Functional Characterization Of Chromatin Remodeling Complex INO80 And Its Subunit Actin-related Protein 5 In Arabidopsis Thaliana

Chromatin remodeling factors are key epigenetic regulators. These factors, usually in the form of protein complexes, modulate chromatin dynamics by utilizing energy from ATP hydrolysis, which include INO80 (Inositol auxotroph 80). INO80 is highly conserved from yeast to human. In these species, INO80 complex is involved in diverse DNA metabolisms as a key regulatory element. However, the function of INO80 in plant development remains unclear. Our lab has previously identified two Atino80 T-DNA insertion mutants. These mutants show pleiotropic phenotype in plant development including dwarf. Through DIC microscopy, I observed the decreased cell size and cell number in leaves of Atino80 mutant. Nevertheless, RT-PCR data revealed that the expression level of cell cycle-related genes were not significantly changed in the Atino80 mutants. Gene Ontology analysis of the mis-regulated genes in Atino80 showed the enrichment of categories related to stress response and defense response. The up and down-regulated genes in Atino80 mutant display statistically significant overlap with H2A.Z body-enriched genes, implying that transcription regulation by AtINO80 may be related to H2A.Z function. Moreover, we also found that the genome integrity in the Atino80 mutant cells is more vulnerable, which is shown by hypersensitivity to genotoxic stress and more DNA damage. In yeast and human, ARP5 is an evolutionarilyconserved nuclear Actin-related protein, and considered as an exclusive core component in INO80 chromatin remodeling complex. Our molecular and genetic analysis showed that AtINO80 and Arabidopsis ARP5 can interact with each other. It was previously reported that Arabidopsis mutants depleting of ARP5 activity display increased stomata in leaves, but the molecular mechanisms remains unknown. RT-PCR analysis revealed that bHLH transcription factor genes SPCH and MUTE, which positively regulate the stomata development, are up-regulated in arp5 mutants. The ARP5 protein can specifically bind histone variant H2A.Z in vitro. Moreover, the enrichment of H2A.Z, but not histone H3, within the SPCH and MUTE chromatin regions is dependent on ARP5 activity, suggesting that ARP5 likely regulates SPCH and MUTE expression by modulating local H2A.Z distribution. All these data suggested that AtINO80 and AtARP5 may exist in a protein complex, and AtARP5 may affect the expression of specific genes by regulating the chromatin distribution of H2A.Z.