engleski

Use of PUB pulse approach to study high order chromatin structure in human cells

Proper organization of the genome is essential for faithful genome function and. In this work a new methodology PUB (Proximity Utilizing Biotinylation) was tested to study chromatin in proximity of the protein of interest in vivo. It is based on co-expression of the protein of interest, fused with the bacterial biotin ligase BirA together with a histone fused with BAP (biotin acceptor peptide), which is specifically biotinylated by BirA. With the incorporation of BAP histones into chromatin, the chromatin located in proximity of the BirA-fusion of protein of interest is preferentially biotinylated and it can be detected by Western blot, Immunofluorescence ect. Protein Emerin and histones H2A and H3.1 were chosen as an experimental model to validate PUB. This approach could provide valuable information about the state of chromatin around the particular protein of interest and it could help in determinating the epigenetic status of the cells on the level of intra-nuclear localization.

chromatin; PUB; BirA; BAP; intranuclear localization

nije evidentirano