Large-scale chromatin movement during cell division; nucleolar reformation as a model
03 / 2006 - 07 / 2008
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
I propose two parallel approaches to identify which chromatin sequences are nucleolar associated, to evaluate whether they are mobile or remain stably associated with nucleoli throughout interphase and also whether the same chromatin regions are associated with nucleoli from one cell cycle to another. First, I will therefore generate cell lines stably expressing both CFP and photoactivatable GFP fused to core-histone H2B. This model system will allow me to specifically activate nucleolar-associated chromatin, while total chromatin can be observed simultaneously. Second, I aim to identify genomic sequences that are consistently associated with nucleoli but lack rDNA arrays, by shotgun cloning DNA sequences from highly purified preparations of isolated nucleoli. I will use FISH on synchronised cell populations to examine whether these chromatin regions are consistently associated with nucleoli in different cell types and whether this association alters during progression through interphase. Subsequently, tandemly repeated lac operons will be inserted in nucleolar-associated chromatin regions, using knock-in technology in mouse ES cells. These cells will be used to generate cell lines co-expressing GFP-lac repressor, allowing these loci to be imaged in living cells. Large-scale movements of nucleolar-associated chromatin will then be studied throughout the entire cell division cycle using quantitative 4D fluorescence imaging.