NAP1-RELATED PROTEIN Research Articles

NAP1-Related Protein 1 (NRP1) has multiple interaction modes for chaperoning histones H2A-H2B

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

NAP1-RELATED PROTEIN1 and 2 negatively regulate H2A.Z abundance in chromatin in Arabidopsis

In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.

The histone methylation readers MRG1/MRG2 and the histone chaperones NRP1/NRP2 associate in fine-tuning Arabidopsis flowering time.

Histones are highly basic proteins involved in packaging DNA into chromatin, and histone modifications are fundamental in epigenetic regulation in eukaryotes. Among the numerous chromatin modifiers identified in Arabidopsis (Arabidopsis thaliana), MORF-RELATED GENE (MRG)1 and MRG2 have redundant functions in reading histone H3 lysine 36 trimethylation (H3K36me3). Here, we show that MRG2 binds histone chaperones belonging to the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) family, including NAP1-RELATED PROTEIN (NRP)1 and NRP2. Characterization of the loss-of-function mutants mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 revealed that MRG1/MRG2 and NRP1/NRP2 regulate flowering time through fine-tuning transcription of floral genes by distinct molecular mechanisms. In particular, the physical interaction between NRP1/NRP2 and MRG1/MRG2 inhibited the binding of MRG1/MRG2 to the transcription factor CONSTANS (CO), leading to a transcriptional repression of FLOWERING LOCUS T (FT) through impeded H4K5 acetylation (H4K5ac) within the FT chromatin. By contrast, NRP1/NRP2 and MRG1/MRG2 act together, likely in a multiprotein complex manner, in promoting the transcription of FLOWERING LOCUS C (FLC) via an increase of both H4K5ac and H3K9ac in the FLC chromatin. Because the expression pattern of FLC represents the major category of differentially expressed genes identified by genome-wide RNA-sequencing analysis in the mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 mutants, it is reasonable to speculate that the NRP1/NRP2-MRG1/MRG2 complex may be involved in transcriptional activation of genes beyond FLC and flowering time control.

Structural Characterization of Arabidopsis thaliana NAP1-Related Protein 2 (AtNRP2) and Comparison with its Homolog AtNRP1.

Nucleosome Assembly Protein (NAP) is a highly conserved family of histone chaperones present in yeast, animals, and plants. Unlike other organisms, plants possess an additional class of proteins in its NAP family, known as the NAP1-related proteins or NRP. Arabidopsis thaliana possesses two NRP isoforms, namely AtNRP1 and AtNRP2, that share 87% sequence identity. Both AtNRP1 and AtNRP2 get expressed in all the plant tissues. Most works in the past, including structural studies, have focused on AtNRP1. We wanted to do a comparative study of the two proteins to find why the plant would have two very similar proteins and whether there is any difference between the two for their structure and function as histone chaperones. Here we report the crystal structure of AtNRP2 and a comparative analysis of its structural architecture with other NAP family proteins. The crystal structure of AtNRP2 shows it to be a homodimer, with its fold similar to that of other structurally characterized NAP family proteins. Although AtNRP1 and AtNRP2 have a similar fold, upon structural superposition, we find an offset in the dimerization helix of the two proteins. We evaluated the stability, oligomerization status, and histone chaperoning properties of the two proteins, for a comparison. The thermal melting experiments suggest that AtNRP2 is more stable than AtNRP1 at higher temperatures. In addition, electrophoretic mobility shift assay and isothermal titration calorimetry experiments suggest histone binding ability of AtNRP2 is higher than that of AtNRP1. Overall, these results provide insights about the specific function and relevance of AtNRP2 in plants through structural and biophysical studies.

AaMps1 protein inhibition regulates the protein profile, nitric oxide, carbohydrate and polyamine contents in embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

The Monopolar Spindle 1 (Mps1) protein is a dual-specificity kinase that plays a critical role in the progression of the cell cycle. Studies on biochemical changes promoted by Mps1 protein inhibition are important for the development of somatic embryogenesis in plants; however, such work has not been previously performed for Araucaria angustifolia. We analyzed the effects of Mps1 protein inhibition on differential protein regulation and the effects of such differential regulation on the carbohydrate, nitric oxide (NO) and polyamine (PA) contents in embryogenic suspension cultures of this species. A proteomic analysis by the shotgun method was performed using mass spectrometry. The PA and carbohydrate contents were determined by high-performance liquid chromatography, while NO was analyzed by fluorescence microscopy. A total of 1518 proteins were identified, and 157 and 162 proteins were down- and up-regulated, respectively, in embryogenic suspension cultures incubated with 10 µM Mps1 inhibitor. Inhibition of the AaMps1 protein affected the abundance of proteins related to cell cycle division by reducing the accumulation of both cell division control protein 48 and NAP1-related protein 2. In addition, the abundance of the sucrose synthase 3 protein was down-regulated, which reduced sucrose contents. Moreover, a significant reduction in endogenous NO and free putrescine contents was observed in the embryogenic suspension cultures treated with the Mps1 inhibitor. These results show that AaMps1 protein inhibition affects the metabolism of proteins, carbohydrates, NO and PAs, which ultimately interferes in the development of somatic embryogenesis in A. angustifolia.

Functional Coordination of the Chromatin-Remodeling Factor AtINO80 and the Histone Chaperones NRP1/2 in Inflorescence Meristem and Root Apical Meristem.

Chromatin structure requires proper modulation in face of transcriptional reprogramming in the context of organism growth and development. Chromatin-remodeling factors and histone chaperones are considered to intrinsically possess abilities to remodel chromatin structure in single or in combination. Our previous study revealed the functional synergy between the Arabidopsis chromatin-remodeling factor INOSITOL AUXOTROPHY 80 (AtINO80) and the histone chaperone NAP1-RELATED PROTEIN 1 (NRP1) and NRP2 in somatic homologous recombination, one crucial pathway involved in repairing DNA double strand breaks. Here, we report genetic interplay between AtINO80 and NRP1/2 in regulating inflorescence meristem (IM) and root apical meristem (RAM) activities. The triple mutant atino80-5 m56-1 depleting of both AtINO80 (atino80-5) and NRP1/2 (m56-1) showed abnormal positioning pattern of floral primordia and enlargement of IM size. Higher mRNA levels of several genes involved in auxin pathway (e.g., PIN1, FIL) were found in the inflorescences of the triple mutant but barely in those of the single mutant atino80-5 or the double mutant m56-1. In particular, the depletion of AtINO80 and NRP1/2 decreased histone H3 levels within the chromatin regions of PIN1, which encodes an important auxin efflux carrier. Moreover, the triple mutant displayed a severe short-root phenotype with higher sensitivity to auxin transport inhibitor NPA. Unusual high level of cell death was also found in triple mutant root tips, accompanied by double-strand break damages revealed by γ-H2A.X loci and cortex cell enlargement. Collectively, our study provides novel insight into the functional coordination of the two epigenetic factors AtINO80 and NRP1/2 in apical meristems during plant growth and development.

Nuclear cytochrome c - a mitochondrial visitor regulating damaged chromatin dynamics.

Over the past decade, evidence has emerged suggesting a broader role for cytochrome c (Cyt c) in programmed cell death. Recently, we demonstrated the ability of Cyt c to inhibit the nucleosome assembly activity of histone chaperones SET/template-activating factor Iβ and NAP1-related protein during DNA damage in humans and plants respectively. Here, we hypothesise a dual concentration-dependent function for nuclear Cyt c in response to DNA damage. We propose that low levels of highly cytotoxic DNA lesions - such as double-strand breaks - induce nuclear translocation of Cyt c, leading to the attenuation of nucleosome assembly and, thereby, increasing the time available for DNA repair. If DNA damage persists or is exacerbated, the nuclear Cyt c concentration would exceed a given threshold, causing the haem protein to block DNA remodelling altogether.

The Histone Chaperone NRP1 Interacts with WEREWOLF to Activate GLABRA2 in Arabidopsis Root Hair Development.

NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) defines an evolutionarily conserved family of histone chaperones and loss of function of the Arabidopsis thaliana NAP1 family genes NAP1-RELATED PROTEIN1 (NRP1) and NRP2 causes abnormal root hair formation. Yet, the underlying molecular mechanisms remain unclear. Here, we show that NRP1 interacts with the transcription factor WEREWOLF (WER) in vitro and in vivo and enriches at the GLABRA2 (GL2) promoter in a WER-dependent manner. Crystallographic analysis indicates that NRP1 forms a dimer via its N-terminal α-helix. Mutants of NRP1 that either disrupt the α-helix dimerization or remove the C-terminal acidic tail, impair its binding to histones and WER and concomitantly lead to failure to activate GL2 transcription and to rescue the nrp1-1 nrp2-1 mutant phenotype. Our results further demonstrate that WER-dependent enrichment of NRP1 at the GL2 promoter is involved in local histone eviction and nucleosome loss in vivo. Biochemical competition assays imply that the association between NRP1 and histones may counteract the inhibitory effect of histones on the WER-DNA interaction. Collectively, our study provides important insight into the molecular mechanisms by which histone chaperones are recruited to target chromatin via interaction with a gene-specific transcription factor to moderate chromatin structure for proper root hair development.

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

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.

NAP1 Family Histone Chaperones Are Required for Somatic Homologous Recombination in Arabidopsis

Homologous recombination (HR) is essential for maintaining genome integrity and variability. To orchestrate HR in the context of chromatin is a challenge, both in terms of DNA accessibility and restoration of chromatin organization after DNA repair. Histone chaperones function in nucleosome assembly/disassembly and could play a role in HR. Here, we show that the NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) family histone chaperones are required for somatic HR in Arabidopsis thaliana. Depletion of either the NAP1 group or NAP1-RELATED PROTEIN (NRP) group proteins caused a reduction in HR in plants under normal growth conditions as well as under a wide range of genotoxic or abiotic stresses. This contrasts with the hyperrecombinogenic phenotype caused by the depletion of the CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) histone chaperone. Furthermore, we show that the hyperrecombinogenic phenotype caused by CAF-1 depletion relies on NRP1 and NRP2, but the telomere shortening phenotype does not. Our analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argues for a role of NAP1 family histone chaperones in nucleosome disassembly/reassembly during HR. Our study highlights distinct functions for different families of histone chaperones in the maintenance of genome stability and establishes a crucial function for NAP1 family histone chaperones in somatic HR.

Arabidopsis NRP1andNRP2Encode Histone Chaperones and Are Required for Maintaining Postembryonic Root Growth

NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) is conserved from yeast to human and was proposed to act as a histone chaperone. While budding yeast contains a single NAP1 gene, multicellular organisms, including plants and animals, contain several NAP1 and NAP1-RELATED PROTEIN (NRP) genes. However, the biological role of these genes has been largely unexamined. Here, we show that, in Arabidopsis thaliana, simultaneous knockout of the two NRP genes, NRP1 and NRP2, impaired postembryonic root growth. In the nrp1-1 nrp2-1 double mutant, arrest of cell cycle progression at G2/M and disordered cellular organization occurred in root tips. The mutant seedlings exhibit perturbed expression of approximately 100 genes, including some genes involved in root proliferation and patterning. The mutant plants are highly sensitive to genotoxic stress and show increased levels of DNA damage and the release of transcriptional gene silencing. NRP1 and NRP2 are localized in the nucleus and can form homomeric and heteromeric protein complexes. Both proteins specifically bind histones H2A and H2B and associate with chromatin in vivo. We propose that NRP1 and NRP2 act as H2A/H2B chaperones in the maintenance of dynamic chromatin in epigenetic inheritance.