility. D, The PSK1 5' region, which contains one CACGTG palindrome and one CACATG motif, shows a Cbf1p-dependent change in MNase accessibility. E, the SHU1-coding region, which contains two CACGTG motifs proximal to the promoters of the divergentSTE20andMRP4genes, shows a Cbf1p-dependent change in MNase accessibility. F, the GAL3 promoter and 5'-coding region, which contain three non palindromic CACATG motifs, does not show a Cbf1p-dependent change in MNase accessibility.

 



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  		FIG. 3.
A 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).

 



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  		FIG. 4.
Destabilizing 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.

 



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  		FIG. 5.
Cbf1p 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 (40) 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 {alpha}-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 (40) 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.

 
ChIP—ChIP was performed using the general method of Meluh and Broach (41) as described in Kent et al. (40). Cultures of yeast strains were fixed in 1% formaldehyde for 2 h at room temperature. Chromatin from 3.0 x 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).



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  		FIG. 1.
ChIP analysis of in vivo Cbf1p binding to CACRTG motifs. A, maps of loci analyzed by ChIP (not drawn to scale). Potential Cbf1p binding motifs are shown with CACGTG as black boxes and with CACATG as gray boxes. Motifs are shown in relation to the 300-bp PCR product used to measure recovery of ChIP DNA and to local genes or centromeres. B, DNA from loci known to bind Cbf1p is specifically recovered by ChIP from a yeast strain expressing C-terminal-tagged Cbf1p-Myc from the CBF1 chromosomal locus. TOT lanes contain DNA amplified from input chromatin and dilutions of 1:10 and 1:20. NoAb and IP lanes contain DNA amplified from Cbf1p-Myc ChIP without or with the addition of anti-Myc antibody, respectively. The No tag row shows a ChIP control experiment for CEN3 DNA using isogenic yeast expressing un-tagged Cbf1p. C, DNA recovery from ChIP (as described above) with Cbf1p-Myc is associated with CACGTG but not CACATG motifs outside of the centromeric context.

 
Chromatin Indirect End-label Analysis—Chromatin was digested with MNase in permeabilized yeast cells according to the general methods (42, 43). All chromatin samples contained DNA from 2.0 x 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 (44). 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 (28). 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 x 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 x 108 cell samples were treated with glass beads to extract total protein into buffer containing 420 mM NaCl as described (28). 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 (28).

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-{alpha}-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.2 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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (5, 19, 37, 45). The remaining loci, DRS2, GDH3, PSK1, FUN30 and ERV46 (46–50), were chosen at random from the left arm of chromosome I with the exception of GAL3 (51) 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.


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  		TABLE I
CACRTG motifs associated with loci analyzed in this study

 
Cbf1p Binds DNA and Modulates Chromatin Structure at CACGTG Palindromes in Promoter-proximal DNA—In vivo binding of Cbf1p was assayed using ChIP from a yeast strain expressing Myc-tagged Cbf1p from the CBF1 locus on chromosome X. This strain is a methionine prototroph and shows wild-type MET gene chromatin structure as assayed by in vivo MNase digestion, indicating that Cbf1p-myc is functional.3 To verify the specificity of the ChIP assay, we first analyzed loci already known to have a functional requirement for DNA-bound Cbf1p (3, 20). Fig. 1B shows that DNA from CEN6, CEN3, and the MET17 promoter was specifically immunoprecipitated with Cbf1p-Myc. In contrast, DNA from the CLB2 5' region, which has no potential Cbf1p binding motif, was not recovered.

ChIP recovery of Cbf1p-Myc-associated DNA from the loci selected for our panel is shown in Fig. 1C; compared with the amounts recovered at the positive control loci in Fig. 1B, a similar amount of DNA recovery was observed for the CACRTG motifs associated with the GAL2, DRS2, PSK1, GSH1, SHU1, and GDH3 loci. Notably, these regions are all promoter-proximal and all contain at least one CACGTG palindrome. A lower level of Cbf1p-Myc-dependent DNA recovery than the positive controls was observed at the FUN30 locus, at which a palindromic CACGTG motif is present but occurs within the open reading frame in a promoter-distal position. Virtually no Cbf1p-Myc-dependent DNA was recovered from the ERV46 and GAL3 regions, which only contain non-palindromic CACATG motifs. We, therefore, conclude that Cbf1p appears to be bound in vivo at a variety of non-CEN/non-MET loci primarily via the palindrome CACGTG when in a promoter-proximal location. This result agrees with genome-wide location experiments which suggested that CACGTG was the optimal Cbf1p binding motif in intergenic DNA (31). The non-palindromic CACATG motif, therefore, seems to be a binding site only within the context of the yeast centromere.

Next, chromatin structure at the loci assayed by ChIP was examined using in vivo MNase digestion and indirect end-label analysis in wild-type and cbf1 null cells (Figs. 2, A–F). Digestion patterns in chromatin were compared with those obtained with de-proteinized naked DNA. Because of the specificity for MNase cleavage at nucleosome linker regions, this technology produces an in vivo footprint of nucleosome positions over a region of about 1 kilobase with a resolution of ±15 bp. This methodology has previously been used to show Cbf1p-dependent changes in nucleosome position at both MET16 and MET17 promoters (28). As expected, no Cbf1p-dependent chromatin structure was observed at the CLB2 5' region (Fig. 2A), which does not possess any sort of CACRTG motif and does not show Cbf1p-specific ChIP. The GAL2, DRS2, PSK1, and SHU1 loci all showed Cbf1p-dependent changes in MNase cleavage pattern in proximity to the CACGTG motifs (Figs. 2, B–E), as did GDH32 and GSH1 (37). No Cbf1p-dependent chromatin structures were observed at GAL3 (Fig. 2F) and FUN30 (Fig. 3A) nor at ERV46.2 Thus, with the exception of FUN30, sites of Cbf1p-dependent chromatin correlated with the sites of Cbf1p association at CACGTG palindromes as assayed by ChIP. This result suggests that efficient binding or activity of Cbf1p at a CACGTG palindrome depends on the context of the motif. The CACGTG motif at FUN30 is located at +1627 bp, deep within the 3395-bp-coding region. This motif is, therefore, in a position unlikely to regulate transcription of either FUN30 itself or of the neighboring genes. However, the other CACGTG motifs described above, which are associated with Cbf1p-dependent chromatin structures, are all promoter-proximal. To test whether Cbf1p may, therefore, require interactions with other transcription factors to function efficiently at CACGTG motifs, we next attempted to manipulate the amount of Cbf1p available for DNA binding at the FUN30 CACGTG, which does not seem to support efficient Cbf1p binding and at the MET16 promoter CACGTG, which does.

Manipulation of the Amount of Cbf1p Alters Its Chromatin-remodeling Potential at Certain CACGTG Motifs—We found that overexpression of Cbf1p from the GAL1-10 promoter leads to a clear change in the MNase accessibility surrounding the CACGTG motif present 1627 bp into the FUN30-coding region (Fig. 3). This result suggests that the CACGTG motif within the FUN30 open reading frame is intrinsically competent to support Cbf1p-dependent chromatin modulation but fails during vegetative growth because the Cbf1p concentration at this site is in some way limiting. We obtain a similar result at the MEC1 locus that contains two CACGTG motifs at +3832bp and +3984bp within a 7107-bp open reading frame.4 These results suggest that Cbf1p normally requires the presence of additional stabilizing or regulatory factors. This appears to be exactly the case at certain MET gene promoters. At the MET16 locus, Cbf1p binding to the CACGTG motif (at -174 bp) has been extensively characterized and shown to be enhanced by formation of a co-operative Cbf1p·Met28p·Met4p complex (24, 25). We, therefore, examined the chromatin structure at MET16 in a met4 mutant in which Cbf1p binds alone to the CACGTG motif with reduced relative affinity. We also examined MET16 chromatin structure under the conditions of Cbf1p overexpression described above. We might predict that the lower affinity of MET16 CACGTG binding by Cbf1p in met4 yeast should decrease the probability of Cbf1p binding and affect Cbf1p-dependent chromatin modulation. Conversely, we might predict that overexpression of Cbf1p in wild-type cells should have no effect on chromatin structure because Cbf1p will already be maximally recruited to MET16. Fig. 4 shows exactly this result; the MNase cleavage pattern at MET16 in the met4 mutant is very similar to a cbf1 mutant but does contain faint bands characteristic of wild-type cells as well (Fig. 4A). This suggests that met4 yeast comprise a mixed population of cells and that Cbf1p is only successful in modulating chromatin structure in a small proportion of them. Overexpression of Cbf1p from the GAL1-10 promoter as predicted has no effect on MET16 chromatin structure (Fig. 4B). To control for the possibility that Met4p is generally required for Cbf1p-dependent chromatin modulation we also tested the MNase accessibility at the DRS2 5' region in met4 yeast (Fig. 4C). At DRS2, loss of Met4p has no effect on the ability of Cbf1p to modulate chromatin; thus, the alteration that we see at MET16 is likely to be specifically related to the trans-acting factor environment at that promoter.

Cbf1p-dependent Chromatin Remodeling Is Only Partially Isw1p-dependent—The experiments described above suggest that Cbf1p must be recruited efficiently to function in chromatin remodeling via the CACGTG palindrome. However, they do not address the mechanism by which Cbf1p modulates chromatin structure. It has recently been shown that both Cbf1p and the chromatin-remodeling ATPase Isw1p are required for repression of the PHO8 gene (which contains a single CACGTG motif at -534bp) and that Cbf1p functions to recruit the Isw1p complex to PHO8 DNA (30). It has also been shown that both Isw1p itself and Isw1p-dependent chromatin structures are present at the Cbf1p-bound MET16 and DRS2 promoter regions (40, 52). Figs. 5, A and B, show that Cbf1p is required for Isw1p-Myc-specific ChIP of the DRS2 promoter. It, therefore, seems likely that Cbf1p-dependent recruitment of Isw1p to CACGTG motif-associated promoters is a general phenomenon. However, although Cbf1p appears to recruit Isw1p to DRS2, the contribution of each factor to the wild-type chromatin structure is not the same. Fig. 5C shows that the loss of Cbf1p causes a much more extensive alteration in the MNase cleavage pattern at DRS2 than loss of Isw1p. In addition, a cbf1 isw1 double mutant shows a chromatin structure that is essentially identical to the cbf1 single mutant. A similar difference in mutant chromatin structures can be observed at the MET16 locus (compare Fig. 4A with Ref 52). Also, the steady-state level of DRS2 RNA, which represents basal expression of the gene in rich media (53), is unaltered in isw1 mutant yeast but abolished in the absence of Cbf1p (Fig. 5D). Taken together, these results suggest that Isw1p is only partially required for the chromatin-remodeling function of Cbf1p.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cbf1p as a Genome-wide Trans-acting Factor—In vitro the palindrome CACGTG is bound by Cbf1p with 10-fold higher affinity than the non-palindromic CACATG motif (3). Despite this, both CACGTG and CACATG motifs occur within centromeric CDEI elements and are known to be functional in Cbf1p binding (3, 54). Indeed, changing the CEN6 CDEI from CACGTG to CACATG, although altering Cbf1p binding affinity in vitro, does not significantly alter centromere function in vivo (2). This binding to both low and high affinity CDEI motifs was also reflected in our ChIP analysis at CEN3 and CEN6, respectively. However, outside of the centromeric context, our ChIP and chromatin results show that on its own non-palindromic CACATG does not provide a significant in vivo Cbf1p binding site. Cbf1p is known to interact strongly with several components of the yeast centromere-kinetochore complex (13–15), and this is likely to explain stable Cbf1p binding to both high and low affinity centromere CDEI elements in vivo (3, 54).

Interactions with other DNA-associated factors may also explain our observation that Cbf1p prefers promoter-proximal CACGTG palindromes. We have shown that a single CACGTG motif 1627 bp into the 3395-bp FUN30-coding region does not normally act as a functional Cbf1p binding site; this CACGTG motif is only weakly recovered by Cbf1p ChIP and shows no change in chromatin structure in the absence of Cbf1p during vegetative growth. However, overexpression of Cbf1p from a plasmid creates a Cbf1p-dependent change in chromatin structure surrounding this CACGTG, suggesting that the motif is intrinsically capable of functional Cbf1p binding but that the concentration of Cbf1p in the yeast nucleus is normally limiting for this site. The functional Cbf1p binding CACGTG palindromes we observe are all in proximity with promoter or gene regulatory sequences. For instance the CACGTG motifs at GAL2, DRS2, and GSH1 occur in upstream locations in a manner analogous to that previously observed in MET gene promoters. The CACGTG motif at GDH3 is found 143 bp into the open reading frame, a location that could constitute a potential downstream regulatory sequence (55). Finally, the two CACGTG motifs within the SHU1-coding region occurs within 600 bp 5' of the start codons of both the MRP4 or STE20 genes. Cbf1p has been suggested to play a role in the regulation of STE20 transcription (45), and the two motifs within the SHU1 open reading frame may, therefore, constitute part of an upstream regulatory region for the STE20 gene. We, therefore, suggest that all these functional Cbf1p binding sites are favored through interactions of Cbf1p with other factors associated with gene regulatory DNA. Exactly this type of stabilizing interaction has been described for Cbf1p with Met28p and Met4p at the MET16 promoter (22, 24, 25). Consistent with this idea, we can show that loss of Met4p, which reduces the affinity for Cbf1p at the MET16 promoter (24), leads to a large proportion of cells exhibiting a cbf1 mutant-like chromatin phenotype. Met4p is not the only example of a factor known to interact with Cbf1p to form stable DNA-bound complexes at promoters. For instance, a number of growth condition-dependent complexes at the CYT1 promoter have been reported (56).

We conclude that Cbf1p has the potential to bind functionally to a large number of CACGTG motifs within the yeast genome where it can function to modulate chromatin structure. However, under normal conditions the concentration of Cbf1p is limiting, and binding occurs only at motifs where it is stabilized by a combination of intrinsic DNA sequence specificity and protein-protein interactions. We note that a similar bias toward binding sites within gene regulatory DNA has also been shown for another yeast DNA-binding protein, Rap1p (57). Our results suggest that, in addition to binding at all yeast centromeres, Cbf1p is likely to bind wherever the CACGTG palindrome occurs in a promoter-proximal context. This could place Cbf1p in the regulatory DNA of ~10% of yeast protein coding genes; i.e. CACGTG motifs occur within 800 bp upstream of 592 yeast genes, and 143 genes have at least 1 CACGTG motif within the first 200 bp of their coding region in a potential downstream regulatory sequence context.

Our analysis of Cbf1p binding is broadly consistent with a genome-wide localization study of transcription factors; Lee et al. (31) used an error model to create p values (a confidence level) for binding of factors to microarrayed yeast intergenic sequences. For loci such as GAL2 and DRS2, which contain CACGTG motifs in intergenic locations and where we observe significant recovery in Cbf1p ChIP, the p values for Cbf1p binding both have a significant value of 1.4 x 10-3. This contrasts with a p value of 7.5 x 10-1 for the CLB2 intergenic region, which does contain any form of CACRTG motif. At GAL3, which contains two CACATG motifs in its intergenic regions and which we fail to recover in Cbf1p ChIP, a similarly non-significant p value of 1.8 x 10-1 is reported. However, we also note that the p values obtained for Cbf1p binding to the MET16 and MET17 intergenic regions are not significant despite verification of Cbf1p binding at these loci by a variety of other studies. As Lee et al. (31) point out, their estimates of binding are likely to be conservative, and we believe this justifies our use of a functional assay for Cbf1p binding in addition to ChIP experiments.

Cbf1p as a Genome-wide Chromatin Modulator and Gene Regulator—Cbf1p has been shown previously to be required for normal chromatin structure at MET genes (28, 29) and several non MET loci (19, 28, 37, 38). Changes in MNase cleavage patterns at MET genes are consistent with a role for Cbf1p in defining the translational position of short tracts of nucleosomes that encompass upstream-regulatory regions. Mapping of MNase cleavage sites in this work shows that this nucleosome-positioning function of Cbf1p appears to be general (Fig. 6).



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  		FIG. 6.
Cbf1p-dependent changes in MNase accessibility are likely to represent changes in the translational position of nucleosomes. Inferred nucleosome positions at DRS2·MNase cleavage sites were measured relative to marker digests, with molecular weight values taken as center points of Gaussian curves fitted to scans of blots using OneDScan (Scanalytics). MNase cleavage sites are plotted relative to the DRS2-coding region for naked DNA, wild-type, and cbf1 mutant yeast. CACRTG motifs are marked as in Fig. 1. Potential nucleosomes are represented by circles ~146 bp in diameter. Short probes to the DRS2 5' region hybridize to nucleosome ladders prepared from both wild-type and cbf1 strains, indicating that nucleosomes are present in both chromatin states.4 However, we cannot rule out the possibility that some nucleoprotein complexes footprinted by MNase in both wild-type and cbf1 yeast represent trans-acting factors of a Cbf1p-dependent composition rather than canonical nucleosomes. Nevertheless, given the specificity of MNase for cleavage in nucleosomal linker regions and that Cbf1p-dependent changes in MNase accessibility extend beyond the immediate vicinity of CACGTG motifs, we conclude that changes in the translational position of nucleosomes are significant in cbf1 yeast.

 
A physical association between Cbf1p and the chromatin remodeling ATPase Isw1p has been shown recently at the PHO8 locus (30), suggesting that the nucleosome sliding activity of the Isw1p enzyme is directly recruited by Cbf1p to modulate chromatin structure. We have shown here that Cbf1p also appears to recruit Isw1p to the DRS2 upstream region. However, by comparing MNase cleavage patterns between normal cells and cbf1 and isw1 mutants, we show that the Cbf1p- and Isw1p-dependent affects on nucleosome position are very different. We conclude that although Cbf1p is likely to be required for the Isw1p-dependent chromatin structure at DRS2 (by recruiting Isw1p to DNA), a significant chromatin remodeling effect due to Cbf1p binding is independent of Isw1p. We have examined promoter-proximal CACGTG-associated chromatin structures in a variety of mutants defective in chromatin remodeling including those that affect SWI/SNF, Isw2p, RSC, SAGA/ADA, Rpd3p, and INO80 (for review, see Ref. 58). None of these mutants re-creates the change in MNase accessibility observed in cbf1 mutants.4 Although we cannot rule out a requirement for some other remodeling complex that we have not yet tested, it is also possible that Cbf1p can define nucleosome position simply by binding DNA. At the yeast centromere, the position of the CDEI motif at one end of the core CEN sequence means that Cbf1p binds in close proximity to the centromere-specific nucleosome-like particle (10, 15). Loss of Cbf1p causes a decrease in centromere efficiency during mitosis and leads to a subtle alteration in the nuclease accessibility surrounding the core particle (59, 19). This is consistent with a role for Cbf1p in stabilizing the centromere core. It is, therefore, possible that a general biochemical property of Cbf1p is the interaction with and stabilization of nucleosome-based complexes. Through this property at gene regulatory sequences, DNA-bound Cbf1p could provide a local positional cue for one or more nucleosomes.

Whatever the mechanism of Cbf1p-dependent nucleosome remodeling, a general nucleosome positioning function of Cbf1p together with its ability to form interactions with other transacting factors is consistent with the known effects of the factor in transcriptional regulation. Loss of Cbf1p or its potential binding motifs within gene regulatory DNA led to varied changes in gene expression depending on the gene. At MET16 and MET14 activated transcription was totally abolished, which explains the methionine auxotrophy of cbf1 mutants (20). We also observed a complete loss of transcription from the DRS2 locus (Fig. 5). However, at other genes the effects are more subtle. At MET17 for instance, activated transcription is not abolished but takes longer to establish (20). Transcript levels at PGK became moderately elevated in the absence of Cbf1p (34). Many other genes, such as GAL2, TRP1, CYT1, RPL45, QCR8, GSH1, and PHO8 showed similar slight perturbation of transcript levels either positively or negatively (19, 32, 33, 35–37, 30). We envisage that Cbf1p acts as a context-dependent transcription factor with the importance of its role depending on the types of interactions made with other transacting factors and the underlying nucleosomal environment. At one extreme Cbf1p may be absolutely required for the formation of particular transcription factor complexes either by direct protein-protein interaction as at MET16 or because the Cbf1p-dependent nucleosome positions create access to cis-acting motifs. At other locations Cbf1p may play a more subsidiary role in maintaining basal or repressed levels of expression. A number of poorly studied phenotypes have been described for cbf1 yeast including growth rate alterations, cell wall defects, reduced sporulation efficiency, and inability to initiate pseudohyphal differentiation (17–19, 45, 60). It is, therefore, likely that Cbf1p has a role in the transcriptional regulation of a large number of genes.


   FOOTNOTES
 
* This work was supported by Wellcome Trust and Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom grants (to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Current address: Institut für Immunologie, Ruprecht-Karls-Universität, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany. Back

To whom correspondence should be addressed. Tel.: 44-1-865-275325; Fax: 44-1-865-275318; E-mail: nicholas.kent{at}bioch.ox.ac.uk.

1 The abbreviations used are: CDEI, centromere DNA element I; MNase, micrococcal nuclease; ChIP, chromatin immunoprecipitation. Back

2 S. M. Eibert and N. A. Kent, unpublished observation. Back

3 N. A. Kent and J. Mellor, unpublished observation. Back

4 N. A. Kent, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We are grateful to Harry Mountain for providing a met4 yeast strain, Daniel Crowther for help in sequence analysis, Tony Spit and Viv Perkins for involvement at the early stages of this project, Anitha Nair for technical help.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ghaemmaghami, S., Huh, W-K., Bower, K., Howson, R. W., Belle, A., Dephoure, N., O'Shea, E. K., and Weissman, J. S. (2003) Nature 425, 737-741[CrossRef][Medline] [Order article via Infotrieve]
  2. Niedenthal, R., Stoll, R., and Hegemann, J.-H. (1991) Mol. Cell. Biol. 11, 3545-3553[Abstract/Free Full Text]
  3. Wilmen, A., Pick, H., Niedenthal, R. K., Sen-Gupta, M., and Hegemann, J-H. (1994) Nucleic Acids Res. 22, 2791-2800[Abstract/Free Full Text]
  4. Robinson, K. A., and Lopes, J. M. (2000) Nucleic Acids Res. 28, 1499-1505[Abstract/Free Full Text]
  5. Bram, R. J., and Kornberg, R. D. (1987) Mol. Cell. Biol. 7, 403-409[Abstract/Free Full Text]
  6. Baker, R. E., Fitzgerald-Hayes, M., and O'Brien T. C. (1989) J. Biol. Chem. 264, 10843-10850[Abstract/Free Full Text]
  7. Cai, M., and Davis, R. W. (1989) Mol. Cell. Biol. 9, 2544-2550[Abstract/Free Full Text]
  8. Jiang, W., and Philippsen, P. (1989) Mol. Cell. Biol. 9, 5585-5593[Abstract/Free Full Text]
  9. Ortiz, J., and Lechner, J. (2000) Protoplasma 211, 12-19[CrossRef]
  10. McAinsh, A. D., Tytell, J. D., and Sorger, P. K. (2003) Annu. Rev. Cell Dev. Biol. 19, 519-539[CrossRef][Medline] [Order article via Infotrieve]
  11. Bloom, K. S., and Carbon, J. (1982) Cell 29, 305-317[CrossRef][Medline] [Order article via Infotrieve]
  12. Funk, M., Hegemann, J. H., and Philippsen, P. (1989) Mol. Gen. Genet. 219, 153-160[Medline] [Order article via Infotrieve]
  13. Meluh, P. B., and Koshland, D. (1997) Genes Dev. 11, 3401-3412[Abstract/Free Full Text]
  14. Baker, R. E., Harris, K., and Zhang, K. (1998) Genetics 149, 73-85[Abstract/Free Full Text]
  15. Hemmerich, P., Stoyan, T., Wieland, G., Koch, M., Lechner, J., and Diekmann, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12583-12588[Abstract/Free Full Text]
  16. Thomas, D., Cherest, H., and Surdin-Kerjan, Y. (1989) Mol. Cell. Biol. 9, 3292-3298[Abstract/Free Full Text]
  17. Baker, R. E., and Masison, D. C. (1990) Mol. Cell. Biol. 10, 2458-2467[Abstract/Free Full Text]
  18. Cai, M., and Davis, R. W. (1990) Cell 61, 437-446[CrossRef][Medline] [Order article via Infotrieve]
  19. Mellor, J., Jiang, W., Funk, M., Rathjen, J. Barnes, C. A., Hinz, T., Hegemann, J. H., and Philippsen, P. (1990) EMBO J. 9, 4017-4026[Medline] [Order article via Infotrieve]
  20. Kuras, L., and Thomas, D. (1995) FEBS Lett. 367, 15-18[CrossRef][Medline] [Order article via Infotrieve]
  21. Patton, E. E., Peyraud, C., Rouillon, A., Surdin-Kerjan, Y., Tyers, M., and Thomas, D. (2000) EMBO J. 19, 1613-1624[CrossRef][Medline] [Order article via Infotrieve]
  22. Kaiser, P., Flick, K., Wittenberg, C., and Reed, S. I. (2000) Cell 102, 303-314[CrossRef][Medline] [Order article via Infotrieve]
  23. Kuras, L., Rouillon, A., Lee, T., Barbey, R., Tyers, M., and Thomas, D. (2002) Mol. Cell. 10, 69-80[CrossRef][Medline] [Order article via Infotrieve]
  24. Kuras, L., Barbey, R., and Thomas, D. (1997) EMBO J. 16, 2441-2451[CrossRef][Medline] [Order article via Infotrieve]
  25. Kuras, L., Cherest, H., Surdin-Kerjan, Y., and Thomas, D. (1996) EMBO J. 15, 2519-2529[Medline] [Order article via Infotrieve]
  26. Blaiseau, P. L., Isnard, A. D., Surdin-Kerjan, Y., and Thomas, D. (1997) Mol. Cell. Biol. 17, 3640-3648[Abstract]
  27. Blaiseau, P. L., and Thomas, D. (1998) EMBO J. 17, 6327-6336[CrossRef][Medline] [Order article via Infotrieve]
  28. Kent, N. A., Tsang, J. S. H., Crowther, D. J., and Mellor, J. (1994) Mol. Cell. Biol. 14, 5229-5241[Abstract/Free Full Text]
  29. O'Connell, K. F., Surdin-Kerjan, Y., and Baker, R. E. (1995) Mol. Cell. Biol. 15, 1879-1888[Abstract]
  30. Moreau, J.-L., Lee, M., Mahachi, N., Vary, J., Mellor, J., Tsukiyama, T., and Goding, C. R. (2003) Mol. Cell 11, 1609-1620[CrossRef][Medline] [Order article via Infotrieve]
  31. Lee, 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. (2002) Science 298, 799-804[Abstract/Free Full Text]
  32. Mellor, J., Rathjen, J., Jiang, W., and Dowell, S. J. (1991) Nucleic Acids Res. 19, 2961-2969[Abstract/Free Full Text]
  33. Oechsner, U., and Bandlow, W. (1996) Nucleic Acids Res. 24, 2395-2403[Abstract/Free Full Text]
  34. Packham, E. A., Graham, I. R., and Chambers, A. (1996) Mol. Gen. Genet. 250, 348-356[Medline] [Order article via Infotrieve]
  35. Kraakman, L. S., Mager, W. H., Grootjans, J. J., and Planta, R. J. (1991) Biochim. Biophys. Acta 1090, 204-210[Medline] [Order article via Infotrieve]
  36. DeWinde, J. H., and Grivell, L. (1995) Eur. J. Biochem. 233, 200-208[Medline] [Order article via Infotrieve]
  37. Dormer, U. H., Westwater, J., McLaren, N. F., Kent, N. A., Mellor, J., and Jamieson, D. J. (2000) J. Biol. Chem. 275, 32611-32616[Abstract/Free Full Text]
  38. De Winde, J. H., Van Leeuwen, H. C., and Grivell, L. (1993) Yeast 9, 847-857[CrossRef][Medline] [Order article via Infotrieve]
  39. Longtine, M. S., McKenzie, A. D., Demarini, J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998) Yeast 14, 953-961[CrossRef][Medline] [Order article via Infotrieve]
  40. Kent, N. A., Karabetsou, N., Politis, P. K., and Mellor, J. (2001) Genes Dev. 15, 619-626[Abstract/Free Full Text]
  41. Meluh, P. B., and Broach, J. R. (1999) Methods Enzymol. 304, 414-430[Medline] [Order article via Infotrieve]
  42. Kent, N. A., Bird, L. E., and Mellor, J (1993) Nucleic Acids Res. 21, 4653-4654[Free Full Text]
  43. Wu, L., and Winston, F. (1997) Nucleic Acids Res. 25, 4230-4234[Abstract/Free Full Text]
  44. Wu, C. (1980) Nature 286, 854-860[CrossRef][Medline] [Order article via Infotrieve]
  45. Nutten, P. R. (2000) Regulation of S. cerevisiae Filamentous Growth in Response to the Nature and Availability of the Extracellular Carbon Source. Ph.D. thesis, University of Oxford
  46. Tang, X., Halleck, M. S., Schlegel, R. A., and Williamson, P. (1996) Science 272, 1495-1497[Abstract]
  47. Avedano, A., Deluna, A., Olivera, H., Valenzuela, L., and Gonzalez, A. (1997) J. Bacteriol. 179, 5594-5597[Abstract/Free Full Text]
  48. Clark, M. W., Zhong, W. W., Keng, T., Storms, R. K., Barton, A., Kaback, D. B., and Bussey, H. (1992) Yeast 8, 133-145[CrossRef][Medline] [Order article via Infotrieve]
  49. Clark, M. W., Zhong, W. W., Keng, T., Storms, R. K., Ouellette, B. F., Barton, A., Kaback, D. B., and Bussey, H. (1993) Yeast 9, 543-549[CrossRef][Medline] [Order article via Infotrieve]
  50. Otte, S., Belden, W. J., Heidtman, M., Liu, J., Jensen, O. N., and Barlowe, S. (2001) J. Cell Biol. 152, 503-518[Abstract/Free Full Text]
  51. Yano, K., and Fukasawa, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1721-1726[Abstract/Free Full Text]
  52. Morillon, A., Karabetsou, N., O'Sullivan, J., Kent, N., Proudfoot, N., and Mellor, J. (2003) Cell 115, 425-435[CrossRef][Medline] [Order article via Infotrieve]
  53. Chen, C. Y., Ingram, M. F., Rosal, P. H., and Graham, T. R. (1999) J. Cell Biol. 147, 1223-1236[Abstract/Free Full Text]
  54. Wieland, G., Hemmerich, P., Koch, M., Stoyan, T., Hegemann, J., and Diekmann, S. (2000) Nucleic Acids Res. 29, 1054-1060
  55. Sinclair, D. A., Kornfeld, G. D., and Dawes, I. W. (1994) Mol. Cell. Biol. 14, 214-225[Abstract/Free Full Text]
  56. Oechsner, U., and Bandlow, W. (1998) Mol. Gen. Genet. 260, 417-425[CrossRef][Medline] [Order article via Infotrieve]
  57. Lieb, J. D., Liu, X., Botstein, D., and Brown, P. O. (2001) Nat. Genet. 28, 327-334[CrossRef][Medline] [Order article via Infotrieve]
  58. Kingston, R. E., and Narlikar, G. J. (1999) Genes Dev. 13, 2339-2352[Free Full Text]
  59. Saunders, M., Fitzgerald-Hayes, M., and Bloom, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 175-179[Abstract/Free Full Text]
  60. Masison, D. C., and Baker, R. E. (1992) Genetics 131, 43-53[Abstract]

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Appl. Envir. Microbiol., October 1, 2007; 73(19): 5990 - 6000.
[Abstract] [Full Text] [PDF]

Home page
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Multiple Basic Helix-Loop-Helix Proteins Regulate Expression of the ENO1 Gene of Saccharomyces cerevisiae
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[Abstract] [Full Text] [PDF]

Home page
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[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
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A Systems Approach to Measuring the Binding Energy Landscapes of Transcription Factors
Science, January 12, 2007; 315(5809): 233 - 237.
[Abstract] [Full Text] [PDF]

Home page
 Eukaryot CellHome page
L.-C. Lai, A. L. Kosorukoff, P. V. Burke, and K. E. Kwast
Metabolic-State-Dependent Remodeling of the Transcriptome in Response to Anoxia and Subsequent Reoxygenation in Saccharomyces cerevisiae.
Eukaryot. Cell, September 1, 2006; 5(9): 1468 - 1489.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Menant, P. Baudouin-Cornu, C. Peyraud, M. Tyers, and D. Thomas
Determinants of the Ubiquitin-mediated Degradation of the Met4 Transcription Factor
J. Biol. Chem., April 28, 2006; 281(17): 11744 - 11754.
[Abstract] [Full Text] [PDF]

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