| In mammals, dosage compensation of X-linked genes between sexes is achieved by X-chromosome inactivation in female cells. X chromosome inactivation is initiated early during development of the embryo and involves many different epigenetic mechanisms resulting in a transcriptionally inactive X chromosome. The inactive state of the X chromosome is normally highly stabile and propagated through many cell divisions. Reactivation of the inactive X chromosome in female cells rarely happens but has been reported in several forms of cancer. In addition, X chromosome aneuploidies are frequently found in different cancer types. However, a clear role for additional copies of an active or partially active X chromosome in the onset and progression of cancer is unclear to date. In this proposal we describe experiments that may shed light on the role of the X chromosome in cancer development. First we will analyze the amount and activity of X chromosomes in breast and ovarian cancer. Secondly, we will generate a mouse model in which we can reactivate the inactive X chromosome. With this mouse model we can determine if there is a causal role for X chromosome reactivation in cancer development. In mammals dosage compensation of X linked genes is achieved by inactivation of one X chromosome in female cells. X chromosome inactivation (XCI) is a very complex process, which is initiated early during development. XCI starts with counting the amount of X chromosomes in a nucleus, if the number of X chromosomes exceeds one, all but one is chosen to be inactivated. This choice process is random and after the choice has been made a non-coding X-linked gene, XIST, is upregulated and coats the future inactive X (Xi). XIST most likely recruits different chromatin components and chromatin modifiers which initiate and establish epigenetic changes rendering the Xi transcriptionally inactive. These changes include modifications of the histone octamer and histone code, like hypermethylation of lysine residues 9 and 27 of histone H3 and hypo-acetylation of histone H4. In addition, the Xi accumulates the histone variant macroH2A and CpG islands on the Xi become hyper-methylated (For review see Plath 2002). In most cells the Xi is visible as a dense stainable structure in the nucleus, called the Barr body. The epigenetic state of the Xi is highly stable and maintained through many cell divisions. Reactivation of the Xi rarely occurs in vivo. Translocation studies and conditional deletion of the Xist gene have demonstrated that Xist is partially dispensable for maintenance of the inactive state, indicating that besides XIST other mechanisms are sufficient to keep the Xi silent (Cattenach 1981, Csankoszki 1999). Define the role of X chromosome aberrations and reactivation of the inactive X chromosome in cancer development, and help establish new diagnostic and therapeutic tools to detect and treat cancer. Female mammalian cells inactivate one X chromosome in order to generate equal expression levels of X linked genes between sexes. X-chromosome inactivation results in a Xi which epigenetic state is stable and maintained through many cell divisions. Reactivation of the Xi rarely occurs in vivo. Recently, several studies have indicated that reactivation of the inactive X chromosome or X chromosome multiplications correlate with cancer development. The experiments described in this proposal allow us for the first time to test a functional relationship between disproportional expression of X linked genes and the development of cancer. Screening of breast cancer and ovarian cancer for X chromosome abnormalities will help establish new diagnostic tools to identify different prognostic subpopulations with distinct clinical implications. In addition, the studies described in this proposal will contribute to the understanding of how epigenetic chromatin domains are assembled and propagated through cell divisions, and may shed more light onto mechanisms of gene activation. The department of Cell Biology and Genetics provides an unique combination of knowledge, tools and facilities to address the questions described in this proposal. Most notably, the department has excellent tissue culture and mouse facilities. The research group of Dr Gribnau has a lot of experience with subjects related to the questions described in this proposal. In addition, the lab has a lot of experience with the different techniques required to manipulate the mouse genome. 1. X inactivation in breast and ovarian cancer cell lines Familial breast cancer patients are predisposed to develop breast cancer due to pre-existence of germ-line mutations in genes that cause breast cancer. Up till now two breast cancer predisposing genes, BRCA1 and BRCA2, have been identified. Mechanistically, however, the function of these genes is much less understood. It is generally thought that loss of specific DNA repair functions causes cancer predisposition in BRCA1 and BRCA2 carriers. Interestingly, BRCA1 has also been shown to colocalize with the inactive X chromosome, and breast and ovarian cancers with BRCA1 mutations lack colocalozation of the XIST RNA to the inactive X chromosome (Ganesan 2002). Furthermore expression analysis of ovarian tumours with BRCA1 or BRCA2 mutations, indicated upregulation of several X linked genes in samples with BRCA1 mutations (Jazaeri 2002). All together this suggests a role for BRCA1 in the maintenance of the inactive X chromosome, and indicates that reactivation of the Xi may result in the development of cancer. |
