H4K20me2 SILAC Nucleosome Affinity Purification

Goal of this project was the identification of chromatin interacting proteins whose binding is differentially regulated by di-methylation of lysine 20 on histone H4 (H4K20me2). To achieve this unodified and H4K20me2-modified histone H4 were generated by native chemical ligation and assembled into recombinant di-nucleosomes. The di-nucleosomes were immobilized on streptavidin-coated beads via the biotinylated di-nucleosomal DNA and used for nucleosome affinity purifications to identify proteins regulated by H4K20me2 from SILAC-labelled HeLaS3 nuclear extracts (Arg10 and Lys8). SILAC affinity purifications were carried out in "forward" (heavy extract on modified nucleosome and light extract on unmodified nucleosome) and "reverse" (light extract on modified nucleosome and heavy extract on unmodified nucleosome) label-swap experiments and protein abundances were quantified by MaxQuant.