Molecular Cell, Vol. 10, 871–882, October, 2002, Copyright 2002 by Cell Press

Interplay of TBP Inhibitors inGlobal Transcriptional Control

characterized interactions in yeast: TBP/NC2, TBP/TAF1, and TBP dimerization.

NC2 is a heterodimer of two histone-fold subunits

Carmelata Chitikila,1,4 Kathryn L. Huisinga,1

Jordan D. Irvin,1 Andrew D. Basehoar,2

and B. Franklin Pugh1,3

(Bur6/DRAP1 and NC2�/DR1). NC2 binds to and stabi-1Department of Biochemistry and Molecular Biologylizes TBP/TATA complexes in mobility shift assays, com-2 Graduate Program in Statisticspetitively inhibiting the association of TFIIA and TFIIBPennsylvania State University(Cang et al., 1999; Goppelt and Meisterernst, 1996; KimUniversity Park, Pennsylvania 16803et al., 1997; Mermelstein et al., 1996). NC2 occupies aregion just under the TBP/TATA interface (see Figure1A) contacting DNA on both sides of TBP (Kamada et

Summary al., 2001). A domain of NC2 reaches up and contactsresidues along the convex surface of TBP and contrib-

The TATA binding protein (TBP) is required for the utes to the steric occlusion of TFIIB. A yeast TBP muta-expression of nearly all genes and is highly regulated tion (F182V) along this interface disrupts TBP/NC2 inter-both positively and negatively. Here, we use DNA mi- actions in vitro and causes increased expression of acroarrays to explore the genome-wide interplay of sev- number of enhancerless genes in vivo (Cang et al., 1999).eral TBP-interacting inhibitors in the yeast Saccharo- NC2 overexpression selectively suppresses phenotypesmyces cerevisiae. Our findings suggest the following: associated with this mutation, providing further evi-The NC2 inhibitor turns down, but not off, highly active dence that TBP (F182V) is primarily defective in NC2genes. Autoinhibition of TBP through dimerization interactions. In addition to acting as an inhibitor, NC2contributes to transcriptional repression, even at re- plays a positive role in transcription, although its basispressive subtelomeric regions. The TAND domain of is not understood (Cang et al., 1999; Geisberg et al.,TAF1 plays a primary inhibitory role at very few genes, 2001; Willy et al., 2000).but its function becomes widespread when other TBP TAF1 inhibits TBP/TATA interactions (Banik et al.,interactions are compromised. These findings reveal 2001; Kokubo et al., 1998; Nishikawa et al., 1997). NMRthat transcriptional output is limited in part by a collab- analysis of the Drosophila TAF1 amino-terminal domainoration of different combinations of TBP inhibitory I (TAND I) indicates that it engages in molecular mimicrymechanisms. of the TATA box (see Figure 1A), occluding the concave

DNA binding surface of TBP (Liu et al., 1998). The yeastTAND I region is smaller, poorly conserved, and func-Introductiontionally dependent upon an adjacent TAND II region(Kokubo et al., 1998; Kotani et al., 1998). Although yeastTranscriptional control of gene expression involves aTAND I also appears to interact with the concave surface

dynamic interplay of positively and negatively actingof TBP (Kokubo et al., 1998), the TBP residues involved

factors, with the relative dominance of one over the otherin binding have not been fully identified. Deletion of the

dictating transcriptional output. Negative regulation is yeast TAND domain is expected to derepress transcrip-generally associated with promoter inaccessibility due tion in vivo. Except in certain artificial situations (Chengto chromatin structure (reviewed in Struhl, 1999). How- et al., 2002), this has not been observed. Therefore, itever, loss of chromatin components, including histone remains unresolved as to whether TAND is a negativeH4, Tup1, or Sir3, has surprisingly modest effects on regulator in vivo.transcription (DeRisi et al., 1997; Wyrick et al., 1999). In the absence of DNA, the conserved core of TBPSince promoter regions are often intrinsically accessible crystallizes as a dimer (see Figure 1A), which occludesto nuclear proteins (Mai et al., 2000, and references its DNA binding surface (Chasman et al., 1993; Nikolovtherein), there are likely to be additional general mecha- et al., 1992). In vitro, this interaction appears to benisms, not based solely on chromatin structure, that weaker in yeast TBP than in human TBP (Campbell etnegatively regulate transcription complex assembly. al., 2000; Coleman and Pugh, 1997; Coleman et al.,

Direct interactions of negative regulators with the gen- 1995). The physiological relevance of TBP self-associa-eral transcription machinery might contribute to tran- tion in both yeast and humans is supported by in vivoscriptional control. In particular, several factors target crosslinking experiments and mutational studies (Jack-the TATA binding protein (TBP) in all eukaryotes, includ- son-Fisher et al., 1999; Taggart and Pugh, 1996). Muta-ing NC2, TAF1 (formerly TAFII145/130 in yeast and tions along the crystallographic concave DNA bindingTAFII250/230 in metazoans), TBP itself, Mot1, Spt3, and and dimerization surface (TBPEB series) inhibit TBP self-the Not-Ccr4 complex. How these factors interrelate to association to varying extents, while TATA binding isregulate transcription through TBP is not known. To equally impaired (Jackson-Fisher et al., 1999). A strongbegin understanding the regulatory network by which correlation exists between dimer instability measuredthese factors function, we first focused on three well- in vitro and elevated basal (EB) transcription in yeast,

as measured by �-galactosidase activity from a lacZreporter gene. Consistent with the notion of autorepres-3 Correspondence: bfp2@psu.edusion, overexpression of wild-type TBP does not lead4 Present address: Johnson and Johnson Pharmaceutical Re-

search & Development, 1000 Route 202, Raritan, New Jersey 08869. to elevated basal transcription. Moreover, in a dose-

Molecular Cell872

dependent manner, TBP overexpression suppresses the Resultselevated basal transcription caused by the dimerization-impaired TBPEB mutants, perhaps by driving unfavorable Yeast TBP/TAND and TBP Dimers Have Overlappingdimer formation with the TBPEB mutants via mass action but Distinct Interfaces(Jackson-Fisher et al., 1999). While TBP is subjected to Amino acids V69, V71, and V161 are located in a tightautoinhibition in vivo, it is not known how broadly this cluster along the concave surface of TBP, forming partmechanism is utilized genome-wide in the context of of the overlapping interfaces with TATA DNA, the TAF1the NC2 and TAND inhibitory mechanisms discussed TAND I domain, and a second molecule of TBP, as de-above. fined by crystallography and NMR (see Figure 1A). Using

Since the concave surface of TBP has the potential a GST pull-down assay, we previously measured theto engage in multiple positive and negative interactions, relative effect of six individual mutations of these aminosuch as with TATA, TBP, and the TAF1 TAND domain, acids (collectively designated “EB”) on TBP dimer stabil-mutations along the concave surface could affect more ity (Jackson-Fisher et al., 1999). The following trend forthan one interaction. However, each of these interfaces dimer stability was observed: (WT, V71E) � V161E �of TBP with its target are chemically distinct and there- N69S � (V71R, V161R, N69R). The trend agreed well withfore could elicit characteristic phenotypes in response the dimer crystal structure, in which the bulky chargedto a series of TBP mutations along the concave surface. arginine substitutions are expected to disrupt dimeriza-It might be possible to correlate specific patterns of tion the most.gene expression with specific interactions defined bio- To compare the relative affinities of the TAF1 TANDchemically. domain for the same mutant TBPs, a GST-TAND pull-

To investigate the potential involvement of different down assay was performed using amino acids 10–88 ofrepression mechanisms functioning through the con- scTAF1 (Kotani et al., 1998). As shown in Figure 1B,cave surface of TBP, we first examined whether the TAND (10–88), but not a mutant version (F23K, D66K),previously characterized “EB” mutations in this region retained wild-type TBP, as previously reported (Kokuboaffect binding to the TAF1 TAND domain. Next, we initi- et al., 1998). A single point mutation (D66K) had an inter-ated a genome-wide study to examine the interplay of mediate effect. A range of affinities of GST-TAND forfactors that interact with TBP’s concave surface (DNA, the TBP mutants was observed, having the followingTAND, TBP homodimerization, and possibly others) to trend: (WT, V71R, N69S) � V161E � N69R � (V71E,regulate transcription. Their relationship with NC2 was V161R).also examined. TAND contribution was assessed by Previously, we reported a tight negative correlationcomparing strains containing and lacking the TAND do- between the relative stability of TBPEB dimers in vitromain. Contributions from DNA binding and TBP dimer- and the level of basal transcription of a reporter geneization were examined through use of the EB mutants. in vivo (Jackson-Fisher et al., 1999). In contrast, less ofThese factors are distinguishable in that DNA plays a a correlation was observed for TBPEB/TAND stability.positive role and dimerization plays a negative role in For example, although V71E destabilized TAND bindingregulating gene expression. NC2 was examined using in vitro, and V71R did not, V71R showed substantiallya TBP mutation (F182V) that abolishes NC2 interactions. higher levels of basal transcription in vivo than V71E.

Through microarray analysis, we find that expression Therefore, the previously observed elevated basal tran-of approximately 40% of the yeast genome is sensitive scription is unlikely to be attributed to a loss of TBP/to either mutations along TBP’s concave surface or a TAND interactions. This conclusion is confirmed by themutation that affects NC2 binding, particularly when the observation that deletion of the TAND domain did notTAND domain of TAF1 is absent. The affected genes significantly enhance basal transcription of the samecluster into four major groups, which show distinct sen- reporter gene (data not shown). As shown below, thesitivities to the various mutations. The first group of

TBPEB mutants lead to transcription derepression evengenes is highly expressed and appears to be sensitive

in the absence of the TAF1 TAND domain. Moreover,to TBP/DNA stability. Interestingly, expression of this

the TBPEB mutants (except V71E) were generally notgroup appears to be attenuated primarily by NC2 anddefective in coimmunoprecipitation of TAF1 in wholepartially by the TAF1 TAND domain. The second groupcell extracts (see Supplemental Figure S1 at http://is positively regulated by TAND, particularly when TBP/www.molecule.org/cgi/content/full/10/4/871/DC1).DNA interactions are compromised. The third group is

negatively regulated by at least two seemingly redun-TBPEB Mutations Are Synthetically Toxic withdant factors: the TAF1 TAND domain and an unidentifieda Deletion of the TAF1 TAND Domainactivity. The fourth group of genes is largely repressed.TBP is inhibited by a variety of factors, including NC2,TBP dimerization and the TAF1 TAND domain contributethe TAF1 TAND domain, and homodimerization. The in-to their repression, with TBP dimerization playing theteraction of these factors along TBP’s surface has beenmore predominant role. These repressed genes aredefined by structural and genetic methods. To examinefound throughout the genome, but are particularly prev-how the function of these various repressors relate toalent in the repressive subtelomeric environment. Theeach other, we sought to selectively eliminate individu-findings presented here suggest that a large portion ofally and in combination their interaction with TBPthe yeast genome is negatively regulated through TBPthrough targeted mutagenesis. The TBPEB mutationsby a collaboration of different combinations of factors,were used to examine TBP’s positive interactions withincluding NC2, TAF1, TBP dimerization, and others.DNA and negative interactions through homodimeriza-Which combination is used might be dependent upon

the expression level of the gene. tion (and potentially TAND interactions). TAND function

Multiple TBP Inhibitory Mechanisms873

Figure 1. Interaction of TBP with Regulatory Factors

(A) Structures of TBP interactions relevant to this study. Shown is the core of yeast TBP interacting with itself (Chasman et al., 1993; Nikolovet al., 1992), the Drosophila TAND I domain (Liu et al., 1998), and TATA DNA plus human NC2 (Kamada et al., 2001). The TFIIA•TBP•TATA•TFIIBstructure is a composite of two structures (Nikolov et al., 1995; Tan et al., 1996). All views are from the same vantage point: upstream of theTATA box looking downstream. Selected amino acids are shown as stick diagrams. The relative affinity of each negative regulator (shown inred) for the relevant TBP mutants is shown below each diagram. Those in parentheses are not significantly different from each other.(B) TBP’s interaction surface with the yeast TAF1 TAND domain. Purified recombinant GST-scTAF1 (10–88) (denoted as TAND), GST-scTAF1(10–88, D66K), or GST-scTAF1(10–88, F23K, D66K) were immobilized on glutathione agarose in the presence of purified his-tagged TBP mutants,as indicated. The resins were washed, and proteins were analyzed for TBP and GST by SDS-PAGE and immunoblotting. 15% of the inputmaterial was loaded where indicated. Relative pull down represents the average of at least three repeats.

was explicitly tested by deleting this domain. NC2 func- basal transcription (Jackson-Fisher et al., 1999). There-fore, the TBP mutants were expressed in the context oftion was probed through use of the TBP F182V mutation.

As an initial indicator of important interactions, we ex- the wild-type chromosomal TBP (SPT15) gene. Sincesome of TBPEB mutants are rapidly degraded in vivoamined the growth properties of cells harboring these

mutations. (Jackson-Fisher et al., 1999), and we wished to expressthem at or near endogenous wild-type levels, they wereThe TBPEB mutants are unable to support cell growth

on their own, but function dominantly to wild-type TBP placed under the control of the highly active inducibleGAL10 promoter on a CEN/ARS plasmid and grown onby inhibiting activated transcription and stimulating

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Figure 2. TBPEB Mutants in Combination with�TAND Display Dominant Synthetic Toxicity

Cultures ([A], wild-type; [B], �TAND), de-scribed in Figure 3, were spotted onto solidmedia containing 2% galactose and incu-bated at 30�C for several days. “Dominantsynthetic toxicity” indicates that the pheno-type was observed in the presence of wild-type TBP and that the growth phenotypeswere severe in the �TAND strain but minor inthe isogenic wild-type strain. The term “toxic-ity” is used since the cells do not die, but dostop growing. Growth was restored when thearrested mutants were transferred to mediacontaining glucose, and it was again inhibitedupon plating on galactose. Similar syntheticgrowth defects have been observed for sev-eral mild TBP mutants that support cell viabil-ity in the absence of wild-type TBP (Koba-yashi et al., 2001).

galactose media. In addition to the mutants, wild-type galactose (Figure 3A). This short window of time wasintended to minimize potential indirect effects, whereand null controls were used. The null mutant is the same

as wild-type except that the first codon was mutated to any initial changes in gene expression lead to subse-quent changes in the expression of other genes. Sincea stop codon.

In galactose media, all the TBPEB mutants except all strains harbor a chromosomal copy of the wild-typeSPT15 (TBP) gene, only dominant effects will be ob-V161E partially inhibited cell growth in the context of

wild-type TAF1 (Figure 2A). Deletion of the TAND domain served.Approximately 2400 genes (�40% of the genome)alone resulted in slow growth and was suppressed by

overexpression of wild-type TBP (Figure 2B, compare changed expression significantly in at least one mutant(see Supplemental Table S1 at http://www.molecule.“null” and “WT”), as previously observed (Bai et al.,

1997; Kotani et al., 1998). Strikingly, complete growth org/cgi/content/full/10/4/871/DC1) when we applied aseries of statistical filters described in the Experimentalinhibition was observed for all TBPEB mutants, except

V161E, when TAND was deleted. Further examination Procedures. The significantly affected genes were clus-tered into four groups by using a K-means algorithm.of V161E revealed that its growth was significantly

slower than wild-type, but not eliminated. Consistent These four groups reflect distinct transcriptional re-sponses to the TBP and TAF1 mutations (Figure 3B, andwith an earlier report (Kobayashi et al., 2001), these

results suggest that TBP’s concave surface and the see Supplemental Table S2 at http://www.molecule.org/cgi/content/full/10/4/871/DC1). Many of the mutationsTAF1 TAND domain serve an overlapping but distinctcaused both an increase and a decrease in transcription.function. Only upon loss of both activities is a severeHowever, F182V (column 20) caused primarily an in-phenotype detected.crease, while V161E (column 1) caused primarily a de-The basis for the synthetic toxicity is not clear. Sincecrease. In total, the pattern of responses suggests thata greater or equal number of wild-type TBP moleculessome of the mutations affect multiple functions of TBP,also are present in the same cell (data presented below),while others are selective.it is unlikely that the TBP mutants are titrating out an

An important concern when interpreting gene expres-important factor that would otherwise work with wild-sion data is assessing indirect effects. Indirect effectstype TBP. However, since the mutants do cause tran-are presumed to arise, in this case, when a mutationscriptional derepression (with V161E being the least ef-directly affects the expression of arbitrary transcrip-fective), it is possible that toxicity is due to inappropriatetional activators and repressors, which subsequentlyexpression of genes.cause increased and decreased expression of othergenes that might not have been directly affected by

Genome-Wide Effects of �TAND and TBP Mutations the mutation. Simultaneous increases and decreases inAs the next step in examining the functional relation- gene expression were not observed for a number ofships among TBP regulatory interactions, we performed mutants, particularly F182V and V161E, indicating thatDNA microarray analysis of TBP mutants in strains har- the expression patterns are likely to be due to directboring either wild-type TAF1 or a �TAND derivative. The effects. Chromatin immunoprecipitation and LexA-strategy involved growing yeast cells in noninducing fusion studies of similar mutations along TBP’s concaveraffinose media and then briefly (45 min) inducing an surface have provided further evidence that these mu-

tants are acting directly on target promoters and engag-HA-tagged version of the TBP mutants by addition of

Multiple TBP Inhibitory Mechanisms875

Figure 3. Microarray Analysis of TBP Mutants in Wild-Type and �TAND Strains

(A) Strains harbored either wild-type or TAF1(�TAND), and the indicated HA-tagged TBP derivatives under control of the GAL10 promoter.TBP expression was induced for 45 min, and equivalent numbers of cells were analyzed for TBP by immunoblotting (Jackson-Fisher et al.,1999). Purified recombinant his-TBP standards are shown.(B) Cluster analysis of gene expression profiles. Cluster and Treeview (Eisen et al., 1998) were used to cluster 2358 significant changes ingene expression (defined in Experimental Procedures) initially into five clusters, using the K-means algorithm. Five clusters were initiallychosen since clusters greater than five were visually redundant. Upon subsequent analysis, it became evident that two of the clustersrepresented similar mechanisms and so were merged to form group 1. Group 1 was then sorted by the values in the F182V column. Eachcolumn represents gene expression changes in the strain designated above each column. Names are colored to signify related mutations.Strains indicated by �TAND contained a deletion of the TAF1 TAND domain, while the remainder were isogenic wild-types. Each rowcorresponds to an expression ratio for a single gene (red � increased expression, green � decreased expression, black � no change, gray �

missing data). The intensity of color correlates to the magnitude of change. The collection of columns were subjected to hierarchical clusteringusing Cluster and Treeview, and the resulting dendrogram is shown above the list of mutants.Several experiments provide a frame of reference. (1) Two independent reference versus reference data sets (null TBP in a wild-type TAF1strain, columns 18 and 19) are indicative of no change. (2) To assess the full reproducibility of the experiments, the V161R experiments (inboth wild-type TAF1 and �TAND strains) were repeated approximately a year apart by different persons (V161RK versus V161RL, columns 4,5, 11, and 12). Of the typically �5700 genes that passed filtering criteria 1 (see Experimental Procedures), correlation coefficients of 0.9 and0.8 for wild-type and �TAND, respectively, were obtained, indicating a high degree of reproducibility.(C) Dendrograms derived from hierarchical clustering of mutants in groups 1 and 4. Portions of the dendrogram that encompass the TBPEB

arginine mutants are boxed in yellow. “�” indicates �TAND.

ing the transcription machinery (Geisberg and Struhl, made from Figure 3B. First, the large number of genesaffected by the TBP mutations indicates that a substan-2000). Further assessment of indirect effects is pre-

sented in the supplemental material at http://www. tial amount of global gene regulation occurs via regula-tion of TBP. Second, the TBPEB mutants behaved simi-molecule.org/cgi/content/full/10/4/871/DC1.

Several general observations and inferences can be larly but not identically, indicating that mutations along

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Figure 4. Dependency of Selected Groups of Genes on the TAF1 TAND Domain

For the indicated groups of genes, log2 ratios of fold changes in gene expression for the indicated mutants in the �TAND strain were plottedas a function of the same mutants in the corresponding wild-type strain. Two groups were plotted in each panel. TAND effects are reflectedas deviations of the data points from the red diagonal.

TBP’s concave surface alter certain interactions but not genes in each group. Models to aid in the discussion ofthe groups are shown in Figure 7. For simplicity, theseothers. Third, since some TBPEB mutations caused both

increased and decreased expression, TBP’s concave models do not include other components of the tran-scription machinery, and they make no inference aboutsurface is likely to be engaged in both positive and

negative interactions. Fourth, deletion of the TAND do- TAF occupancy at the promoters.main had very modest effects on transcription (column15), unless coupled to defects along TBP’s concavesurface (columns 8–14). This type of behavior is indica-tive of functional redundancy between the TAND domainand other factors that interact with the concave surfaceof TBP. Fifth, overexpression of wild-type TBP (column17) caused �0.5% of the genome to significantly changeexpression. The concentration of TBP, per se, thereforeis not limiting for gene expression. Additional genome-wide comparisons of the mutants can be found in Sup-plemental Figure S4 at http://www.molecule.org/cgi/content/full/10/4/871/DC1.

Distinct Gene Expression Groups RevealCombinatorial Interactions of TBP RegulatorsSince the four expression groups shown in Figure 3Breflect distinct response patterns to the mutations, weinvestigated their underlying mechanisms by examiningthem separately. The data were examined in a numberof ways, as shown in Figures 3C, 4, 5, and SupplementalFigure S5 (at http://www.molecule.org/cgi/content/full/10/4/871/DC1). First, the mutants in each group werereclustered separately using a hierarchical method. If

Figure 5. Expression Level of Various Gene Groupsmultiple factors interact along TBP’s surfaces, then TBP

Fold changes in gene expression (log2 ratio) for representative mu-mutations that disrupt certain interactions but not otherstants in each indicated group of genes were plotted as a function

might generate a characteristic transcriptional re- of log10 expression intensity in the reference state (null TBP in asponse. Comparison of this pattern with patterns of in wild-type TAF1 strain). Intensities represent an average from 12

reference hybridizations. The entire nullL dataset is plotted in blackvitro interactions of the mutants might shed light onto provide a frame of reference for the distribution of gene expres-the underlying mechanism. Second, the specific TANDsion intensities. Group 1, represented by the V161RK mutant in thedependency of representative mutants from each groupwild-type TAF1 strain, is plotted in the lower half of the panel (green).was examined quantitatively to assess the influence ofGroup 4, represented by the V161RK mutant in the �TAND strain, is

TAND on gene expression. Third, to examine whether plotted in the upper half of the panel (red). Plots of other groups canparticular regulatory mechanisms direct absolute output be found in Supplemental Figure S6 at http://www.molecule.org/cgi/

content/full/10/4/871/DC1.levels, we examined the absolute expression level of

Multiple TBP Inhibitory Mechanisms877

Group 1 genes decreased in expression upon muta- in group 3), suggesting that positive TBP/DNA interac-tion of the concave surface of TBP (Figure 3B, columns tions are not rate limiting for these genes. TAND nega-1–7 of group 1). Hierarchical clustering of the mutants tively regulated this group only in the presence of the(columns) in group 1 indicated that all TBPEB mutations TBPEB mutants, as evidenced by a leftward deviationin the wild-type TAF1 strain had equivalent negative from the diagonal of group 3 genes in Figure 4A (showneffects on TBP function (Figure 3C, group 1). Previously, in red). All the TBPEB data sets derived from the �TANDwe had shown that all six of these TBPEB mutants are strain appeared to be very similar (reflected by the shal-similarly impaired for TATA binding in vitro, but show low dendrogram branches in Supplemental Figure S5,large differentials in dimerization and TAND binding group 3, at http://www.molecule.org/cgi/content/full/(Jackson-Fisher et al., 1999) (Figure 1B). Therefore, ex- 10/4/871/DC1). This is not the behavior expected frompression of this group of genes correlated best with impaired TBP dimerization. V161R, N69R, and V71R dis-TBP/DNA stability. play severe dimerization defects when compared to the

Deletion of the TAF1 TAND domain by itself had little other EB mutants (V161E, N69S, and V71E), and thuseffect on group 1 expression (Figure 3B, column 15 in they should cluster separately from them. Since this wasgroup 1) but partially suppressed the decreased tran- not observed, we suspect that an additional unidentifiedscription caused by the TBPEB mutations (Figure 4A, negative regulator that interacts with TBP’s concaveshown as a deviation of the green data points from the surface might be in play (Figure 7, model 3), althoughdiagonal). Thus, the TAF1 TAND domain might play a other interpretations are not excluded.negative role at group 1 genes, but this only becomes Group 4 genes clustered furthest to the left in thedetectable when TBP’s positive function (i.e., DNA bind- intensity profiles (Figure 5, group 4 shown in red). Theing) is compromised. median expression level of this group was 20% of the

Strikingly, most of the genes in group 1 appear to be group 1 level, and thus appeared to be generally re-negatively regulated by NC2, since the F182V mutation pressed or only weakly active. Mutations along the con-leads to increased expression (Figure 3B, column 20 in cave surface of TBP lead to increased transcription ingroup 1). Inhibition by NC2 is detectable in the presence both the wild-type and �TAND TAF1 strains (Figure 3B,of TAND, whereas the reciprocal is not true (column group 4). Therefore, positive TBP/DNA interactions do20 versus 15). Therefore, NC2 appears to be a more not appear to be rate limiting for these genes. Like grouppredominant inhibitor than TAND at these genes. Inter- 3, deletion of the TAND domain exacerbated the in-estingly, group 1 genes that are the most sensitive to creased transcription of group 4 (Figures 3B and 4B,the F182V mutation tended to be less sensitive to the red), indicating that TAND is playing an inhibitory role.TBPEB mutations. This suggests that NC2 stabilizes TBP The transcriptional response from the V161R, V71R, andat many group 1 promoters, despite playing a negative N69R mutants clustered very tightly (Figure 3C, grouprole. 4, and see Supplemental Figure S5 at http://www.

We next looked at the absolute expression level of molecule.org/cgi/content/full/10/4/871/DC1), regard-group 1 genes in the reference state (wild-type TAND, less of whether TAND was present, and showed a much“null” TBP overexpression). Fold changes in gene ex- greater transcriptional effect than V161E, V71E, andpression for a representative group 1 mutant (V161R) N69S. The pattern of response is very similar to thewere plotted as a function of absolute expression inten- pattern of impaired dimerization displayed by these mu-sity (Figure 5, green). As a guide for comparison, the tants in vitro (Jackson-Fisher et al., 1999). Therefore, wedistribution of expression intensities for all genes in a suspect that TBP dimerization is contributing signifi-null mutant were also plotted (black). Of the four groups, cantly to the repression of group 4 genes (Figure 7,group 1 represented the most highly expressed set, model 4).clustering to the far right of the expression intensityprofile and having a median expression level �50%

Repressive Subtelomeric Regions Are Intrinsicallyhigher than the next highest group (see SupplementalAccessible to the General Transcription MachineryFigure S6 at http://www.molecule.org/cgi/content/full/Subtelomeric regions as far as 15 kb from chromosomal10/4/871/DC1). Together, the group 1 data suggest thatends tend to be quite repressive for resident genesNC2 is an inhibitor of highly expressed genes (Figure 7,(Aparicio et al., 1991; Grunstein, 1998; Kurtz and Shore,model 1).1991; Kyrion et al., 1993; Loo and Rine, 1995; Zakian,Group 2 genes were also highly active (see Supple-1996). Sir proteins direct subtelomeric silencing out tomental Figure S6 at http://www.molecule.org/cgi/about 3–4 kb, but in regions out to �15 kb, histone H4content/full/10/4/871/DC1). The genes in this groupand presumably other histones direct Sir-independentwere equivalently sensitive to mutations along TBP’srepression (Hecht et al., 1996; Wyrick et al., 1999). How-DNA binding surface (see Supplemental Figure S5 atever, �10% of telomere-proximal genes are dere-http://www.molecule.org/cgi/content/full /10/4/871/pressed upon deletion of SIR3, and �50% are dere-DC1).Unlike group 1 genes, TAND functioned positivelypressed upon depletion of histone H4 (Wyrick et al.,on group 2 genes (Figures 3B and 4B), particularly when1999). Therefore, many telomere-proximal genes mightthe DNA binding surface of TBP was compromised (Fig-be subjected to other modes of repression.ure 7, model 2).

Genes that belong to groups 3 and 4 appear to beGroup 3 genes appeared to be less active than thoseweakly active or repressed and are sensitive to TBPin groups 1 and 2 (see Supplemental Figure S6 at http://inhibition, rather than being sensitive exclusively towww.molecule.org/cgi/content/full /10/ 4/871/DC1).chromatin structure. Therefore, we expected that genesMutations along the concave surface of TBP, in general,

lead to increased transcription (Figure 3B, columns 1–14 in these two groups would not be found in presumably

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(TAND), we also examined the TBP mutants in strainslacking the TAND domain.

The microarray data are interpreted within the contextof established properties of these regulators. Thus, ac-tive genes generally have TBP bound to their promoters;inactive genes generally do not (Kuras and Struhl, 1999;Li et al., 1999). Therefore, transcriptional output gener-ally can be interpreted as a reflection of TBP occupancy.When TBP is bound to active RNA polymerase II promot-ers, where tested, it invariably also has NC2 bound(Geisberg et al., 2001). NC2 stabilizes TBP/TATA interac-tions in vitro and competitively inhibits TFIIB and TFIIAbinding (Cang et al., 1999; Goppelt and Meisterernst,1996; Kim et al., 1997; Mermelstein et al., 1996). WhenTBP is not bound to DNA, evidence suggests that itsDNA binding surface is complexed with inhibitors, suchas the TAND domain of TAF1 (Banik et al., 2001; Kokuboet al., 1998; Nishikawa et al., 1997) or a second moleculeof TBP (Coleman and Pugh, 1997; Jackson-Fisher et al.,1999). All of these observations are well supported bycrystallographic or NMR structures of these interactions(Figure 1A), and these physical structures have been

Figure 6. Subtelomeric Frequency Profile of Group 3 and 4 Genesvalidated using mutagenesis and interaction assays in

Shown is a composite profile of all 32 subtelomeric regions. Thevitro and in vivo.frequencies of nonrepetitive genes that increased in expression in

The main conclusion of this work is that expressiona 50 gene window, tiled every 10 genes, were plotted as a functionof a substantial portion of the yeast genome is regulatedof their average distance from the telomere (Wyrick et al., 1999).

Group 3 is shown in blue and group 4 in red. Also plotted (open in part by the concerted action of a variety of TBP inhibi-circles) is the percentage of genes in the same 50 gene window tors (Figure 7). In particular, we find that NC2 attenuatesthat are in the lowest tenth percentile of genome-wide expression transcriptionally active genes. The TAND domain oflevels.

TAF1 has both positive and negative functions, but thecell does not fully depend upon these functions unlessother TBP interactions are compromised. For repressedchromatin-repressed subtelomeric regions. To addressor lowly expressed genes, our results suggest that TBPthis, the frequency of group 3 and 4 genes was plotteddimerization plays a substantial repressive role. TBPas a function of distance from chromosomal ends (Fig-self-association might keep an otherwise monomericure 6). As a measure of the boundary of the repressiveTBP from binding to repressed genes located withinsubtelomeric region, the frequency of genes in the low-accessible chromatin (including the normally repressiveest tenth percentile of genome-wide expression inten-subtelomeric environment). Our results also indicatesity was also plotted. The weakly expressed genes ofthat an unidentified activity that functions through TBP’sgroup 3 appeared to be generally absent from subtelo-concave surface also inhibits TBP, particularly whenmeric regions, as expected. Surprisingly, group 4 genesTAND function is absent.were quite prevalent and were as frequent as the lowest

tenth percentile of expressed genes throughout the ge-nome. Expression of as much as 30% of the genes in NC2 Attenuates Highly Active Genes

NC2 plays both a negative and positive role in transcrip-the subtelomeres appeared to be sensitive to negativeinteractions along TBP’s concave surface. These find- tion (Cang et al., 1999; Geisberg et al., 2001; Willy et al.,

2000). Its histone fold domain binds to the bent DNAings indicate that the repressive subtelomeric environ-ment is intrinsically accessible to the general transcrip- beneath the TBP/TATA complex (Kamada et al., 2001)

and is required for NC2’s positive and negative functiontion machinery.(Willy et al., 2000). Alpha helices H4 and H5 protrudefrom the core of NC2 (see Figure 1A) and are requiredDiscussionfor NC2’s inhibitory activity (Willy et al., 2000). The H5helix binds TBP’s convex surface and interacts withThe Yeast Genome Is Negatively Regulated in Part

by a Variety of TBP Inhibitors amino acid F182 on TBP, positioning H4 to block TFIIBaccess (Kamada et al., 2001). Consistent with the inhibi-To investigate how TBP interactions contribute to the

global gene regulatory network in yeast, we have cre- tory function of H5, mutation of F182 to valine disruptsNC2 binding and causes an increase in transcription.ated a number of mutations in TBP that differentially

affect TBP binding to DNA, NC2, the TAF1 TAND domain, The properties of NC2 raise a number of questionsabout seemingly paradoxical behavior. First, as a re-and a second molecule of TBP. These mutants were

briefly expressed in an otherwise wild-type TBP strain, pressor, NC2 might be expected to operate at lowlyexpressed or repressed genes, not at highly activeand effects on the expression of individual genes

throughout the genome were examined using DNA mi- genes. If NC2 prevents essential transcription factorslike TFIIA and TFIIB from assembling at a promoter, thencroarrays. To more directly examine the potentially sub-

tle contribution of TAF1’s N-terminal inhibitory domain how can genes that have NC2 bound at their promoters

Multiple TBP Inhibitory Mechanisms879

Figure 7. Models for the Interplay of TBP Effectors in Regulating the Four Groups of Genes Identified in This Study

Positively acting functions are shown in green, and negatively acting functions are shown in red. The thickness of the black equilibrium arrowsreflects the tendency of one interaction to dominate over another.

be actively transcribed? Second, how can a factor act Multiple Inhibitory Interactions along TBP’s ConcaveSurface Provide Redundant Mechanisms fornegatively on one hand and positively on the other, par-

ticularly if the same structural interactions are involved in Preventing Unregulated TranscriptionIn contrast to NC2’s modulation of the accessibility andboth? The latter question is of general interest because

many transcription factors, including TBP, TAF1, and stability of the TBP/DNA complex, TBP dimerization ap-pears to serve a repressive role by keeping TBP off ofNC2, play both positive and negative roles in tran-

scription. the DNA (Figure 7, model 4). Genes that have accessiblepromoter regions are susceptible to being turned onThese apparent contradictions might be reconciled in

the context of a model where transcriptional output is when the dimerization function of TBP is eliminatedthrough mutation, or by the positive action of transcrip-dictated not by an all-or-none binding of factors, but by

the net effect of a dynamic and continuous interplay of tional activators. The buffering effect of dimerizationprovides one explanation as to why overexpression ofpositively and negatively acting factors. While compo-

nents of the transcription machinery may be making wild-type TBP does not lead to increased gene ex-pression.similar interactions regardless of gene expression lev-

els, the relative stability of these interactions may limit The TAND domain of TAF1 exhibits limited inhibitoryeffects on transcription, which might be attributed to atranscriptional output. In particular, dynamic competi-

tion between negatively acting NC2 and positively acting number of mechanisms. First, some genes might not beregulated by TAF1 (i.e., are TAF independent), and thusTFIIA and/or TFIIB (and hence RNA polymerase II holo-

enzyme) for binding to a TBP/DNA complex might limit they are not sensitive to deletion of the TAND domain.Second, TBP must first dissociate from DNA beforetranscriptional output at highly active genes. For other

genes, where weak TBP/DNA interactions might be lim- TAND I can bind TBP (Banik et al., 2001; Kokubo et al.,1998). If TFIIA, TFIIB, and other factors stabilize TBP/iting transcriptional output, NC2 could make a net posi-

tive contribution by stabilizing the binding of TBP to DNA binding, particularly at highly active promoters,then the TAND domain cannot inhibit TBP binding. Muta-DNA (in addition to being antagonistic to TFIIA/B in a

non rate-limiting way). Indeed, NC2 plays a positive role tions along the concave DNA binding surface of TBPthat destabilize TBP/DNA interactions could lead to in-at TATA-less promoters (Willy et al., 2000). Interestingly,

we find that highly expressed genes that are inhibited creased dissociation of TBP (manifested as a decreasein transcription) and increased susceptibility to the in-by NC2 tend to be less sensitive to mutations along

TBP’s DNA binding surface, which suggests that NC2 hibitory action of the TAND domain (Figure 7, models1, 3, and 4). Thus, for group 1 genes, deletion of themight be stabilizing TBP/DNA interactions while none-

theless inhibiting the expression of these genes. If NC2’s TAND domain partially suppresses mutations that impairTBP/DNA interactions (Figure 3B). A third explanationpositive and negative contributions are mutually off-

setting at some promoters, then NC2 might not appear for a lack of a dominant TAND effect is applicable togroup 3 and 4 genes. For these genes, alternative TBPto regulate these promoters despite being bound to

them. Consistent with this, NC2 appears to be bound repressors (an unknown factor for group 3, and possiblydimerization for group 4) might dominate the repressionto all mRNA promoters tested that are also occupied

by TBP (Geisberg et al., 2001). While TFIIB also appears of TBP that is not bound to DNA. Only in the context ofmutations that destabilize these inhibitory interactionsto be bound at the same promoters, the two might not

be bound at the same time and could be in dynamic does the inhibitory function of the TAND domain affecttranscriptional output. Thus, TAND’s potential as a neg-competition.

Molecular Cell880

ative regulator may be widespread, but largely redun- Coleman et al., 1999; Kamada et al., 2001; Kim et al.,1997; Kokubo et al., 1998; Kotani et al., 2000; Mer-dant with other TBP inhibitors.melstein et al., 1996). Activators might also play a directrole in alleviating TBP repression. For example, c-junThe Repressive Subtelomeric Environment Isinteracts with the TAND domain of hsTAF1 to alleviateAccessible to the General Transcription Machinerytranscriptional repression (Lively et al., 2001). In thisHistones and other chromosomal proteins are importantstudy, we have described the interplay of several inhibi-negative regulators of gene expression. Subtelomerictors of TBP. There are likely to be multiple inhibitors atregions are particularly repressive (Aparicio et al., 1991;all stages of the gene activation process. Peeling backGrunstein, 1998; Kurtz and Shore, 1991; Kyrion et al.,each layer of this complex network of regulation should1993; Loo and Rine, 1995; Zakian, 1996). Active geneshelp illuminate some of the underlying mechanisms gov-placed within these regions are often silenced. Silencingerning gene regulation.is due in part to Sir proteins, which are thought to gener-

ate an inaccessible heterochromatin structure emanat-Experimental Proceduresing from the telomeres and extending inward about 3–4

kb along the chromosome (Hecht et al., 1996; Wyrick etGST Pull-Down Assays

al., 1999). Less than 10% of the telomere-proximal genes Pull-down assays were performed as described (Kotani et al., 1998),fall under Sir regulation (Wyrick et al., 1999). Sir-indepen- using 300 nM of the following purified recombinant proteins: GST-dent nucleosomal repression extends to about 15 kb TAF1(10–88), GST-TAF1(10–88, F23K, D66K), GST-TAF1(10–88,

D66K), or GST, and his-yTBP derivatives. TBP derivatives were puri-and is much more prevalent (Wyrick et al., 1999). Consis-fied as described (Jackson-Fisher et al., 1999). Reactions containedtent with this, we find that as many as 40% of the nonre-150 mM KCl, 20 mM Tris-Cl (pH 8.3), 12.5 mM MgCl2, 10% glycerol,petitive genes near the telomeres are in the lowest tenth50 �g/ml bovine serum albumin, and 1 mM dithiothreitol in 100 �l;

percentile of genome-wide expression levels. resins were washed three times, each with 500 �l of reaction buffer.Surprisingly, as many as 30% of the genes in regions TBP was probed by immunoblotting and detected by enhanced

close to the telomeres were classified as group 4. Group chemiluminescence. All reactions were performed at least threetimes, and representative data are shown. TBP was quantitated by4 genes are characterized as being repressed or lowlydensitometric scanning of autoradiograms. Relative pull down wasexpressed due in part to inhibition of TBP. The frequencydetermined by subtracting local background and normalizing to aof occurrence of group 4 genes within subtelomeric re-wild-type TBP pull down present on the same gel.

gions is four times higher than the genome-wide averageof 7% and is about the same as the frequency of the Assay for Synthetic Toxicitylowest tenth percentile of genome-wide expression. The yeast plasmid shuffle strain Y13.2 (MAT� ura3-52 trp1-�63This suggests that many repressed promoters in leu2,3-112 his3-609 �taf145 pYN1/TAFII145), and plasmids pRS314/

TAFII145(WT) and pRS314/TAFII145 (�10–73) were a gift from T. Ko-subtelomeric regions and throughout the genome arekubo (NIST, Japan) (Kokubo et al., 1998). Plasmid shuffling was usedaccessible to TBP/TFIID and the general transcriptionto exchange the endogenous pYN1/TAFII145 plasmid with either themachinery. If repressive nucleosomes nevertheless re-wild-type or �TAND TAF1 plasmids. Plasmids expressing various

side at these promoters, then accessibility might also TBP derivatives under control of the GAL10 promoter have beenrequire chromatin remodeling activities associated with described (Jackson-Fisher et al., 1999) and were transformed intoTFIID and/or the general transcription machinery. We both wild-type and �TAND TAF1 strains. Transformants were se-

lected on CSM-Leu-Trp plus 2% glucose, and subsequently grownfind that lowly expressed genes of groups 3 and 4 showin CSM-Leu-Trp plus 2% raffinose liquid media. At OD600 � 1.0, 10a general sensitivity toward deletion of the TAF1 TAND�l of washed cells, or serial 10-fold dilutions, were plated onto CSM-domain, which might reflect a requirement for TFIID inLeu-Trp plus 2% galactose agar.

delivering TBP to their promoters. Alternatively, if thecore promoters are intrinsically accessible, then the re-

Microarray Analysispressive nature of the subtelomeres might be directed Amplification of 6188 open reading frames (99.4% coverage) of S.at steps upstream of TBP/TFIID recruitment, such as cerevisiae strain S288C was performed as described at http://

cmgm.stanford.edu/pbrown/. All PCR products were confirmed topreventing key gene-specific activator proteins frombe the correct size by gel electrophoresis. Microarray fabricationbinding.was performed on aminosilane glass slides at the Penn State Univer-One study has suggested that the Sir-repressedsity Microarray facility.HMRa1 promoter is occupied by TBP and is repressed

All experiments were performed in derivatives of S. cerevisiaedownstream of TBP recruitment (Sekinger and Gross, strain Y13.2 (described above). The reference strain for all experi-2001). We find that the HMRa1 gene is unaffected by ments contained wild-type TAF1 and a galactose-inducible null de-

rivative of TBP (having a stop codon at position 1): pCALF-T(M1stop)any of our TBP mutants, which is consistent with the(GAL). Strains were grown in CSM-Leu-Trp plus 3% raffinose atnotion that this promoter and many others are not regu-30�C to an OD600 � �0.8. Galactose (2%) was then added for 45lated at the point of TBP access. However, our data domin. Samples for immunoblotting were taken before and after induc-not distinguish whether HMRa1 is regulated before ortion, and equivalent numbers of cells were analyzed (equivalent to

after TBP recruitment. 0.5 ml of OD 1.0). The remainder of the culture was harvested byUltimately, sequence-specific activators control the centrifugation at room temperature, washed, and frozen in liquid

nitrogen over a period of 15 min. Total RNA was extracted by theexpression of most genes. They do so by regulatinghot acidic phenol method (Holstege et al., 1998). Poly(A) RNA waspromoter accessibility, factor recruitment, and competi-isolated using Oligotex resin (Qiagen) according to the manufactur-tion between positive and negative regulators. Part ofer’s instructions. It was then treated with DNase I as describedthe activation process involves removal of TBP inhibi-(Ausubel et al., 1994) and stored in water at 80�C. Poly(A) RNA

tors. Indeed, TFIIA counteracts TBP dimerization and (2 �g) was then reverse-transcribed with aminoallyl-dUTP, followedTBP/TAND, TBP/Mot1, and TBP/NC2 interactions (Au- by incorporation of Cy3 or Cy5. Microarrays were scanned and

quantitated with a GenePix 4000A scanner and GenePix 3.0 softwareble et al., 1994; Cang et al., 1999; Chicca et al., 1998;

Multiple TBP Inhibitory Mechanisms881

(Axon Instruments). All experiments were performed in duplicate Auble, D.T., Hansen, K.E., Mueller, C.G., Lane, W.S., Thorner, J.,and Hahn, S. (1994). Mot1, a global repressor of RNA polymerasefrom independent transformants in which the dyes for the reference

and test samples were swapped. Additional details can be found in II transcription, inhibits TBP binding to DNA by an ATP-dependentmechanism. Genes Dev. 8, 1920–1934.the supplemental material at http://www.molecule.org/cgi/content/

full/10/4/871/DC1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G.,Smith, J.A., and Struhl, K. (1994). Current Protocols in Molecular

Statistical Filtering Biology (New York: John Wiley & Sons).All gene expression ratios from a single array of test versus reference Bai, Y., Perez, G.M., Beechem, J.M., and Weil, P.A. (1997). Structure-were normalized by mode centering, which sets the peak of a function analysis of TAF130: identification and characterization ofsmoothed frequency histogram of the log2 ratios to zero. This a high-affinity TATA-binding protein interaction domain in the Nmethod of normalization assumes that the most frequent ratios re- terminus of yeast TAF(II)130. Mol. Cell. Biol. 17, 3081–3093.flect an unchanging population of mRNAs. Unlike the more common

Banik, U., Beechem, J.M., Klebanow, E., Schroeder, S., and Weil,method of normalizing to total signal, mode centering is insensitive

P.A. (2001). Fluorescence-based analyses of the effects of full-to changes in gene expression. This includes asymmetric changes

length recombinant TAF130p on the interaction of TATA box-bindingwhere only increases or decreases in gene expression are observed.

protein with TATA box DNA. J. Biol. Chem. 276, 49100–49109.Normalization by mode centering was validated in every experiment

Campbell, K.M., Ranallo, R.T., Stargell, L.A., and Lumb, K.J. (2000).by taking an equal number of test and reference cells (measuredReevaluation of transcriptional regulation by TATA-binding proteinby OD600) and spiking in equal amounts of externally generated poly-oligomerization: predominance of monomers. Biochemistry 39,adenylated unique mRNAs (B. subtilis LysA, PheB, ThrC, TrpE,2633–2638.DapB) (Holstege et al., 1998). The spiked controls were processedCang, Y., Auble, D.T., and Prelich, G. (1999). A new regulatory do-along with the total yeast mRNA and hybridized to cognate featuresmain on the TATA-binding protein. EMBO J. 18, 6662–6671.on the arrays. The Cy3/Cy5 ratios of the spiking controls were nor-

malized using the same factor used to normalize the yeast mRNA Chasman, D.I., Flaherty, K.M., Sharp, P.A., and Kornberg, R.D.data. In all cases, their ratios were within 10% of 1.0, which validates (1993). Crystal structure of yeast TATA-binding protein and modelthe assumption that the most frequent ratios reflect an unchanging for interaction with DNA. Proc. Natl. Acad. Sci. USA 90, 8174–8178.population of mRNAs. Cheng, J.X., Nevado, J., Lu, Z., and Ptashne, M. (2002). The TBP-

To assess the intrinsic error from all sources in the microarray inhibitory domain of TAF145 limits the effects of nonclassical tran-experiments, we used 13 independently derived reference versus scriptional activators. Curr. Biol. 12, 934–937.reference hybridizations to assess gene-specific and overall varia-

Chicca, J.J., 2nd, Auble, D.T., and Pugh, B.F. (1998). Cloning andtion when no changes were taking place. Typical results from abiochemical characterization of TAF-172, a human homolog of yeastsingle hybridization are shown in Supplemental Figure S2 at http://Mot1. Mol. Cell. Biol. 18, 1701–1710.www.molecule.org/cgi/content/full/10/4/871/DC1. The standardColeman, R.A., and Pugh, B.F. (1997). Slow dimer dissociation ofdeviation was uniform (�10%) throughout the entire dynamic rangethe TATA binding protein dictates the kinetics of DNA binding. Proc.of all homotypic hybridizations. The greater value of either the gene-Natl. Acad. Sci. USA 94, 7221–7226.specific or overall standard deviation was used to filter each gene for

significant changes in expression. Typically, in any one experiment, Coleman, R.A., Taggart, A.K., Benjamin, L.R., and Pugh, B.F. (1995).�5700 genes gave measurable transcriptional output (passing crite- Dimerization of the TATA binding protein. J. Biol. Chem. 270, 13842–rion 1, below). Fold changes in gene expression were considered 13849.significant if they met all of the following filtering criteria: (1) raw Coleman, R.A., Taggart, A.K., Burma, S., Chicca, J.J., 2nd, and Pugh,gene expression intensities were greater than one standard devia- B.F. (1999). TFIIA regulates TBP and TFIID dimers. Mol. Cell 4,tion above local background in both the test and reference samples 451–457.in both replicates; (2) ratios changed in the same direction in each

DeRisi, J.L., Iyer, V.R., and Brown, P.O. (1997). Exploring the meta-replicate; (3) ratios in each replicate were greater than two standard

bolic and genetic control of gene expression on a genomic scale.deviations above 1.0; (4) p values of the arithmetic average of the

Science 278, 680–686.log2 ratios were �0.005; and (5) fold changes in gene expression

Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998).were �1.5. We chose to use an arithmetic average of log values soCluster analysis and display of genome-wide expression patterns.as to blunt the effect of any potential large variations between theProc. Natl. Acad. Sci. USA 95, 14863–14868.two replicate experiments. Applying these filters to independentlyGeisberg, J.V., and Struhl, K. (2000). TATA-binding protein mutantsderived homotypic hybridizations typically resulted in no genes be-that increase transcription from enhancerless and repressed pro-ing reported as falsely significant. The p value cut-off of 0.005 wasmoters in vivo. Mol. Cell. Biol. 20, 1478–1488.assigned arbitrarily and conservatively. To examine the percentage

of the genome that changes as a function of p value, see Supplemen- Geisberg, J.V., Holstege, F.C., Young, R.A., and Struhl, K. (2001).tal Figure S3 at http://www.molecule.org/cgi/content/full/10/4/871/ Yeast NC2 associates with the RNA polymerase II preinitiation com-DC1. plex and selectively affects transcription in vivo. Mol. Cell. Biol. 21,

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