Functional Identification Of Plant Nucleosome Assembly Protein 1 (nap1) Family Members

Eukaryotic genomes are packaged into chromatin,a regularly repeated structure whose fundamental building block is the nucleosome.Each nucleosome core particle is composed of a histone octamer consisting of two molecules each of the core histones H2A,H2B,H3,and H4,around which approximately 146-147 bp of DNA is wrapped.The assembly of nucleosome is the first step of chromatin assembly which is important for the chromatin structure affecting a broad ranges of biological events including DNA replication,repair,recombination,transcription,cell differentiation, proliferation,and organism development,and is fulfilled with the help of histone chaperones,which are important for the organization and dynamics of chromatin templates,and are involved in the storage,translocation to the nucleus and exchange of histones and their deposition onto the DNA for replication-dependent chromatin assembly.Nucleosome Assembly Protein 1(NAP1) represents the primary chaperone of H2A and H2B and is highly conserved from yeast to human.Through H2A-H2B dimer removal or exchange,or causing nucleosome sliding,NAP1 can change chromatin structure,and thus affects chromatin metabolism,which provides a mechanism for controlling cell differentiation and development properly.Consistent with this view,genetic studies reveal important functions of NAP1 in Drosophila and mouse,in which deletion of a NAP1 gene leads to embryonic lethality.NAP1 is conserved in plants.However,few studies have been done on its property and biological function.My thesis work aimed to characterize NAP1 to reveal its molecular and cellular activities and its biological functions in higher plants.The first part of my thesis focused on the functional analyses of NAP1 family proteins in rice(Oryza sativa) and tobacco(Nicotiana tabacum).The previous work in our labs described the isolation of three cDNAs encoding NAP1 family proteins from the monocotyledon rice(Oryza sativa) and four from the dicotyledon tobacco (Nicotiana tabacum)(Dong et al.,2003.Planta 216,561-570).Intracellular localization analyses showed that some NAP1 family proteins localize in the nucleus whereas others are cytoplasm-localized.I further investigated the details about intracellular localization of rice and tobacco NAP1 family proteins firstly by identification of interacting partners and by examination of the localization of green fluorescent protein-tagged proteins.Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal,we demonstrated that Orysa;NAP1;land Nicta;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B,our results support the current model and provide additional evidence that the function of NAP1 as histone chaperones appears to be conserved in plants.In addition, we showed that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCB1;1,suggesting a role for NAP1 in microtubule dynamics.Interestingly, in spite of their high homology with the above NAP1 proteins,the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm.Moreover,Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence is localized in both the cytoplasm and the nucleus. Finally,we showed that only Orysa;NAP1;3 could be phosphorylated by casein kinase 2αin vitro.However,this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies.Together,our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants.These results described above were published in the journal "Plant Journal".Because of the difficulties of functional analyses of NAP1 proteins in rice and tobacco,and great advantages of the model plant Arabidopsis in genetic analysis,we switched to Arabidopsis for further studies.The second part of my thesis focused on the functional analyses of NAP1 family proteins in Arabidopsis.The Arabidopsis genome encodes 4 NAP1 genes.We found that one of them,AtNAP1;4,is specifically expressed in stamen and a small part of the elongation zone of the root,while the other three NAP1-subgroup genes are universally expressed and their encoded proteins are mostly localized in the cytoplasm.Further,we showed that Arabidopsis NAP1 proteins form homo- and heterodimeric complexes,and bind plant histone H2A and H2B.Although triple mutants of the three universally expressed NAP1 genes, Atnap1;1-1Atnap1;2-1Atnap1;3-1 and Atnap1;1-1Atnap1;2-1Atnap1;3-2,show a normal growth phenotype under our laboratory standard growth conditions,both triple mutants exhibit a moderate UV-C sensitivity as well as reduced efficiency of in vitro repair of UV-damaged DNA.Transcription profilings and ChIP analyses of the mutants showed that AtNAP1 proteins directly targeted to the promoter regions of some nucleotide excision repair(NER) pathway genes and activated expression of these genes.These results suggest that NAP1 family proteins in Arabidopsis act as H2A/H2B chaperones and are involved in modulating NER pathway.Interestingly,transcription profilings of triple mutants also showed that,in addition to some NER pathway genes,several phytohormone and stress responsive genes were misregulated,stimulating us to study the function of NAP1 proteins in the plant responses to diverse phytohormones and stresses.We reported that loss-of-function mutations in AtNAP1;1,2,and 3 resulted in a slight abscisic acid (ABA)-sensitive phenotype.By the constrast,the triple mutant Atnap1;1-1Atnap1;2-1Atnap1;3-2 exhibited pleiotropic and strong ABA-insensitive phenotypes in seedling growth and stomatal movement and led to decreased tolerance of the mutant seedlings to salt stress.Furthermore,the ABA-insensitive phenotypes of Atnap1;1-1Atnap1;2-1Atnap1;3-2 were observed in all Atnap1;3-2 mutant allele-containing mutants and Atnap1;3-2 heterozygote plants.Detailed study revealed that a stable truncated AtNAP1;3 protein(named as AtNAP1;3T) with C-terminal 34 aa of the full-length protein lost was present in all Atnap1;3-2 mutant allele-containing mutants and Atnap1;3-2 heterozygote,and overexpressing this AtNAP1;3T protein in wild-type plants conferred the plant with ABA hyposensitivity, and caused the downregulation of some genes which are involved in ABA signaling. Taken together,our results demonstrated the dominant role of this truncated AtNAP1;3 protein on Arabidopsis responses to ABA and revealed the significant role of the C-terminal acidic region in NAP1's structure and function,providing informations about AtNAP1;3T protein which might be useful for future biotechonological applications.In conclusion,my thesis work has gained no-previously obtained knowledge about the molecular,cellular and functional properties of NAP1 family proteins in higher plants.My results indicate that AtNAP1 proteins can function as modulators between environmental stress and chromatin structure and function.