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. 2007;35(1):193-202.
doi: 10.1093/nar/gkl1059. Epub 2006 Dec 7.

Genome-wide analysis of transcriptional dependence and probable target sites for Abf1 and Rap1 in Saccharomyces cerevisiae

Arunadevi Yarragudi  1 , Laura Wegener Parfrey, Randall H Morse
Affiliations

Genome-wide analysis of transcriptional dependence and probable target sites for Abf1 and Rap1 in Saccharomyces cerevisiae

Arunadevi Yarragudi et al. Nucleic Acids Res. 2007.

Abstract

Abf1 and Rap1 are general regulatory factors (GRFs) that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing and telomere structure in yeast. In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined. Here, we use microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37 degrees C. We then combine this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1. We also identify a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding. Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1. Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1. Finally, we show for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar 'memory effect' that allows continued transcription after loss of Abf1 binding.

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Figures

Figure 1
Figure 1
Microarray analysis of gene expression in rap1 ts or abf1 ts yeast compared to wild-type strains at 37°C. (A) Scatter plots of gene expression at 37°C in single mRNA preparations from wild-type or ts yeast strains, as indicated, plotted against average gene expression from three independent preparations from wild-type strains. Each point represents the expression of an individual gene. Only genes indicated by Affymetrix software as being present (i.e. expressed above background levels of detection) are shown. (B) Number of genes showing changed expression with FDR <0.05 and subsets with increased or decreased expression by 2-fold (left), and the total number of genes showing 2-fold increased or decreased expression without regard for FDR (right). (C) Major functional categories, consensus motifs, and transcription factors associated with differentially expressed genes from ChIP-chip data (6), derived from T-profiler (22). Only motifs or factors having E-value <0.01 are shown, and all were associated with decreased expression for Rap1.
Figure 2
Figure 2
Identification of gene targets of Abf1 and Rap1. (A) Flow chart for identification of direct functional targets. (B) Logo for Rap1 site. (C) Logo for Abf1 site. Total height of letters in each column reflects the total information content for that position, and the height of each individual letter reflects the relative frequency of a specific base at that position. (D) Summary of identified targets, as discussed in text and listed in Supplementary Tables 1–6.
Figure 3
Figure 3
Frequency of Abf1 and Rap1 sites in target genes as a function of distance from ATG. Positions are given for the first nucleotide in the recognition sequence as defined in Figure 2; for promoters with more than one binding site, only the closest site to the ATG was used, so that each promoter was counted only once. Sites are grouped according to the categories discussed in the text and listed in Supplementary Tables 1–6. (A) Rap1 targets; (B) Abf1 targets.
Figure 4
Figure 4
Loss of Abf1 binding in abf1-1 yeast does not result in decreased RPS28A expression. (A) ChIP of FLAG-tagged wild-type or ts Abf1 after 1 h at 37°C. Immunoprecipitated and input DNA were amplified using primers spanning the Abf1 site in the RPS28A promoter (upper panel) and the PCR fragments visualized by gel electrophoresis followed by Southern blotting (middle panel). Lanes are labeled as containing PCR products amplified from input DNA (In), immunoprecipitated DNA (IP), or mock IP without antibody (No Ab). The lane labeled ‘−’ is empty. Quantified results from this and an independent ChIP experiment are depicted at the bottom; SDs (too small to be visible for the FLAG-abf1-1 sample) are indicated. (B) RNA was prepared from wild-type or abf1-1 ts yeast grown at 25°C or for 1 h at 37°C (upper panel), and also from yeast having the Abf1 binding site in the RPS28A promoter mutated (lower panel). RPS28A and PYK1 mRNA were reverse transcribed and amplified by PCR, electrophoresed and visualized by ethidium bromide staining. These experiments were performed at least three times with similar results.
Figure 5
Figure 5
Transcript levels of Abf1-dependent genes at 25°C and after 1 h at 37°C in yeast expressing Abf1(1–592) or the corresponding ts mutant. (A) Growth of yeast expressing Abf1(1–592) or the corresponding ts mutant after 2 days at the indicated temperature on YPD plates. (B) Transcript abundance, measured by northern analysis and normalized to PYK1 mRNA, of TCM1/RPL2, QCR8 and PRO3, from yeast expressing full-length Abf1 (wt) or Abf1(1–592) or the corresponding ts mutants, as indicated, grown at 25°C or for 1 h at 37°C. SDs(n = 2–4) are indicated. Note that relative abundances of transcripts from yeast expressing full-length Abf1 (top) or Abf1(1–592) (lower panel) can be directly compared, as transcript levels were measured on the same blots (see Materials and Methods).
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