Mechanisms of H2A.Z and its Chromatin Remodelling Complexes during the Cell Cycle
Creators: name: Dijkwel, Yasmin
Publisher: name: The Australian National University
Year: 2020

description: The DNA in a cell is packaged into a structure called chromatin. Chromatin structure and function is regulated by epigenetic marks such as the histone H2A.Z. H2A.Z deposition in the genome is regulated by the chromatin remodelling complexes SRCAP and p400. Most research focusses on their roles in transcription, however, the essential roles of H2A.Z go beyond that, as it has roles in the maintenance and inheritance of distinct chromatin states. H2A.Z localises to centromeres, where in mouse TS cells, it becomes enriched during mitosis. Its function at the centromere and deposition mechanism are unknown. The aim of this thesis was to gain further insights into the dynamic roles of H2A.Z and its remodelling complexes during the cell cycle. The first aim was to identify changes in the protein interactome of H2A.Z. I found that ANP32e, an H2A.Z chaperone, preferentially interacts with H2A.Z in the nucleoplasm during G1. ANP32e has been implicated in H2A.Z removal from chromatin, however, in U2OS cells ANP32e does not regulate the chromatin association of H2A.Z. Further, ANP32e is not a stable component of the p400 complex, and is not associated with the chromatin. Instead, ANP32e is mainly localised in the cytoplasm where it maintains the stability of H2A.Z protein. This suggests ANP32e acts as a histone chaperone required for maintaining H2A.Z protein stability in U2OS cells, rather than as a chromatin remodeller. The second aim was to investigate H2A.Z PTM dynamics during the cell cycle. I found that H2A.Z is deacetylated at lysines 4, 7, and 11. This occurs specifically from metaphase to early G1 and may play a role in chromosome condensation or segregation. It is known that histone deacetylation occurs during mitosis. However, H2A.Z de- and re-acetylation occurs later than H3K9ac, suggesting H2A.Z deacetylation may not occur as part of global histone deacetylation. Also, in contrast to H3K9ac, H2A.Zac is partially retained on mitotic chromosomes, suggesting H2A.Zac may play roles in mitotic progression. The third aim was to elucidate the functions of H2A.Z, p400, and SRCAP at centromeres. H2A.Z becomes enriched at centromeres during mitosis in mouse TS cells, and the p400 component TIP60 functions at centromeres during early mitosis. I found that upon TIP60 depletion there is a defect in mitotic progression, however, upon p400 depletion this defect does not occur. p400 does not localise to centromeres during early mitosis, suggesting a TIP60 subcomplex exists independent of p400. But, both p400 and TIP60 are required for H2A.Z acetylation. p400 becomes enriched at centromeres during early G1, but the p400 component and H2A.Z chaperone YL1 does not. This suggests p400 may not deposit H2A.Z but plays a novel role at centromeres. My model is that three subcomplexes of p400 and TIP60 exist. The first complex contains p400, TIP60, and YL1 and is involved in H2A.Z deposition and acetylation. The second complex contains TIP60 but neither p400 nor YL1, and functions at centromeres during early mitosis. The third complex contains p400, but not TIP60 or YL1, and becomes enriched at centromeres during early G1. It does not deposit H2A.Z but remarkably, H2A.Z is required for its deposition. The final aim was to characterise genome-wide binding patterns of H2A.Z and its remodelling complexes during the cell cycle. A significant number of genomic sites are enriched with H2A.Z, YL1, TIP60, or ACTR6 specifically during G1 or G2-M. Interestingly, the promoter regions where H2A.Z is dynamic during the cell cycle overlap strongly with occupancy of YL1. Thus YL1 may be involved in regulating the relocalisation of H2A.Z at promoters during the cell cycle. This thesis has provided new insights into the roles of H2A.Z and its chromatin remodelling complexes in regulating cell cycle progression, and furthers our understanding of the diverse roles of H2A.Z, p400, and SRCAP in the maintenance of chromosomes and inheritance of chromatin states.