Histone H2A.Z is a highly conserved H2A histone variant, which plays a key role in maintaining genomic integrity, the regulation of gene expression, chromosome segregation, and DNA double strand break repair. It does this by recruiting proteins such as transcription factors and remodelling complexes to reorganise the local chromatin structure [1]. We have observed that during G1 phase of the cell cycle, H2A.Z is primarily located at promoters. However, during S and M phase, there is a global shift of H2A.Z to centromeres [2]. Currently, the mechanism by which this process is regulated is unknown.

To investigate the dynamics of the re-distribution of H2A.Z during the cell cycle we used U-2 OS FUCCI-expressing cell lines [3]. Asynchronous U-2 OS FUCCI cells were fractionated into four cell cycle stages: early G1, late G1, Early S, and S/G2/M phase, based on their individual fluorescent biomarker. This system will now enable us to 1) determine which remodelling complexes are involved in evicting and depositing H2A.Z at promoters and centromeres, and 2) identify changes in these complexes or H2A.Z-containing chromatin during the cell cycle which may regulate its differential localisation. We have optimised the affinity purification of One-Strep-tagged H2A.Z and remodeller proteins. Mass spectrometry will be used to identify novel proteins and post-translational modifications associated with H2A.Z or with those known remodellers in complexes identified during each stage of cell cycle. In parallel, ChIP-seq will be performed to determine the localisation of H2A.Z-specific remodelling complexes during the cell cycle, and to pin point the timing of H2A.Z deposition at promoters during G1 phase.

Determining how H2A.Z localisation is regulated will further expand our understanding of the mechanisms of gene regulation by H2A.Z during the cell cycle, and provide a better insight into modes of epigenetic inheritance.