Transcription regulation by the human Ccr4-Not proteins

Abstract

Transcription by RNA polymerase II is a highly regulated process. Multiple protein complexes are involved in the regulation of mRNA synthesis. The Ccr4-Not complex regulates transcription at a global level and, most likely, requires other proteins to associate with promoters. The complex is evolutionary conserved from yeast to human. Originally Ccr4-Not subunits have been identified in yeast and it was shown that the yeast core complex is composed of at least nine subunits. Also, nine potential orthologs, called CNOT1-4 and CNOT6-10, have been identified in mammalian cells. This thesis describes two aspects of the human Ccr4-Not complex. For the first time we show the ability of the complex to directly regulate transcription, and we have been determined interactions within the human complex. In chapter 2 we investigated whether the human Ccr4-Not subunits can repress transcription when they were targeted to the HSV TK-promoter. We have set up an artificial promoter recruitment system, because direct gene targets for the human Ccr4-Not complex are not known yet. In this way we were able to measure direct transcriptional effects of the Ccr4-Not components. We observe repression by the CNOT2 and CNOT9 subunits. The repression domain of CNOT2 has been determined and this conserved protein domain was named the Not-Box. This domain is conserved throughout evolution and is also present in CNOT3 and its orthologs and paralogs. The repression is sensitive to the histone deacetylase inhibitor, trichostatin A. The CNOT2 mediated repression can be enhanced in reporter assays when expression constructs for the nuclear hormone receptor co-repressors N-CoR and SMRT or histone deacetylases were added as described in chapter 3. These results suggest that the CNOT2 mediated repression is due to the recruitment of histone deacetylase activity via a N-CoR/SMRT complex. This is supported by the interaction between the Not-Box of CNOT2 and N-CoR. This hypothesis is strengthened by the observation that CNOT1 and N-CoR domains can interact in the yeast two-hybrid assay (Chapter 4). Together this indicates that the N-CoR/SMRT complex functions as a cofactor for the Ccr4-Not complex. Chapter 4 also describes the interactions within the core Ccr4-Not complex and potential interactors of CNOT1.