Keynote Speeches (1)Mapping genome-wide nucleosome dynamics

Eukaryotic gene expression occurs in the context of chromatin, and maintaining a region accessible to DNA-binding proteins for transcriptional regulation requires active processes that mobilize nucleosomes. Our approach to studying these processes has been to map nucleosome dynamics genome-wide, and we have introduced several different strategies to achieve this goal: (1) To measure relative levels of histone replacement across the genome, we have followed incorporation of the replication-independent histone variant, H3.3, which replaces replication-coupled H3 over the course of the cell cycle. (2) To map histone turnover kinetics directly we have developed a novel method based on metabolic labeling of proteins followed by affinity purification of newly synthesized histone core particles. (3) To map classical 'active' chromatin genome-wide we have applied salt fractionation to intact micrococcal nuclease-treated nuclei. (4) To effectively profile chromatin landscapes at single base-pair resolution, we have developed a simple sequencing library preparation protocol and data display method that we have applied to mapping transcription factors, nucleosome remodelers, nucleosomes and kinetochores from a single dataset. (5) To extend genome-wide chromatin profiling to tissues, we have introduced an affinity-based method for purification of nuclei expressing a nuclear envelope protein under control of a cell-type-specific promoter. Application of these methods to model organism genomes suggests that nucleosome turnover is crucial for epigenetic inheritance of gene activity and for maintaining a single centromere on a chromosome.