Cells need to inherit their chromosomes accurately. Errors in segregation cause aberrant chromosomal composition and thus disease. The centromere, a discrete locus on each chromosome, is vital in this process, but its composition and structure remain largely unknown. How does a cell know where to form a centromere? What is the architecture of this large molecular assembly? Molecular and genomic studies of the centromere have been hampered by the occurrence of repetitive a-satellite DNA on which centromeres normally form.
This problem does not exist in neocentromeric cell lines which contain additional chromosomes that lack a-satellite DNA. This implies that epigenetic modifications such as histone methylation may determine centromere identity. Neocentromeres are thus an excellent model to identify what determines centromere identity. We will generate high-resolution genomic microarrays (CHIPs) covering neocentromeric regions. Using chromatinimmunoprecipitations (ChIP), we will produce a map of centromeric components and epigenetic modifiers at a resolution closeto that of individual nucleosomes. We will use RNA interference assays to alter the protein levels of key centromeredeterminants, which will allow us to probe the hierarchical organization of centromere assembl y, and use the CHIPs to detect novel RNA transcripts from the centromeric regions.
There is no epigenetic map and no insight into the molecular organization of a human centromere. Our novel project may finally get at these important questions. Our results have implications for the design of novel anticancer drugs through the targeting of centromeres (rather than microtubules), and for generating shuttle vectors for livestock and crop engineering, as well as human gene therapy. Further, we will establish novel Ch IP on CHIP technology of immediate appeal for human genome research within the European Research Area.
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