Investigation of the chromatin characteristics driving murine RIF1s association with and compartmentalisation of late-replicating chromatin

Abstract

The mammalian genome is packaged in a highly organised manner to maintain genome integrity over multiple generations. A key aspect of this organisation is the separation of early- and late-replicating chromatin. RIF1 plays a vital role in this by compartmentalising late-replicating chromatin. However, RIF1s role in the mechanisms underlying this chromatin organisation is poorly understood. This study investigates the principles underlying RIF1s association with and compartmentalisation of late-replicating chromatin. Firstly, identifying the chromatin features driving RIF1s association with late-replicating chromatin. Through in vitro investigations of RIF1's affinity for heterochromatin substrates using pull-down and electrophoretic mobility assays, I demonstrate that RIF1 interacts with chromatin through heterochromatin-specific proteins, namely HP1α and Histone H1. Secondly, the study examines the types of molecular interactions that assemble RIF1 foci—revealing that RIF1 and HP1α colocalise in mouse embryonic stem cells (mESCs) during the establishment of the replication-timing programme. Furthermore, biophysical examinations of fluorescently tagged RIF1 and HP1α in mESCs, demonstrate that similar forces maintain RIF1 and HP1α foci. Lastly, this study investigates how RIF1’s interactions change across replication. Analysing changes in the distribution and dynamics of RIF1 across the cell cycle relative to HP1α reveal a loss of heterochromatin-specific RIF1 distribution and an increase in RIF1 mobility following replication. This study furthers the molecular understanding of RIF1’s nuclear distribution, the processes contributing to that distribution and the compartmentalisation of late-replicating chromatin. Importantly, it advances our knowledge of chromatin reorganisation after mitosis and the influence of chromatin compartmentalisation on replication timing.