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Title: Characterisation of KRAB-ZFPs in the mouse
Author: Briers, Stephanie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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In 2001, Sutherland et al. performed a gene-trap screen in mouse ES cells for nuclear proteins. Seven novel KRAB-ZFPs were identified. I investigated the sub-nuclear distribution of the trapped fusion proteins and found that all except one co-localised with KAP-1 at pericentromeric heterochromatin in a subpopulation of cells. Furthermore this sub-population of cells increased when induced to differentiate with retinoic acid. I further investigated the localisation of these fusion proteins by making full-length GFP-tagged constructs with KRAB-ZFP 492, and by producing an antibody to the endogenous protein. KRAB-ZFP 492 mis-localised in the gene-trap screen as AP492 antibody identifies both nuclear diffuse and nucleolar staining in ES cells. This suggests that the zinc finger domain of KRAB-ZFP 492 is required for correct localisation of this protein in vivo. Furthermore, endogenous KRAB-ZFP 492 is not recruited to pericentromeric heterochromatin via KAP-1 upon differentiation. However in differentiated cells, endogenous KRAB-ZFP 492 and NT-2 is found associated with KAP-1 and HP1 proteins in smaller foci. This suggests that KRAB-ZFP target genes may not be recruited to heterochromatin for repression. To gain insight into the functions of KRAB-ZFPs, I generated two mutant KRAB-ZFP mouse lines using gene-trap ES cells. From these mice I was able to detect the tissues in which each KRAB-ZFP was expressed. In 113+/- embryos, fusion protein expression is initially restricted to the heart and thymus. In adults, expression is observed in the thymus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available