Distinct roles of the histone chaperones NAP1 and NRP and the chromatin‐remodeling factor INO80 in somatic homologous recombination in Arabidopsis thaliana

Abstract

Homologous recombination (HR) of nuclear DNA occurs within the context of a highly complex chromatin structure. Despite extensive studies of HR in diverse organisms, mechanisms regulating HR within the chromatin context remain poorly elucidated. Here we investigate the role and interplay of the histone chaperones NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) and NAP1-RELATED PROTEIN (NRP) and the ATP-dependent chromatin-remodeling factor INOSITOL AUXOTROPHY80 (INO80) in regulating somatic HR in Arabidopsis thaliana. We show that simultaneous knockout of the four AtNAP1 genes and the two NRP genes in the sextuple mutant m123456-1 barely affects normal plant growth and development. Interestingly, compared with the respective AtNAP1 (m123-1 and m1234-1) or NRP (m56-1) loss-of-function mutants, the sextuple mutant m123456-1 displays an enhanced plant hypersensitivity to UV or bleomycin treatments. Using HR reporter constructs, we show that AtNAP1 and NRP act in parallel to synergistically promote somatic HR. Distinctively, the AtINO80 loss-of-function mutation (atino80-5) is epistatic to m56-1 in plant phenotype and telomere length but hypostatic to m56-1 in HR determinacy. Further analyses show that expression of HR machinery genes and phosphorylation of H2A.X (γ-H2A.X) are not impaired in the mutants. Collectively, our study indicates that NRP and AtNAP1 synergistically promote HR upstream of AtINO80-mediated chromatin remodeling after the formation of γ-H2A.X foci during DNA damage repair.

Emerging roles of chromatin in the maintenance of genome organization and function in plants

“…ChIP assays have revealed that the increase in gene expression occurs concomitantly with the increase in H3K4me3 levels, particularly around the TSS and gene bodies, without changes in the DNA methylation levels [115]. The gene expression changes in response to DNA damage are not affected, even after knocking out the six genes encoding NAP1 and NRP histone chaperones [116]. This indicates that they participate downstream in the pathway, probably during nucleosome remodeling associated with DNA repair.…”

“…This indicates that they participate downstream in the pathway, probably during nucleosome remodeling associated with DNA repair. It has been shown that NAP1 and NRP are required to trigger homologous recombination (HR) before chromatin is remodeled at damaged sites, once γ-H2A.X foci are formed and in an INO80-dependent manner [116]. Recent results show that NRP1 accumulates in chromatin after DNA damage and binds cytochrome c [117] through the NRP1 histone-binding domain [118].…”

Disruption ofNAP1genes supresses thefas1mutant phenotype and enhances genome stability

“…NAP1;1-3 proteins are ubiquitously expressed, form homodimers and heterodimers and localize abundantly to the cytoplasm (Liu et al, 2009). NAP1;4 encodes a shorter protein lacking canonical sequences and is only weakly expressed in roots and pollen grains (Liu et al, 2009; Zhou et al, 2016). Loss-of-function mutations of individual NAP1 genes do not affect plant growth and development under standard laboratory conditions (Gao et al, 2012).…”

“…NRP s are related to the yeast VACUOLAR PROTEIN SORTING (Vps75 and human SET/TAF proteins and participate in nucleosome assembly and histone binding (Zhu et al, 2006; Tang et al, 2008; Bowman et al, 2011; Bowman et al, 2014; Kumar et al, 2019). NAP1 and NRPs act in a synergetic molecular pathway to regulate HR, however, there is a lack of information on which step/s of it they participate in (Zhu et al, 2006; Zhou et al, 2016). The multiple mutants of nap1;1-4nrp1-2 are largely sensitive to genotoxic stress, but are otherwise viable (Zhou et al, 2016).…”

“…NAP1 and NRPs act in a synergetic molecular pathway to regulate HR, however, there is a lack of information on which step/s of it they participate in (Zhu et al, 2006; Zhou et al, 2016). The multiple mutants of nap1;1-4nrp1-2 are largely sensitive to genotoxic stress, but are otherwise viable (Zhou et al, 2016). Unlike NAP1, which is considered to be an H2A/HB histone chaperone shuttling between the cytoplasm and nucleus, NRP proteins are nuclear factors forming homo-and heterodimers and binding all histones in vitro (Liu et al, 2014; Gonzalez-Arzola et al, 2017).…”