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Originally published In Press as doi:10.1074/jbc.M405156200 on June 22, 2004 Originally published In Press as doi:10.1074/jbc.M405156200 on June 10, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34865-34872, August 13, 2004
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Genome-wide Analysis of ARS (Autonomously Replicating Sequence) Binding Factor 1 (Abf1p)-mediated Transcriptional Regulation in Saccharomyces cerevisiae*

Tsuyoshi Miyake, Justin Reese, Christian M. Loch, David T. Auble, and Rong Li{ddagger}

From the Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908-0733

Received for publication, May 10, 2004 , and in revised form, June 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autonomously replicating sequence-binding factor-1 (Abf1p) is an essential sequence-specific transcription factor in Saccharomyces cerevisiae that participates in multiple nuclear events including DNA replication, transcription activation, and gene silencing. Numerous gene-specific analyses have implicated Abf1p in the transcriptional control of genes involved in a diverse range of cellular functions, leading to the notion that Abf1p acts as a global transcriptional regulator. Here we report findings from a genome-wide comparison of the gene expression profiles in the wild-type and abf1-1 temperature-sensitive mutant. The study identifies a total of 86 Abf1p-regulated genes (1.4% of the genome) of which 50 are activated and 36 are repressed by Abf1p. Interestingly, Abf1p binds to its own promoter in vivo and strongly represses its own transcription, suggesting a potential negative regulatory loop in Abf1p-mediated gene regulation. A comparison of our microarray data with the available databases reveals a significant overlap of genes regulated by Abf1p and those by several general transcription factors such as Mot1p and TAFs (TATA-binding protein-associated factors). Different mutant alleles of abf1 affect Abf1p-mediated transcription in a gene-dependent manner. Furthermore, Abf1p in vivo is associated with the promoter region of most Abf1p-activated but not with that of most Abf1p-repressed genes. Taken together, these results strongly suggest distinct underlying mechanisms by which Abf1p regulates gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autonomously replicating sequence (ARS)1-binding factor-1 (Abf1p) is an abundant, essential DNA-binding protein in Saccharomyces cerevisiae (1–4). Its DNA-binding sites have been found at numerous locations in the yeast genome including the silent mating type loci, ARSs, telomeric X-regions, and the promoter regions of over 80 genes (5, 6) (also reviewed in Ref. 7). Indeed, many of these Abf1p-binding sites have been implicated in mediating multiple nuclear events. For example, the Abf1p-binding site in ARS1 serves as an auxiliary element that supports efficient initiation of DNA replication from the best-characterized origin of replication in S. cerevisiae (8, 9). In addition, the Abf1p-binding site at the HMR-E silent mating-type locus is one of the three important cis-acting elements that collectively confer gene silencing (10, 11). Furthermore, Abf1p-binding sites within many transcriptional promoters have been shown to mediate transcriptional activation of genes that are involved in disparate processes including carbon source regulation, meiosis and sporulation, mitochondrial and ribosomal functions, and more (12–16).

Similar to many site-specific transcription factors, Abf1p can be divided structurally into an N-terminal DNA-binding domain (DBD) (approximately aa 1–500) (17) and a C-terminal activation domain (AD) (aa 604–731) (18). Although the DBD of Abf1p is responsible for recognizing specific DNA sequences, the C-terminal AD confers the stimulatory activity of Abf1p in all three chromosomal events (19). Detailed mutagenesis of the AD indicates the presence of two critical regions named C-terminal sequence 1 (CS1) (aa 624–628) and CS2 (aa 639–662) (19). Although both regions are important for the essential function of Abf1p in supporting cell viability, they seem to play distinct roles in various Abfp1-mediated nuclear events. CS2 is capable of inducing chromatin remodeling, which most likely accounts for its positive role in multiple nuclear events such as transcription and DNA replication (19). In contrast, recent study indicates that CS1 plays an important role in the nuclear localization of Abf1p.2 Despite these findings, the impact of different functional domains of Abf1p on global gene expression remains to be examined.

Abf1p also distinguishes itself from most of the known yeast transcription factors in several aspects. First, as reviewed above, Abf1p stands out as a multifunctional protein that stimulates diverse chromosomal events. Second, unlike many transcription factors that are present in low intracellular concentrations and are dedicated to gene expression under specific physiological conditions (e.g. Gal4p for galactose metabolism), Abf1p is very abundant and apparently involved in gene regulation of diverse metabolic activities. Given the presumed role of Abf1 in global gene regulation, it is intriguing that Abf1p only displays weak trans-activation potential (20, 21) but acts synergistically with other trans-acting factors to stimulate transcription (14). Of note, many of these features of Abf1p are shared by another abundant, essential, and multifunctional protein Rap1p despite their apparent non-overlapping functions in yeast (22).

In this study, we employed a whole genome approach to identify Abf1p-regulated genes in the yeast genome. Our results uncover a surprisingly small number of Abf1p target genes, one of which is ABF1 itself. Despite the general importance of the various functional domains of Abf1p in gene regulation, our finding also indicates a gene-specific effect of these domains on transcriptional regulation. Finally, the Abf1p-controlled gene expression profile shares a significant overlap with those of Mot1p and certain TATA-binding protein (TBP)-associated factors (TAFs), suggesting a functional cooperation between Abf1p and these general transcription factors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Media, Genetic Analysis, and Strains—TMY86 (MATa ade2–1 his3–11,15 leu2–3,112 trp1–1 ura3–1 can1–100 abf1::HIS3MX6 pRS416-ABF1) (19) and its derivatives carrying various abf1 mutant plasmids were used in this study. Standard techniques were used for preparation of yeast media, genetic analyses, and yeast transformation (23).

Plasmids—The K625I mutation was introduced into pRS415-ABF1-(1–662) by site-directed mutagenesis as described previously (24). Plasmids expressing the FLAG-tagged Abf1p and its derivatives were constructed in the following manner. First, a SalI site was introduced between the first and second codon of ABF1 on the shuffling plasmid pRS415-ABF1-(1–662) (19). Second, three tandem repeats of an oligonucleotide (5'-TCG ATT ATA AAG ATG ATG ACG ATA AAG GTG GTG-3') encoding the FLAG tag were introduced at the SalI site of the pBluescript vector (Stratagene). A fragment containing the three tandem repeats was excised by the XhoI-SalI digestion and subsequently cloned into SalI site of the re-engineered shuffling plasmid. To generate the other FLAG-tagged ABF1 mutant constructs, the modified 5'-coding region of the full-length ABF1 shuffling vector was used to replace that of various mutants of ABF1 as described previously (19).

Microarray Hybridization Experiments and Data Analysis—Microarray hybridization and data analyses were described previously (25). Wild-type and mutant strains were cultured in YPAD medium at 30 °C until A600 reached 1.0–1.2. The culture was split into two, and each was mixed with the same volume of YPAD preheated at 30 and 42 °C, respectively, to reach the final temperature of 30 or 36 °C. After a 45-min incubation, the cultures were harvested and processed for further analyses. RNA was isolated, and microarray hybridization was performed in quadruplicate as described previously. The complete ABF1 microarray dataset is available at dir.niehs.nih.gov/microarray/datasets/home-pub.htm. Note that four genes were eliminated from the original microarray data for the following reasons. YOL053C-A and YCLX10C most probably do not represent genuine yeast genes. YGL032C (AGA2) was deleted because the fold of change was <1.8. YCL018W (LEU2) was omitted, because LEU2 was used as a marker for shuffling and it was unclear whether the microarray result of LEU2 reflects chromosomal gene expression.

To measure the overlap between genes affected by Abf1p and genes affected in previously published experiments as shown in Figs. 4 and 5, published data sets were taken from several sources (25–27). A PERL program was written to match hybridization data for genes affected by Abf1p with the hybridization data for those same genes in other published experiments. Clusters and trees were produced from hybridization data using protocols available at slcview.stanford.edu/.



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  		FIG. 4.
Correlations of Abf1p-regulated gene set with those controlled by other transcription regulators. A, a tree view of the results of Abf1p-activated and repressed genes with the change of expression level in abf1-1, taf1, taf9, and mot1 mutants. Red and green represent an increase and decrease of the expression of each gene, respectively, following the shift to the restrictive temperatures. The data of taf1-MT1, taf1-MT2, taf9-MT1, and taf9-MT2 were retrieved from previously published work (26). MT1 and MT2 are independent data from the same mutant. The mot1–42 and mot1–14 data were retrieved from previously published work (25). B, Venn diagram of the Abf1p-activated (upper) and repressed genes (bottom). In all of the Abf1p-activated genes, fractions of TAF1 and/or TAF9-activated genes (>2-fold) are shown. Also shown in the Abf1p-repressed genes is the fraction of those repressed by Mot1 (>2-fold).

 



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  		FIG. 5.
Correlation between Abf1p-regulated gene set and the gene expression profile in cells at stationary phase. Clustering and tree views were made from the abf1-1 and YPD stationary growing cell data sets of Abf1p-regulated genes. YPD stationary data were retrieved from previously published work (27). The lengths of the culturing time following inoculation at A600 = 0.3 are indicated. Clustering is applied only to the genes.

 
RNA Isolation, Northern Blotting, and Chromatin Immunoprecipitation—RNA was isolated by following the methods described previously (25). Fifteen micrograms of total RNA were resolved by formaldehyde-containing agarose gel electrophoresis, transferred to a positively charged nylon membrane, and probed with radiolabeled random-primed DNA probes corresponding to specific genes. Signals were detected by autoradiography. Chromatin immunoprecipitation (ChIP) was performed as described previously (28).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Whole Genome Analysis of Abf1p-regulated Genes—To conduct a genome-wide search for genes that are regulated by Abf1p, total RNA was isolated from wild-type ABF1+ cells and cells harboring a temperature-sensitive abf1 mutant (abf1-1) (29). This mutant allele was chosen, because it causes a complete loss of the DNA binding ability of the protein and thus abolishes the protein function in all of the Abf1p-mediated nuclear events at the restrictive temperature (29). Fluorescence-labeled cDNA was used to co-hybridize microarray chips that contain 6,024 yeast open reading frames. Four independent co-hybridization experiments were performed, and the data were analyzed. 86 genes displayed over a 1.8-fold difference in expression between the wild-type and mutant strains at the 99% confidence level (Supplemental Table I). Among these genes, 50 were expressed at lower levels in the mutant background and thus scored as Abf1p-activated genes (Supplemental Table IA), whereas the remaining 36 were expressed more robustly in the abf1-1 strain and therefore were considered as Abf1p-repressed genes (Supplemental Table IB). The list includes some (e.g. ARO3 (30) and TRP3 (31)) but not all of the previously identified Abf1p-regulated genes (e.g. ADH1 (32) and TUB2 (4)). The Abf1p-controlled gene set represents 1.4% of the genome and entails genes involved in a diverse range of cellular and biological processes in budding yeast including signal transduction (e.g. MFA1), protein biosynthesis (e.g. RIX7, HBS1, RPL5, and RPP1A), stress response (e.g. HSP12 and HSP26), amino acid biosynthesis (e.g. ARO3, PRO3, and TRP3), and phosphate metabolism (IPP1). Intriguingly, the expression of the ABF1 gene itself is significantly increased in the abf1-1 mutant background, indicating a negative feedback loop involved in the ABF1 gene expression.

Effects of Multiple Mutant Alleles of abf1 on Abf1p-mediated Gene Expression—Using Northern blot analysis, we confirmed the effect of ABF1 on the expression of seven Abf1p-activated and five Abf1p-repressed genes. RNA was isolated from the wild-type and abf1-1 mutant cells grown at either the semi-permissive (30 °C) or restrictive temperature (36 °C). As shown in Fig. 1A, the message levels of the genes in the first group were increased to various degrees in the abf1-1 background (compare lane 1 with 2 and lane 8 with 9), consistent with them being Abf1p-repressed genes. In contrast, the transcripts of the second group of genes were significantly reduced in the mutant strain (Fig. 1B), thus corroborating the stimulatory effect of Abf1p on expression of these genes. As controls, the transcription of those genes in the third group (TUB2, ACT1, and SPT15) remained relatively constant in the wild-type and mutant cells at both temperatures (Fig. 1C). It is also worth noting that the effect of abf1-1 on some genes (e.g. TRP3 and ARO3) only manifested at the restrictive temperature (compare the difference between lanes 1 and 2 with that between lanes 8 and 9). This observation is correlated with the microarray findings of relatively small differences in gene expression at these loci between the wild-type and mutant strains (Supplemental Table I).



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  		FIG. 1.
Northern blot analysis of Abf1p-regulated genes. RNA was isolated from log phase cells either growing continuously at 30 °C or shifted to 36 °C for 45 min. Fifteen micrograms of RNA were resolved by agarose gel electrophoresis and transferred to a nylon membrane. The membrane was incubated with the probes indicated on the left side. Groups A and B are Abf1p-repressed and activated genes, respectively (see Supplemental Table I). Group C represents several control genes.

 
Abf1p can be divided structurally into an N-terminal DBD (roughly aa 1–500) and a C-terminal AD (aa 604–731) (17, 18). The severe phenotype of abf1-1, which carries a point mutation in the zinc finger motif and therefore impairs the DNA binding ability of the protein, is in full agreement with the essential role of the DBD in Abf1p-mediated gene regulation (29). The abf1-5 mutant, which harbors a point mutation that leads to a P357L change in the second DNA-binding domain of Abf1p (aa 323–496) (17), displays thermolabile binding to DNA in vitro (29). As expected, Abf1p-dependent gene expression was also significantly affected in the abf1-5 mutant background (Fig. 1, lanes 3 and 10). Detailed mutagenesis of the AD conducted in our laboratory indicates the presence of two critical regions for Abf1p function named CS1 (aa 624–28) and CS2 (aa 644–48) (19). However, the GAL4-tethering approach used in the previous work precluded the assessment of the importance of the C-terminal AD in the expression of native Abf1p-controlled genes. Therefore, as described in detail below, we also examined the impact of several AD mutants on the expression of Abf1p-regulated genes.

Consistent with the previous finding that the last 69 amino acids of Abf1p is dispensable for cell growth (19), Abf1p-(1–662), which lacks these sequences, behaved nearly equivalently to the full-length protein in mediating gene repression and activation (Fig. 1, compare lane 1 with 4 and 8 with 11). A larger C-terminal deletion that includes the CS2 region of the AD, as in the case of Abf1p-(1–643), led to the elevated expression of some Abf1p-repressed genes such as HSP12 and HSP26 as well as overall reduced levels of the Abf1p-activated genes (compare lane 1 with 6 and lane 8 with 13). Truncation of the entire activation domain (aa 1–607) severely impaired the regulation of all of the Abf1p-mediated genes examined (compare lane 1 with 7 and lane 8 with 14), consistent with the previous conclusion that this part of the protein contains an important trans-activation domain (19). Among all of the abf1 mutant alleles examined in the study, a point mutation in CS1-(1–662/K625I) that resulted in impaired nuclear localization exhibited the most striking phenotypes at both temperatures (lanes 5 and 12).2 For instance, the Abf1p-repressed genes in the Abf1p-(1–662/K625I) mutant background were expressed at levels even higher than those in abf1-1. On the other hand, Abf1p-activated gene expression was significantly compromised in the 1–662/K625I mutant strain with some of the genes in this group expressed below detectable levels (e.g. PRO3 and SEC53). This finding clearly demonstrates the significance of the CS1 region in Abf1p-dependent gene regulation.

It is noteworthy that a given mutant allele of abf1 does not appear to have a uniform impact on the different Abfp1-regulated genes within the same group. For example, abf1-1 reduced the expression of PRO3 to a lower level than did abf1-5, whereas the opposite was true for ARO3 and SEC53 (Fig. 1, compare lanes 9 and 10). Likewise, although abf1-1, 1–662/K625I, and 1–607 mutants had similar effects on the expression of IMD1, RPP1A, TRP3, and ARO3, the 1–662/K625I mutant clearly gave rise to a more prominent increase in the steady-state levels of MFA1 and ABF1. The gene-specific effect of the different abf1 mutant alleles may result from distinct interactions of Abf1p with specific promoter sequences and other trans-acting factors.

Distinct Modes of Abf1p Action in Transcriptional Activation and Repression—Given the DNA sequence-specific binding nature of Abf1p, it was of importance to determine whether the Abf1p-mediated gene regulation was due to a direct or an indirect impact of the protein at the corresponding promoters. The Yeast Genome Pattern Matching in the Saccharomyces genome data base indicates a total of 1,787 consensus Abf1p-binding sites (RTCRYNNNNNACG) (29) that are located within the 1,000-base pair region upstream of the initiation codons of 1,497 yeast genes. Among the 50 Abf1p-activated genes revealed by the current microarray study, 39 genes contain a total of 42 putative ABF1-binding sites (Fig. 2A). Furthermore, the majority of the putative binding sites (35) are located between 100 and 250 bp upstream of the initiation codon (Fig. 2B). In contrast, only 6 of the 36 Abf1p-repressed genes contain the consensus Abf1p-binding sites within the 1,000-base pair upstream region examined. A recent genome-wide chromatin immunoprecipitation analysis ("ChIP on Chip") of yeast transcription factors has led to the prediction of 275 (p < 0.001) and 458 (p < 0.005) Abf1p-bound genes, the largest numbers among all of the known site-specific transcription factors in yeast (6). Interestingly, only 5 (p < 0.001) and 12 (p < 0.005) of the predicted genes were identified as the 50 Abf1p-activated genes in the current microarray study. Furthermore, only one of the 36 Abf1p-repressed genes is predicted to be Abf1p-associated at the p < 0.005 value.



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  		FIG. 2.
Analysis of ABF1-binding sites in the 5' -untranslated regions of the Abf1p-activated genes. A, Venn diagram of Abf1p-activated genes. Among all of the Abf1p-activated genes listed in Supplemental Table I, the fractions of those that also have the consensus Abf1p-binding sequence (RTCRYNNNNNACG) within 1,000 base pairs of the 5'-untranslated region are shown. Also shown are the genes that possess Abf1p-binding sites at their promoter regions (with p value <0.005 or 0.001) as reported previously (6). B, diagram illustrating the positions of all of the consensus Abf1p-binding sequences within 1,000 bp of 5'-untranslated regions of the Abf1p-activated genes. The Abf1p consensus sequence (RTCRYNNNNNACG) was identified within 1,000 bp of the 5'-untranslated regions of the Abf1p-activated genes listed in Supplemental Table I using Yeast Genome Pattern Matching in the Saccharomyces genome data base. The position of the center of the consensus sequence is fractioned by the distance from the first codon. The first nucleotide upstream of the initiation codon is set as -1. The height of the column reflects the total number of consensus sequence in each fraction (50 bp). The open columns are the number of the consensus sequences in the Abf1p-activated genes that are also reported by Lee et al. (6) with a p value <0.005.

 
To directly assess the physical association of Abf1p with its target promoters, we performed ChIP using yeast strains harboring FLAG-tagged full-length Abf1p or its derivatives. As shown in Fig. 3, four of five Abf1p-activated promoters examined were associated with the FLAG-tagged wild-type Abf1p (lane 2) and those derivatives carrying the intact DBD (lane 4 for aa 1–662 and lane 5 for aa 1–643). As controls, cells harboring untagged wild-type Abf1p (lane 1) or the tagged DNA-binding defective mutant abf1-1 (lane 3) did not give rise to any significant signals for the specific PCR products. Furthermore, PCR amplification of the ChIP samples for the coding region of the corresponding genes (e.g. ARO3 and IPP open reading frames) did not yield any specific signals, strongly suggesting that the amplified promoter fragments were due to an association of Abf1p with the corresponding promoter regions. Interestingly, the promoter region of the Abf1p-activated gene PRO3, which does not contain any Abf1p consensus binding sites in its promoter region, is not associated with Abf1p in vivo (Fig. 3). In addition, most of the Abf1p-repressed promoters examined did not show any indication of in vivo association with Abf1p in the ChIP assay (Fig. 3). The only exception in this category is the ABF1 promoter, which contains multiple consensus Abf1p-binding sites and is apparently associated with Abf1p to a similar extent as the Abf1p-activated promoters. This finding lends additional support to the notion that Abf1p negatively autoregulates its expression by directly binding to its own promoter. Lastly, Abf1p was clearly associated with the promoter regions of both the TUB2 and ACT1 genes, yet neither gene was identified as the Abf1p-controlled gene in the microarray or Northern blot analysis. Taken together, the ChIP results suggest different modes of Abf1p action in gene activation and repression. Whereas most of the Abf1p-mediated transcriptional activation may be carried out upon direct binding of the regulator protein to the transcriptional promoters, many of the Abf1p-repressed genes may be targeted by Abf1p through an indirect mechanism (also see "Discussion"). In addition, Abf1p association with a given promoter is apparently not sufficient for the transcription factor to exert a major impact on the promoter activity.



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  		FIG. 3.
Chromatin immunoprecipitation assay of FLAG-tagged Abf1p. Following incubation for 40 min at 36 °C, the cultures were harvested and cross-linked. Sheared chromatin from various abf1 allele backgrounds as indicated on the top was immunoprecipitated with an anti-FLAG antibody. The Input and Bound lanes represent the PCR amplification products of the indicated regions (pro., promoter; ORF, open reading frame; DS, downstream of the gene) before and after the immunoprecipitation, respectively. The presence of the FLAG tag in the Abf1p construct is indicated by a plus sign on the top.

 
Correlations with the Actions of Other Transcription Regulators—To determine whether the Abf1p-dependent gene expression profile is correlated with the gene expression profiles of other transcription regulators, the Abf1p-regulated gene sets were compared with a total of 492 different available gene profiles. Figs. 4 and 5 represent comparisons of the microarray data from the current study and the data from previously published work (25–27). We found that many Abf1p-repressed genes (e.g. MFA1, RNP1, HSP12, HSP26, and SSA4) are also repressed by Mot1p, a global transcription regulator that binds TBP and dissociates its interaction with DNA (correlation coefficient 0.367 for mot1–42 and 0.390 for mot1–14) (25). On the other hand, the Abf1p-activated gene set is significantly correlated with genes that are dependent upon several TAFs (26). Even higher correlation coefficients are obtained when only those genes listed with >99% confidence in the ABF1 microarray are used for comparison. For instance, 18 of the 36 Abf1p-repressed genes are also negatively regulated by Mot1p (Fig. 4B; correlation coefficient 0.664 for mot1–14 and 0.653 for mot1–42). In contrast, only 3 of the 50 Abf1p-activated genes are also activated by Mot1p. On the other hand, 18, 20, 26, and 13 of the Abf1p-activated genes have been previously shown to be regulated by TAF1, TAF6, TAF9, and TAF10, respectively (Fig. 4B; correlation coefficients 0.43–0.50 for taf1; 0.45–0.46 for taf6; 0.46–0.48 for taf9; and 0.37–0.38 for taf10). For example, IPP1, SEC53, DPS1, and RPL5 are all activated by Abf1p, TAF1, and TAF9. Taken together, the correlation between the Abf1p-dependent genes and those genes regulated by TBP-associated transcription regulators raises the interesting possibility of a functional link between Abf1p and the regulation of TBP activity.

In addition to the correlations described above, the Abf1p-dependent gene expression profile also shares significant overlaps with several sets of genes that are regulated by specific physiological conditions (27), including yeast extract-peptone ethanol (correlation coefficient 0.301), nitrogen depletion (0.316 for 8 h; 0.319 for 12 h; and 0.305 for 1 day), and yeast extract-peptone-dextrose stationary phase (0.298–0.348 for 12 h to 28 days). In the case of yeast extract-peptone-dextrose stationary culture, the correlation coefficient for the abf1-1-affected genes (>99% confidence) at the log phase is -0.492, whereas it increases with time in the stationary phase and reaches 0.516 at day 28 (Fig. 5). This finding indicates that the gene expression profile in the abf1-1 mutant background shares a significant correlation with that in cells cultured at stationary phase, thus consistent with the notion that Abf1p may lose its transcriptional regulation function in the stationary phase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The essentiality of ABF1 to cell viability, the cellular abundance of the protein, the functional diversity of known Abf1p-regulated genes, and the large number of its consensus binding sites in the yeast genome all argue for a global role of Abf1p in gene regulation. This notion is further bolstered by a recent genome-based ChIP analysis, which shows that, among all of the known site-specific transcription factors in budding yeast, Abf1p is associated with the largest number of promoter regions (6). In this regard, it is somewhat surprising that the microarray experiment only identified a relatively small number of Abf1p-activated genes. The most likely explanation for this apparent discordance is the functional redundancy between Abf1p and other transcription factors bound at the same promoter regions. It is well documented that Abf1p has a relatively weak transcriptional activation potential on its own and that its trans-activation capability manifests in conjunction with other site-specific transcription factors (12). For example, transcription of many genes encoding ribosomal proteins is promoted by Abf1p and two other global regulators, Rap1p and Reb1p, and the combinatorial actions of these transcription factors contribute to the activation of gene expression from these loci (33, 34). Analogous cases for functional redundancy involving Abf1p can be found in initiation of DNA replication from ARS1 and gene silencing at the mating-type loci. In both cases, the stimulatory functions of the Abf1p-binding site become apparent only when one or more of the other cis-acting elements are crippled (9, 35).

In addition to functional redundancy, several other possible scenarios could also account for the disparity between the large number of Abf1p-binding sites in the genome and the relatively few Abf1-controlled genes as revealed in our study. For example, it is conceivable that some Abf1p-regulated genes may not require stable binding of Abf1p to the promoter regions. Such a "hit-and-run" mode of action has been previously suggested for Abf1p (36).3 In such an event, the corresponding genes might have escaped the detection under the current microarray condition. Furthermore, the genes that are only subject to Abf1p regulation under specific physiological contexts or in response to specific environmental cues might not have been uncovered in the current study. It is also possible that the 99% confidence level set for the microarray data analysis is excessively stringent that it excludes additional bona fide Abf1-regulated genes. Alternatively, some of the Abf1p-binding sites located proximal to transcription promoters may actually play a role in other Abf1p-regulated chromosomal events such as initiation of DNA replication from nearby origins of replication. Lastly, the abf1-1 mutant allele may still retain some transcription function at the restrictive temperature, although this is not very likely because the mutant protein completely loses its DNA binding ability at the restrictive temperature and causes rapid cell cycle arrest (29).

In contrast to the Abf1p-activated gene set, most of the Abf1p-repressed genes do not harbor consensus Abf1p-binding sites in their promoter regions. Furthermore, the ChIP analysis failed to detect any significant association of Abf1p with these promoters. These data indicate that Abf1p represses transcription from these promoters through an indirect mechanism, perhaps by up-regulating the expression of a transcription repressor. However, the presence of such putative repressors was not obvious in the Abf1p-activated gene set. In addition, it is worth noting that expression of the Abf1p-repressed genes responds very rapidly to the temperature shift in the abf1-1 mutant background (i.e. 45 min following the shift). Therefore, it is still formally possible that Abf1p directly acts upon the Abf1p-repressed promoters via its interaction with another site-specific transcription factor but that such association is too weak or transient to be detected under the ChIP assay condition. Alternatively, Abf1p may be associated with genomic regions distal to the Abf1p-repressed promoters, thus eluding detection by the ChIP assay that focused on the proximal promoter regions.

Both microarray and Northern blot analyses show that the expression of ABF1 is significantly elevated in multiple abf1 mutant backgrounds, strongly suggesting the existence of a negative autoregulatory loop. Moreover, consistent with a direct role of Abf1p in negatively regulating its own expression, the ChIP experiment indicates that Abf1p is associated in vivo with the ABF1 promoter. In further support of Abf1p-mediated negative feedback regulation, several hypomorphic mutants of Abf1p were expressed at higher levels than the wild-type protein (data not shown). For instance, the K625I mutation in the context of aa 1–662 results in a major defect in Abf1p nuclear localization and the corresponding mutant protein is grossly overexpressed.2 In addition, the observation that more Abf1p-(1–643) is associated with endogenous promoters than the wild-type protein (Fig. 3) is also consistent with Abf1p-mediated negative autoregulation. The exact physiological importance of this negative regulatory loop remains to be elucidated. Because of the multifunctional nature of Abf1p and its global regulatory role in gene expression, an excess amount of Abf1p may cause an imbalanced distribution of Abf1p among the multiple Abf1p-mediated nuclear events. In this regard, it is notable that overexpression of ABF1 in yeast gives rise to a lethal phenotype (29, 37).4

The Abf1p-dependent gene expression profile shows significant correlations with gene sets that are controlled by several other transcription regulators. More specifically, Abf1p-activated gene sets display a strong correlation with those regulated by several TAFs, raising the possibility that Abf1p and these TAFs may stimulate transcription initiation from a common set of promoters. In this regard, it is interesting that the gene expression profile in the abf1-1 mutant shares significant correlations with cells cultured at stationary phase (Ref. 27 and this study) and that depletion of several TAFs also occurs at high cell density or following nutrient deprivation (38). Our results also suggest that the Abf1p-repressed gene subset shares a substantial overlap with the genes that are down-regulated by the global regulator Mot1p. Obviously, this cannot be due to an effect of Abf1p on Mot1p gene expression. Although Mot1 is localized in vivo to the Mot1p-repressed promoters (25), our ChIP experiment did not provide evidence for such a physical association for Abf1p. However, as discussed above, it is still possible that Abf1p acts directly on the repressed promoters over a long range. Under such a scenario, Mot1p and Abf1p could cooperate to repress transcription from the same set of promoters. In this regard, it is worth noting that a relatively large number of the Abf1p-repressed genes contain the consensus TATA element in the promoter regions (42% for the Abf1p-repressed genes versus 13% in the entire genome and 12% among the Abf1p-activated genes) (39). Interestingly, 12 of the 15 TATA-containing Abf1p-repressed genes are also repressed by Mot1p (25). This finding is consistent with the possibility that a functional cooperation between Abf1p and Mot1p is displacing TBP from the TATA-containing promoters.


   FOOTNOTES
 
The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession numbers GSM25879 [NCBI GEO] , GSM25880 [NCBI GEO] , GSM25881 [NCBI GEO] , GSM25882 [NCBI GEO] , and GSE1492 [NCBI GEO] .

* The work was supported by National Institutes of Health Grants GM57893 (to R. L.) and GM55763 (to D. T. A.). 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

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Tables 1, A and B. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, P. O. Box 800733, Charlottesville, VA 22908-0733. Tel.: 434-243-2727; Fax: 434-924-5069; E-mail: rl2t{at}virginia.edu.

1 The abbreviations used are: ARS, autonomously replicating sequence; Abf1p, ARS-binding factor-1 protein; DBD, DNA-binding domain; aa, amino acid; AD, activation domain; CS1 and CS2, C-terminal sequences 1 and 2; TBP, TATA-binding protein; TAF, TBP-associated factor; ChIP, chromatin immunoprecipitation; ABF1, ARS-binding factor 1. Back

2 C. M. Loch, submitted for publication. Back

3 R. Morse, personal communication. Back

4 T. Miyake and R. Li, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Cynthia A. Afshari and Jenny Collins at the NIEHS, National Institutes of Health for excellent technical support in conducting the microarray experiments. We also thank Russell P. Darst and Arindam Dasgupta for their help of data analysis.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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