Investigating epigenome dynamics the RITE way

Eukaryotic DNA is packaged into chromatin to fit in the limited dimensions of the nucleus. The basic unit of chromatin is the nucleosome, which consists of DNA and an octamer of histones proteins. Chromatin is a dynamic structure that has a profound influence on gene expression. The cell has several ways to alter chromatin structure. Histones can be modified by post-translational modifications, and canonical histones can be replaced by histone variants. A relatively unexplored layer of chromatin dynamics is histone exchange, that is, the replacement of a histone with the same type of histone without a prerequisite change in occupancy. Histone exchange could have several critical functions in the cell (Introduction and general discussion). It can provide the cell with a means to renew the chromatin. To measure histone exchange it is necessary to be able to distinguish between existing and newly synthesized proteins (Chapter 1). Recombination-Induced Tag Exchange (RITE, Chapter 3) is a recently developed genetic pulse-chase assay that is used to detect histone exchange. RITE is compatible with biochemical analyses, genomics studies, and single-cell fluorescent applications. In Chapter 4 RITE is used to investigate the exchange of histone variant H2A.Z. The results suggested that resident H2A.Z in non-replicating cells transiently leaves the chromatin and is recycled. In Chapter 5 accumulation of methylation on aging histone H3 lysine 79 is detected using RITE. The results suggested that this methylation can be used by the cell as a timer mechanism to couple cell cycle-length to changes in modifications on nucleosomes.