Functional Study Of Histone Chaperone AtASF1 In Arabidopsis Thaliana

Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone chaperone. Studies in yeast and animals indicate that ASF1 proteins play important roles in various chromatin-based processes, including gene transcription, DNA replication and repair. While two genes encoding ASF1 homologues, AtASF1A and AtASF1B, are found in the Arabidopsis genome. We find that both AtASFIA and AtASF1B proteins specifically bind histone H3, and are localized both in the cytoplasm and the nucleus. Loss-of function of either AtASFIA or AtASFIB does not show obvious defects, whereas simultaneous knockdown of both genes in the double mutant Atasflab drastically inhibit plant growth and cause abnormal vegetative and reproductive organ development. The Atasflab mutant plants exhibit cell number reduction, S-phase delay/arrest, and reduced polyploidy levels. Selective up-regulation of expression of a subset of genes, including those involved in S-phase checkpoints and the CYCB1;1 gene at the G2-to-M transition, was observed in Atasflab. Furthermore, the Atasflab-triggered replication fork stalling also constitutively activates the DNA damage checkpoint and repair genes, including ATM, ATR, PARP1 and PARP2 as well as several genes of the homologous recombination (HR) pathway. In spite of the activation of repair genes, an increased level of DNA damage are detected in Atasflab, suggesting that defects in the mutant largely exceed the available capacity of the repair machinery.Meanwhile, we prove that AtASFIA and AtASF1B are involved in gene transcription activation in response to heat stress. The Atasflab mutant displays defective basal as well as acquired thermotolerance phenotypes. Heat-induced expression of several key genes, including the HEAT SHOCK PROTEIN (HSP) genes Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, and the HEAT SHOCK FACTOR (HSF) genes HsfA2 are drastically impaired in Atasflab as compared with that in wild type. We find that AtASFlA/B proteins are recruited onto chromatin, and their enrichment is correlated with nucleosome removal and RNA polymerase II accumulation at the promoters and coding regions of HsfA2 and Hsa32. Moreover, AtASF1A/B facilitate H3K56 acetylation (H3K56ac), which is associated with HsfA2 and Hsa32 activation, suggesting an important function of AtASF1A/B in transcription activation via nucleosome removal and H3K56ac stimulation.Taken together, our results show that each of ASF1 homologues in Arabidopsis has histone chaperone activity. The AtASF1-dependent nucleosome assembly and disassembly processes play important roles in multiple chromatin processes, such as DNA replication, DNA repair and heat stress induced gene transcription regulation.