How does physical organisation of chromatin regulate gene expression?
Final Report Abstract
We have set out to analyse the impact of the Kcnq1ot1 RNA and various protein factors on the three dimensional structure of the Kcnq1ot1 domain. As ES cells recapitulate well the Kcnq1ot1 silencing profile of the embryonic lineage and provide a model system amenable to manipulation we have derived and characterised a hybrid ES cell line which bears allelic information for the Igf2/H19 and Kcnq1ot1 imprinted domains. We have also established efficient shRNA mediated knock‐down of Ezh2, Rnf2, Ctcf and SatBI. Chromatin modifiers such as G9a or PRC1 and PRC2 complexes, as well as structural proteins like Ctcf and SatB1 have been implicated in the domain‐wide silencing of the domain. With the 4C‐Seq technique fully established in an ES cell based system, we want to compare the physical organisation of the Kcnq1ot1 domain with other regions in the genome. By adapting the assay to an available full genomic hybrid between Mus musculus domesticus and Mus musculus molossinus (B6xMSM) we aim to analyse genome wide and allele‐specific regions monoallelically silenced by coating RNA (e. g. Kcnq1ot1, Igf2r), by other imprinting mechanisms (e. g. Igf2/H19, Gnas) or biallelically expressed regions, particularly with respect to transallelic interactions.
