Specific roles for the Ccr4-Not complex subunits in expression of the genome

Abstract

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit's function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome.

FIGURE 1.

Genome wide analysis of gene expression reveals specific roles for subunits of the Ccr4-Not complex. Total cellular RNA was extracted from the indicated wild-type or mutant cells growing exponentially or 30 min after glucose depletion (post-diauxic) and was analyzed by micro-array experiments using the Affymetrix system. (A) The number of target genes defined in each case is indicated. (B) Hierarchical clustering of gene expression in exponentially growing cells (left panel) or cells grown to the post-diauxic phase (right panel). Each line represents a gene, and the color value indicates the fold change (in log2 scale) between the mutant and the wild type in the same growth condition, except in the left lane of the left panel, where we show changes in expression of the genome in wild-type cells after the diauxic shift.

FIGURE 2.

S1 analysis of specific transcripts confirms specific roles for the Ccr4-Not complex subunits in expression of mRNAs. Total cellular RNA was extracted from the indicated wild-type and mutant strains grown to exponential phase or 30 min after glucose depletion (post-diauxic) as indicated, and 30 μg were analyzed by S1 digestion for the levels of the indicated mRNAs in the different indicated strains. All hybridizations were internally controlled by cohybridization with DED1 and all hybridizations with a given probe were analyzed on the same gel even though not always in the same order, such that in the figure signals for each strain have been in some cases cut and pasted such that the order for each of the strains is in relatively the same position.

FIGURE 3.

Genes involved in specific functions are overrepresented in the pool of genes deregulated in specific mutants of the Ccr4-Not complex. This figure lists a selection of subpopulations of genes identified as families that are significantly overrepresented in target genes for given mutants. Black shows genes that are not affected, red shows genes that are overexpressed, and green shows genes that are underexpressed.