The Ccr4-Not Complex: Bifunctional roles in Repression of Steroid Receptor-Mediated Transcription and mRNA Decay?

Cells can respond to environmental factors such as hormones by changing the instructions that are used. These instructions, or genes, are contained in the heritable material, the DNA. When genes are actively expressed, mRNA copies are produced by transcription in some, but not all cells. At a given time, only a sub-set of genes is expressed by a highly regulated and complex process that requires thousands of different protein factors. The precise number of factors involved in transcription is unknown, and the mechanism by which they work is often unclear. Because different cells contain different transcription factors, a characteristic pattern of genes is expressed for each cell type in the human body. Equally important to the activation of transcription, is the switching off of genes. This can be achieved in different manners. For example, factors can bind to DNA and repress transcription. Alternatively, factors can inactivate gene expression by destroying the mRNA copies of genes. The Ccr4-Not factor can potentially influence gene expression in both ways: by repressing transcription and by destruction of mRNA. Recently, we showed that this factor can interact with estrogen receptor. Estrogen receptor is a transcription factor that mediates the cellular effects of the steroid hormone estrogen. Binding of the hormone to estrogen receptor causes a structural change of the receptor which leads to activation of the receptor. As a result, the expression of several hundreds of genes is altered. Some genes are switched on and are actively transcribed in response to the hormone, such as several genes that promote cell division. Other genes are switched off in response and become repressed, including genes that inhibit cell division. The proposed work will investigate how Ccr4-Not will influence cellular gene expression in response to estrogen. It will do so by specifically engineer cells that lack subunits of Ccr4-Not. Using DNA microarray technology, the expression of >30,000 genes, the majority of human genes, will be determined in a single experiment. The resulting large data sets obtained using normal cells and cells lacking functional Ccr4-Not will be compared using bioinformatics. This will allow to define how important Ccr4-Not is in the cellular response to estrogen. In addition, the proposed work will determine to what extent Ccr4-Not uses its capacities to inhibit transcription and destroy mRNA to influence gene expression in response to estrogens. This will be done in cell lines that lack either the transcriptional repression function, or the mRNA destruction function of Ccr4-Not. Finally, because it is known that estrogens can stimulate cell proliferation, it will assess how Ccr4-Not can have an effect on cell division. The results will increase our basic understanding of the molecular machinery in cells. Furthermore, they may be of interest to the pharmaceutical industry, because there are several drugs that can bind to estrogen receptor and influence its activity in different ways as compared to the natural hormone estrogen. These drugs are successfully used for the treatment of osteoporosis, severe post-menopausal symptoms and cancer, but concerns about efficacy and safety remain. A better understanding of the workings of estrogen receptor may eventually result in the development of safer drugs.

Estrogen receptor alpha (ER) is a nuclear receptor (NR) that mediates the cellular effects of estrogens as a ligand-regulated transcription factor. Mechanisms by which ER activates gene transcription are relatively well understood. While it is known that NRs can associate with transcriptional repressors in the absence of ligand, it is becoming increasingly clear that these factors also associate in a ligand-dependent manner with negative cofactors. The significance of this remains elusive and the mechanisms by which these corepressors function are poorly understood. Recently, we identified the human Ccr4-Not complex as a ligand-dependent repressor of ER. The Ccr4-Not complex is a conserved regulator of transcription and cytoplasmic mRNA decay. The proposed work will focus on functional interactions between ER and the Ccr4-Not complex. Using human MCF7 cells, a well-characterised ER-expressing cell line that requires the hormone estradiol for efficient proliferation, knockdown cell lines for several Ccr4-Not subunits will be generated. These cells will be used to assess the influence of Ccr4-Not components on: estradiol-dependent gene expression; the assembly of protein-DNA complexes and the presence of chromatin modifications on estradiol target genes; the assembly of protein-RNA complexes on ER-induced transcripts; and estradiol-dependent cellular proliferation. This work will result in an understanding as to whether the Ccr4-Not complex is generally required for gene expression by ER, or for a particular sub-set of genes. In addition, it will dissect the functions of the Ccr4-Not complex in transcription and RNA decay. Also, it will provide insight into the mechanisms by which the complex acts as a ligand-dependent repressor of estrogen-mediated gene expression. Finally, the study will demonstrate the requirement for Ccr4-Not in cell proliferation. The findings will comprehensively describe the role of the Ccr4-Not complex as a repressor of estrogen signalling.