Functional and genetic analysis of the Ccr4-Not complex in Yeast

Abstract

Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription factors and RNA polymerase II. One of the major obstacles for the transcription machinery is chromatin, which consists of DNA wrapped around an octamer composed of the histone core particle (two molecules of H2A, H2B, H3 and H4) and the linker histone H1. One of the complexes involved in regulation of transcription is the nine-subunit Ccr4-Not complex. Initially, components of this complex were identified as negative regulators of the non-consensus TATA box driven transcription of the HIS3 gene. In addition, some data also suggest an additional positive role in transcription. Recently, Ccr4p and Caf1p were identified as the major cytoplasmic deadenylases in yeast, indicating that his complex functions in both transcription and mRNA turnover. In addition, human CNOT4 displays RING-finger mediated UbcH5B-dependent ubiquitylation activity. To address the physiological cones-quences of the ubiquitylation activity of Not4p, we used Saccharomyces cerevisiae (bakers' yeast) as a model organism. First, site-directed mutagenesis of the RING-finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. Next, we performed synthetic genetic array (SGA) analyses using not4Δ and not4L35A alleles. This indicates involvement of the RING-finger of Not4p in transcription, ubiquitylation and DNA damage responses. In addition, we found overlapping phenotypes for deletions of UBC4 and mutants encoding single amino acid substitutions of the RING-finger of Not4p. Together, the results presented in chapter 2 show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p mediated stress responses in vivo. Furthermore, genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU)-induced replication stress and ionizing radiation. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4, and degradation of its inhibitor, Sml1p. In chapter 3, we investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU-induced Sml1p degradation. We showed, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or MMS treatment. Chromatin immuno-precipitation experiments showed that TBP, RNA polymerase II and Set1p recruitment to the activated RNR3 locus was defective in cells lacking NOT4. Moreover, RNR3 promoter activity was not induced by HU treatment of these cells. Together, these experiments show that induction of RNR gene transcription is defective in ccr4-not mutant strains, providing an explanation for their sensitivity to HU and implicating the Ccr4-Not complex in positive regulation of transcription. Chapter 4 elaborates on this positive role as we found that Ccr4-Not components display genetic interactions with BUR1 and BUR2. These genes were previously shown to be involved in transcription elongation. We found that the genes encoding the Not-proteins are essential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 or H3K79me2/3 levels. In addition, we show that NOT4 is important for ubiquitylation of histone H2B via recruitment of the PAF complex without affecting Bur1/2 recruitment, providing evidence that the Ccr4-Not complex facilitates H3K4 tri-methylation by functioning downstream of the Bur1/2 kinase. Finally, the proteomic and electron microscopy (EM) study of the Ccr4-Not complex described in chapter 5 provides insight in possible regulation of its functions. Multiple phosphorylation sites on Not4p, Not1p and Caf1p were identified. The functional consequences of these modifications are the subject of current work. Overall, the work presented here contributes to our understanding of the function of the Ccr4-Not complex and act as a basis for future experiments to further explore the exact function(s) of this complex in regulation of mRNA deadenylation and transcription