H2B chaperones: from molecules to chromatin-based functions in plant growth and development.

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Received Date: 15-Feb-2015 Revised Date: 10-Mar-2015 Accepted Date: 11-Mar-2015 Article Type: Review Article Special Issue: Chromatin and Development

Histone H2A/H2B chaperones: from molecules to chromatin-based functions in plant growth and development

Wangbin Zhou1,#, Yan Zhu1,#, Aiwu Dong1 and Wen-Hui Shen1,2,*

1

State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and

Development, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 20043, PR China 2

Institut de Biologie Moléculaire des Plantes, UPR2357 CNRS, Université de Strasbourg, 12 rue du

Général Zimmer, 67084 Strasbourg Cédex, France

# These authors contributed equally to this work. * To whom correspondence should be addressed: [email protected]

Running head: Histone H2 chaperones

Key words: chromatin remodeling / nucleosome assembly / histone chaperone / histone variant / DNA repair / homologous recombination / epigenetic regulation / plant development

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12830 This article is protected by copyright. All rights reserved.

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Summary Nucleosomal core histones (H2A, H2B, H3 and H4) must be assembled, replaced or exchanged to preserve or modify chromatin organization and function according to cellular needs. Histone chaperones escort histones and play key functions during nucleosome assembly/disassembly and in nucleosome structure configuration. Because of their location at the periphery of nucleosome, histone H2A-H2B dimers are remarkably dynamic. Here we focus on plant histone H2A/H2B chaperones, particularly members of the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) and FACILITATES CHROMATIN TRANSCRIPTION (FACT) families, to expose and discuss their molecular features, properties, regulation and

function.

Covalent

histone modifications (e.g.

ubiquitination,

phosphorylation, methylation, acetylation) and H2A variants (H2A.Z, H2A.X, H2A.W) are also discussed in light of their crucial importance to modulate nucleosome organization and function. We further discuss roles and mechanisms of NAP1 and FACT in chromatin-based processes, such as transcription, DNA replication and repair. Specific functions of NAP1 and FACT become increasingly evident when their distinct roles are discussed in regulation of plant growth and development and in plant response to environmental stresses. Future major challenges remain to define in more detail the overlap/specific roles of different members of the NAP1 family as well as between NAP1 and FACT, and to identify and characterize their partners as well as new family of chaperones to understand histone variant incorporation and chromatin target specificity. This review is part of a special issue: Chromatin and epigenetics at the nexus between cell division, differentiation and development.

Introduction In every eukaryotic cell, genomic DNA is packaged in the form of chromatin within the nucleus. The basic structural unit of chromatin is the nucleosome, consisting of about 1.65 turns of DNA (roughly 145-147 bp in length) wrapped around an histone octamer that is composed of two molecules of each of the core histones H2A, H2B, H3 and H4 (Luger et al., 2012). Within the nucleosome, the (H3-H4)2 tetramer organizes the central ~80 bp of the nucleosomal DNA, and at its periphery the two H2A-H2B dimers bind and further wrap each about 30-40 bp of DNA, constituting the entry/exit points of the nucleosomal DNA access. Histones have diverse post-translational modifications and variant isoforms; together with DNA sequence variations and methylation patterns they contribute to form a plethora of

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nucleosome structure diversities (Fransz and de Jong, 2011; Zentner and Henikoff, 2013; Zhu et al., 2012). Neighboring nucleosomes are connected by short linker DNA segments, forming a fiber structure appeared like a “beads-on-a-string” array under electron microscopy examination. Histone H1 binds at internucleosomal linker DNA and participates in higher-order chromatin compaction. The nucleosome is one of the most stable protein-DNA complexes under physiological conditions. Meanwhile, studies in different organisms, including yeast, plants and animals, have established that histones are in continuous turnover within the chromatin and that the exchange is more rapid for the nucleosomal periphery-located H2A/H2B than for the more inside H3/H4 (Dion et al., 2007; Jamai et al., 2007; Kimura and Cook, 2001; Rosa et al., 2014). The dynamic exchange process can be regulated by and meanwhile impact on histone variant incorporation and/or histone covalent modifications of the chromatin.

Chromatin composition and structure determine all DNA-based processes and mutually dynamic nucleosome assembly/disassembly/reassembly is associated with transcription, DNA replication and repair (Avvakumov et al., 2011; Desvoyes et al., 2014; Ransom et al., 2010; Raynaud et al., 2014; Van Lijsebettens and Grasser, 2014; Zhu et al., 2012). Because of the intrinsic strong electrostatic interactions between DNA and histone molecules, the ordered formation of the nucleosome structure at physiological ionic strength conditions requires the participation of histone chaperones. Histone chaperones shield nonspecific interactions between the positively charged histones and the negatively charged DNA, playing key roles in nucleosome assembly/disassembly processes. Most histone chaperones are conserved in eukaryotes and are classified as either the H3/H4-type or the H2A/H2B-type chaperones. These different types of histone chaperones play specific roles in distinct steps of nucleosome assembly/disassembly (Avvakumov et al., 2011; De Koning et al., 2007; Ransom et al., 2010). During nucleosome assembly, the H3/H4-type histone chaperones first escort two dimers of H3/H4 into an (H3-H4)2 tetramer on DNA and then the H2A/H2B-type chaperones escort H2A-H2B dimers in further assembly. Notably, H3/H4 but not H2A/H2B histones can be deposited directly onto DNA, providing a key control point for the ordered structure formation of the nucleosome. During nucleosome disassembly, the eviction of H2A/H2B occurs first and then H3/H4 can be removed. Recent studies (Furuyama et al., 2013; Rhee et al., 2014) on nucleosome asymmetry and subnucleosomal structures, such as hemisomes/half-nucleosomes (one


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copy of H2A/H2B/H3/H4) and hexasomes (two copies of H3/H4 and one copy of H2A/H2B), imply existence of multiple assembly/disassembly pathways operating to modulate nucleosomal structures.

Based on their nucleosomal positioning, dynamics of H2A/H2B could play several critical roles, including: (1) to accomplish octamer nucleosome assembly; (2) to initiate nucleosome disassembly;

(3)

to

favor

nucleosome

'breathing’ and

position

sliding

via

loosening

nucleosomal/internucleosomal histone-histone and histone-DNA interactions; (4) to exchange nucleosomal H2A-H2B with free histones carrying similar or different modifications or variant histones. Here we focus on the H2A/H2B-type histone chaperones, including NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1), NAP1-RELATED PROTEIN (NRP), and FACILITATES CHROMATIN TRANSCRIPTION (FACT), to expose their characteristic features, molecular activities and functions, and their roles in plant growth and development.

The NAP1-family histone chaperones Features, evolutionary conservation and multiplication of plant NAP1-family proteins Distinct from other well-known chromatin-related factors such as ATP-dependent chromatin remodeling factors or histone methyltransferases that contain specifically recognizable catalytic domains, histone chaperones have no uniform domain or a common sequence structure. Yet, most of histone chaperones are largely conserved during evolution, and NAP1 belongs to such a type. NAP1 was first identified and purified from eggs of Xenopus laevis as a factor that can facilitate assembly of nucleosome structures in vitro (Laskey et al., 1978). Later, homologues of NAP1 have been identified and characterized in various organisms ranging from yeast to human (Zlatanova et al., 2007). The first cDNA encoding a NAP1 homologue in plants was reported in soybean (Yoon et al., 1995). A systematic survey of NAP1-encoding sequences was performed later in several plant species including tobacco (Nicotiana tabacum), rice (Oryza sativa), and Arabidopsis thaliana (Dong et al., 2003; Liu et al., 2009a). In nowadays, it becomes clear that NAP1 is present broadly in the green lineage and there is a multi-amplification of NAP1 isoforms in higher plants (http://www.chromdb.org; Table 1). The single-cell green alga, Ostreococcus.tauri, contains only one NAP1, whereas Arabidopsis or rice each contains four NAP1 homologues. Multi-amplification of NAP1 occurs likely at different times during green lineage evolution, and the multicellular moss Physcomitrella patens


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dominant negative factor (Liu et al., 2009b), we believe that AtNAP1;4 does not have a canonical NAP1 function. In human and mouse, NAP1L1 and NAP1L4 are ubiquitously expressed, whereas NAP1L2, NAP1L3 and NAP1L5 are specifically expressed in neuron cells (reviewed in Attia et al., 2013). Loss of mouse NAP1L2 leads to embryonic lethality from mid-gestation onwards, which is associated with severe neural tube defects and overproduction of neural stem cells (Rogner et al., 2000). While NAP1L2, NAP1L3 and NAP1L5 each have only one exon (Attia et al., 2013), the AtNAP1;4 gene contains several introns but without 5’ and 3’-UTR, suggesting a different evolutionary strategy of mechanisms used for tissue-specific expression of NAP1 genes between animals and plants.

Core histones including H2A are robustly expressed in S-phase of the cell cycle to meet the huge demand of histones during chromatin replication. The investigation of the histone chaperone gene expression during the cell cycle is relevant to its regulatory mechanism for proper histone incorporation. The analysis of expression of some tobacco NAP1 genes in the highly synchronizable tobacco BY2 (Bright Yellow 2) cells revealed that the transcript levels of NAP1 genes as well as the overall NAP1 protein levels are largely constant during the cell cycle progression (Dong et al., 2003). Other uncharacterized tobacco NAP1/NRP genes as well as NAP1/NRP genes in other plant species remain to be examined for their expression during the cell cycle.

Subcellular localization and regulation of NAP1-family proteins Nucleosome assembly/disassembly occurs within the nucleus; localization of NAP1/NRP proteins to different subcellular compartments could have important implication. So far subcellular localizations of tobacco, rice and Arabidopsis NAP1/NRP proteins have been investigated by using fluorescent protein (GFP/YFP) fusion technology or western blot analysis of protein extracts from subcellular fractionation. Interestingly, different NAP1/NRP proteins show varied subcellular localization, and accordingly may be grouped into three categories: cytoplasm, cytoplasm & nucleus, and nucleus (Figure 2). Among the Arabidopsis proteins, all the NAP1-subfamily members are predominantly located in the cytoplasm, whereas the NRP-subfamily members are majorly localized to the nucleus (Gallichet and Gruissem, 2006; Iglesias et al., 2013; Liu et al., 2009a; Zhu et al., 2006). Nevertheless, western blot analysis of subcellular fractions of the Arabidopsis protein extract from young seedlings


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showed that small quantities of endogenous AtNAP1 proteins are detectable in the nucleus (Liu et al., 2009a). Furthermore, AtNAP1;2-GFP was found to accumulate in the nucleus when the Arabidopsis plants had been treated by phosphate deprivation (Iglesias et al., 2013). Cytoplasm localization of YFP-NRP1 and YFP-NRP2 was also detectable in some of the Arabidopsis root cells (Zhu et al., 2006), and retention of NRPs in the nucleus was recently reported to be influenced by elevated temperatures (Ayaydin et al., 2015). Taken together, it becomes clear that the classification of NAP1/NRP proteins into different cytoplasm/nucleus localization categories (Figure 2) is useful for description of their different specificities but should not be taken as an exclusive function definition in different subcellular compartments. Moreover, it is evident that the subcellular localization of at least some plant NAP1/NRP proteins is subjective to environmental growth condition and developmental or cell-type specific regulations. Like the yeast NAP1, plant NAP1-subfamily proteins contain both NLS and NES sequences (Figure 1a). By sequence mutagenesis and treatment of cells with leptomycin B (LMB), a specific nuclear export inhibitor, several plant NAP1 proteins were shown to shuttle between the cytoplasm and the nucleus (Figure 2), including the Arabidopsis AtNAP1;4 (Liu et al., 2009a), the tobacco Nicta;NAP1;1 and the rice Orysa;NAP1;1 (Dong et al., 2005). The yeast NAP1 is also a nucleocytoplasmic shuttling protein (Miyaji-Yamaguchi et al., 2003), and its nucleocytoplasmic shuttling is critical for the histone nuclear import and chromatin assembly (Keck and Pemberton, 2013). Despite of high sequence similarities, nucleocytoplasmic shuttling activity could not be detected for AtNAP1;1, AtNAP1;2 and AtNAP1;3 in LMB-treatment assay (Liu et al., 2009a) and also not for Nicta;NAP1;2, Nicta;NAP1;3, Nicta;NAP1;4, and Orysa;NAP1;2 in both LMB-treatment and NES-mutagenesis assays in tobacco BY2 cells (Dong et al., 2005). This might be caused by assay limitations, e.g. the visual detection of GFP/YFP fluorescence used in the assay might not be sensitive enough and/or the tobacco BY2

cell line used in the assay could not sufficiently represent varied

cell types and physiological states of an entire plant. Nevertheless, the differences of nucleocytoplasmic shuttling activity observed among different plant NAP1 proteins do suggest that the function of NES and NLS depends on other sequence and/or posttranslational modification context of the protein. In line with this idea, nuclear localization of Orysa;NAP1;1 largely depends on an NLS located at C-terminal region but not the highly conserved NLS located at central region of the protein (Dong et al., 2005).


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During the cell cycle, Drosophila NAP1 shows nuclear localization at S phase and predominantly cytoplasmic localization at G2 phase (Ito et al., 1996). Human NAP1L1 and NAP1L4 can be phosphorylated by CASEIN KINASE 2 (CK2), which is associated with their cell-cycle distribution (Rodriguez et al., 2000). They are phosphorylated at the G0/G1 boundary but not in S-phase; the phosphorylated NAP1 proteins remain in the cytoplasm in a complex with histones during the G0/G1 transition, whereas their dephosphorylation triggers their transport into the nucleus at the G1/S-boundary (Rodriguez et al., 2000). In contrast, CK2 phosphorylation promotes import of the yeast NAP1 protein into the nucleus (Calvert et al., 2008). The rice Orysa;NAP1;3 protein also can be phosphorylated by CK2 kinase in in vitro assays; however, mutagenesis analysis fails to establish a role of this phosphorylation in the cytoplasmic and nuclear localization of OsNAP1;3 (Dong et al., 2005). Nevertheless, plant NAP1/NRP proteins contain additional multiple potential phosphorylation sites by CK2 or Cyclin Dependent Kinases (CDKs), and thus possible roles of their phosphorylation remain to be investigated. In line with this, tobacco NAP1 proteins were shown to interact with the mitotic cyclin Nicta;CYCB1;1 (Dong et al., 2005).

Among other types of modifications, farnesylation has been reported for both the human NAP1L1 (Kho et al., 2004) and the Arabidopsis AtNAP1;1 (Gallichet and Gruissem, 2006). Farnesylation can facilitate protein-membrane association and/or modulate protein-protein interaction (Hemsley, 2015), and potential farnesylation sites (CKQQ motif) can be found at the C-terminus sequences of many NAP1 proteins. Yet, farnesylation of AtNAP1;1 has no effect on the subcellular localization of the protein (Gallichet and Gruissem, 2006), and a role of farnesylation on NAP1 function in general remains to be investigated.

The FACT complex Features and evolutionary conservation of FACT subunits FACT represents a distinct family of H2A/H2B-type histone chaperones. FACT activity was initially detected from purified human nuclear extracts that could facilitate productive transcript elongation on assembled chromatin templates (Orphanides et al., 1998). In nowadays, it is known that FACT acts as


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a protein complex composed of two core subunits, SUPPRESSOR OF TY 16 (SPT16) and STRUCTURE-SPECIFIC RECOGNITION PROTEIN 1 (SSRP1), which are evolutionarily conserved in eukaryotes including the green lineage (Table 1 and Figure 3).

SPT16 protein is highly conserved and is composed of an N-terminal domain (NTD), a dimerization domain (DD), a middle domain (MD), and a negative-charged and intrinsically disordered C-terminal domain (CTD). The structures of several of these domains have been determined primarily using the yeast SPT16 (reviewed in Formosa, 2012; Winkler and Luger, 2011). The SPT16 NTD has a 'pita-bread' fold and can bind with various proteins including N-terminal tails of histones H3/H4, but yeast cells can tolerate its deletion, indicating that NTD of SPT16 is not absolutely required for FACT function. The DD domain is required for hetero-dimerization between SPT16 and SSRP1. The structure of SPT16 MD has been more recently revealed in complex with the H2A-H2B dimer, and is involved in preventing histone-DNA aggregate formation (Hondele et al., 2013). The acidic SPT16 CTD is also important for the interaction with histones, and its deletion abrogates H2A-H2B binding, chaperone activity and cellular viability (Formosa, 2012; Winkler and Luger, 2011).

SSRP1 proteins in animals and plants and their counterpart Pob3 in yeast show more varied degree of sequence conservation and domain organization (Figure 3a). The SSRP1/Pob3 proteins have high degree of sequence homologies at three conserved domains, NTD/DD, MD and CTD/IDD (Intrinsically Disordered Domain). The NTD/DD domain of SSRP1/Pob3 displays a single 'pleckstrin homology' (PH) fold and is the domain forming dimerization with the DD domain of SPT16 (Formosa, 2012; Winkler and Luger, 2011). SSRP1/Pob3 MD shows two PH folds, shares some sequence homologies with SPT16 MD, and is likely involved in dimer stabilization. Like SPT16 CTD, SSRP1/Pob3 CTD/IDD contains a high percentage of acidic residues, which is a common feature of histone chaperones. Deletion of either SPT16 CTD or SSRP1/Pob3 CTD/IDD can cause yeast lethality (Belotserkovskaya et al., 2003; Schlesinger and Formosa, 2000). While the human SSRP1 contains, in addition to the three conserved domains, a HMG (High Mobility Group) domain and a CID (C-terminal Intrinsically Disordered) domain, the Arabidopsis SSRP1 has conserved only HMG but not CID domain, and the yeast Pob3 lacks both HMG and CID domains. HMG domain can bind


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DNA and is found broadly in many proteins (Antosch et al., 2012). The HMG domain of SSRP1 can bind to nucleosomal DNA and may help FACT to recognize, bind, and reorganize chromatin (Winkler and Luger, 2011). In yeast, the HMG domain is provided by the small HMG-box protein Nhp6, separately from Pob3 (Formosa, 2012; Winkler and Luger, 2011). Thus, the yeast FACT complex has three components, SPT16, Pob3 and Nhp6, in comparison to the SPT16-SSRP1 dimer FACT complexes in metazoan (Figure 3b). The Arabidopsis FACT complex consisting of AtSPT16 and AtSSRP1 has also been identified (Duroux et al., 2004; Figure 3). While complete loss of AtSSRP1 function is lethal (Lolas et al., 2010), a mutant (ssrp1-4) carrying truncation of the conserved HMG domain at the C-terminus of AtSSRP1 is viable (Ikeda et al., 2011), indicating that the HMG domain is not absolutely required for all FACT functions or that another plant protein may provide HMG domain for the truncated AtSSRP1-4 function (in a manner similar to yeast Nhp6). It is worthy to note that while FACT is essential for yeast cell viability, without Nhp6 yeast cells can also survive though grow slowly (Formosa, 2012). The SPT16 and SSRP1 proteins with structural domain organizations similar to AtSPT16 and AtSSRP1 are conserved in the green lineage (Table 1). Interestingly, rice contains three homologues of SPT16 and two homologues of SSRP1, indicating possible formation of multiple different FACT complexes in this plant species.

Models of FACT function Human FACT was first demonstrated to specifically interact with nucleosomes and histone H2A-H2B dimers (Orphanides et al., 1999). Later, the FACT interaction with nucleosomes was shown to be via the SPT16 subunit (Belotserkovskaya et al., 2003). Similar to NAP1, FACT can also deposit core histones onto free DNA in vitro (Belotserkovskaya et al., 2003). The crystal structure of the evolutionarily conserved SPT16 MD from Chaetomium thermophilum showed that this domain makes several interactions with H2A-H2B dimer, which may help the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA (Hondele et al., 2013). In plants, the 71-kDa maize SSPR1 protein interacts with mononucleosome particles and can be released from chromatin upon limited micrococcal nuclease treatment. It can bind non-sequence-specifically to DNA and bend DNA to facilitate the formation of higher-order nucleoprotein structures (Lichota and Grasser, 2001; Röttgers et al., 2000). Both the Arabidopsis AtSPT16 and AtSSRP1 proteins associate


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with actively transcribed chromatin regions (Duroux et al., 2004). These studies in plants are thus in agreement with the FACT histone chaperone activity in chromatin reorganization.

Two major models exist in the literature for FACT function: the “dimer eviction” model and the “global access” model. FACT binds H2A-H2B dimer and mediates in vitro H2A-H2B displacement in immobilized nucleosome resulting in hexasome; thus the first model proposed for FACT function is on H2A-H2B dimer eviction in nucleosome disassembly (Belotserkovskaya et al., 2003; reviewed in Formosa, 2012; Winkler and Luger, 2011). The “eviction model” is further supported by the findings that disruption of FACT activity inhibits nucleosome reformation after passage of the RNA polymerase and that FACT prevents the accumulation of free histones evicted from transcribed chromatin, which is likely toxic to other regions of chromatin DNA (Formosa et al., 2002; Mason and Struhl, 2003; Morillo-Huesca et al., 2010). However, this model is challenged by the observation in which FACT induces global accessibility of nucleosomal DNA without significant loss of H2A-H2B in vivo (Xin et al., 2009). Thus, the “global access” model proposes that FACT induces/maintains nucleosomes in a more open configuration in which the components of the nucleosome are tethered together but capable of moving so that different regions of the DNA are available at different times (Formosa, 2012). In line with existence of a more open nucleosome configuration, an intermediate nucleosome structure, in which the distance between H2B and the nucleosomal dyad is increased as compared to a canonical nucleosome, was detected through single molecule FRET (Fluorescence Resonance Energy Transfer) experiments (Böhm et al., 2011). The multiple contact interfaces between FACT subunits, between FACT and H2A/H2B, between FACT and H3/H4, as well as between FACT and DNA, may provide supports for tethering together the components of a loosening nucleosome.

The “global access” model and the “dimer eviction” model could be both truly operational, likely depending on chromatin context/circumstances. The “global access” model depicts FACT loosening internal contacts within the nucleosome including interface between the H2A-H2B dimer and the (H3-H4)2 tetramer, which can lead to loss of the dimer from nucleosome. The difference is that in the “global access” model H2A-H2B loss is an optional outcome whereas in the “dimer eviction” model the remove of H2A-H2B is the obligate outcome of FACT function. Current models


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are based on in vitro studies and in vivo investigations in yeast and animals. Considering exist of sequence and domain organization differences from plant SPT16 and SSRP1 (Figure 3) as well as possible plant-specific features of nucleosome/chromatin modification structures, to which extent the “global access” model and the “dimer eviction” model apply to plant FACT function requires future investigations.

Regulation of FACT In both animals and plants, expression of SPT16 and SSRP1 genes is generally high in undifferentiated cells such as tumor cells, germ cells and stem cells in mammals (Garcia et al., 2013; Safina et al., 2013) and root apex and lateral primordial cells, gametophytic cells, and embryo cells in Arabidopsis (Duroux et al., 2004; Ikeda et al., 2011; Lolas et al., 2010). Interestingly, the amount of AtSPT16 protein was found reduced in the Atssrp1-2 mutant and vice versa the amount of AtSSRP1 protein was reduced in the Atspt16 mutants, suggesting a co-regulation of the two Arabidopsis FACT subunits (Lolas et al., 2010). Because the AtSSRP1 transcript level was not detectably affected in the Atspt16 mutants, it was proposed that the observed co-regulation might be explained by protein degradation when a heterodimer FACT complex cannot be formed (Lolas et al., 2010). A more recent study in mammals showed that the FACT complex can bind SPT16 and SSRP1 mRNAs, which stabilizes both mRNAs and the FACT proteins, and this can explain the interdependence of regulation of the SPT16 and SSRP1 protein levels (Safina et al., 2013). It thus appears that in both plants and mammals FACT activity is stringently controlled by amplified co-regulation mechanisms of its subunits.

Biochemical modifications of SPT16 and SSRP1 can provide additional levels of control of FACT activity. Protein kinase CK2 phosphorylates several Ser/Thr residues of the maize SSRP1 and modulates its DNA-binding activity (Krohn et al., 2003). Phosphorylation of SSRP1 by CK2 in Drosophila inhibits nucleosomal DNA binding and such a regulatory mechanism has been proposed to create a storage pool of inactive FACT that can be rapidly activated through dephosphorylation to facilitate chromatin transactions (Tsunaka et al., 2009; Winkler and Luger, 2011). Human SPT16 is poly(ADP-ribosyl)ated by PARP1 (Poly(ADP-Ribose) Polymerase 1), resulting in physical interaction between these two proteins, and causing inability of FACT to bind nucleosomes (Huang et al., 2006).


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Remarkably, automodification of PARP1 can switch its function into a histone-chaperone-like function, e.g. to sequester histones (in vitro and in cells) and to assemble nucleosomes efficiently in vitro (Muthurajan et al., 2014). Although absent in yeast, PARP1 is broadly conserved in plants and plays important roles in plant stress tolerance (Feng et al., 2015; and references therein). It will be interesting to investigate plant PARP1 function in relation with FACT in chromatin reorganization.

Histone modifications, variants and H2A/H2B chaperones Histone modifications in regulation of nucleosome dynamics Besides functioning as building blocks of nucleosomes, histones are also subjective to extensive and intensive post-translational modifications (PTMs); so far over 100 distinct histone PTMs have been reported, and among others the best characterized ones include methylation, acetylation, ADP ribosylation, phosphorylation, and ubiquitination at various histone amino acid residues (Tessarz and Kouzarides, 2014; Zentner and Henikoff, 2013). The majority of identified histone PTMs are located within the N-terminal tails of core histones protruding from the globular domain of nucleosome. PTMs are thought to contribute to the regulation of chromatin dynamics by altering the charge of modified residues or by altering the inter-nucleosomal interactions or by recruiting PTM-specific recognition proteins via specialized structures (Tessarz and Kouzarides, 2014; Zentner and Henikoff, 2013). Thus, in vivo histone chaperones are regulated under different histone PTM contexts and their action can contribute to modulate PTM composition of a nucleosome via histone exchange (Figure 4).

Histone H2B mono-ubiquitination (H2Bub1) is highly conserved and is generally associated with active transcription in eukaryotes ranging from yeast, plants and animals (Feng and Shen, 2014; Fuchs and Oren, 2014). H2Bub1 enhances FACT-mediated transcription in a reconstituted chromatin system in vitro (Pavri et al., 2006). In yeast, H2Bub1 is involved in SPT16 recruitment to GAL1. SPT16 regulates the formation of H2Bub1 at GAL1, and H2Bub1 and SPT16 function in a cooperative manner in nucleosome reassembly during GAL1 transcription (Fleming et al., 2008). The human SPT16 binds H2B and H2Bub1 with similar efficiency, and it can also bind H2A but not H2Aub1 (Zhou et al., 2008). In contrast to H2Bub1, H2Aub1 is generally involved in transcription repression. Interestingly, H2Aub1 represses transcription initiation by blocking FACT recruitment to


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the GAL4 promoter (Zhou et al., 2008). In Arabidopsis, AtSSRP1 and AtSPT16 genetically interact with HUB1/HUB2 (HISTONE MONOUBIQUITINATION 1/2), which encode enzymes responsible for H2Bub1 formation, in regulation of some aspects of plant development (Lolas et al., 2010). Yet, so far, molecular mechanisms linking FACT and H2Bub1 or H2Aub1 remain uncharacterized in plants.

The interaction of histone H2A/H2B with its chaperone FACT can also be affected via methylation on H2AQ105 by the Nop1 methyltransferase in yeast and on H2AQ104 by fibrillarin in human (Tessarz et al., 2014). The methylation of the glutamine residue weakens interaction between H2A and FACT, and in yeast H2AQ105 mutation reduces histone incorporation and increases RNA polymerase I (RNA Pol I) transcription at the ribosomal DNA (rDNA) locus. This H2A glutamine residue looks specifically important for physical interaction with FACT, since its methylation does not affect NAP1 binding (Tessarz et al., 2014). In addition to PTMs on H2A/H2B, PTMs on H3/H4 can also affect H2A/H2B dynamics within the nucleosome. Modifications are found at several amino acid residues at interface of histone-DNA and histone-histone interactions, e.g. methylation on H3R42, acetylation on H3K56, H3K64, H3K122 or H4K91, and phosphorylation on H3T118 (Tessarz and Kouzarides, 2014; Ye et al., 2005). H4K91 residue forms a salt bridge via its positive charge with a glutamic acid residue of H2B (H2BE75 in yeast); its acetylation disrupts the salt bridge by removing the positive charge and meanwhile introducing a bulkier side chain, and thus triggers the instability of nucleosomes and peripheral H2A/H2B dissociation (Ye et al., 2005). Nevertheless, a role of FACT or NAP1 in this interaction remains to be examined. Furthermore, it is currently unknown whether or not these latter described PTMs exist on plant histones.

Higher plants contain multiple variants of H2A Histone variants display differences in amino acid sequence with canonical histones and can confer distinct properties to the nucleosome via histone exchange (Figure 4). While the H2B family of histones lacks a functional identified variant, the H2A family comprises several types of H2A variants, including H2A.X, H2A.Z, H2A.W, H2A.Bbd and macroH2A (Bönisch and Hake, 2012; Hu and Lai, 2014; Zhu et al., 2012). Among H2A variants, H2A.Z has been most extensively studied in various organisms including Arabidopsis, whose functions have been implicated in transcriptional regulation, chromatin boundaries and stability (March-Diaz and Reyes, 2009). Four genes in Arabidopsis (HTA4,


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HTA8, HTA9 and HTA11) and three genes in rice (HTA705, HTA712 and HTA713) encode H2A.Z proteins, while each plant species contains four canonical H2A genes. The three Arabidopsis H2A.Z genes HTA8, HTA9 and HTA11 have redundant function in transcription regulation of responsive genes (Coleman-Derr and Zilberman, 2012; Kumar and Wigge, 2010). Two genes in Arabidopsis (HTA3 and HTA5) and two in rice (HTA704 and HTA711) encode H2A.X proteins, which have conserved SQEF motif at C-terminal and H2A.X is known in general as involved in DNA repair (Lang et al., 2012). More recently, three Arabidopsis H2A variant (HTA6, HTA7 and HTA12) containing conserved SPKK motif at C-terminal have been defined as a novel group named H2A.W, and have been shown to index heterochromatin (Bönisch and Hake, 2012; Yelagandula et al., 2014). Four rice proteins (HTA701, HTA706, HTA707 and HTA710) belong to the H2A.W group, and this group seems to be plant specific because the SPKK motif cannot be found from mammal, insect or yeast H2A proteins. Otherwise, H2A.Bbd and macroH2A identified in vertebrates cannot be found in plants. The two rice H2A variant genes HTA714 and HTA715 are considered as pseudogenes because their transcripts have not been found (http://www.chromdb.org).

Chaperones of H2A variants Differences exist at the small interface for the interaction of two H2A/H2B or H2A.Z/H2B dimers within the nucleosome, and the H2A.Z-containing nucleosomes are more stable and wrap DNA more tightly (Kumar and Wigge, 2010). H2A.Z is mainly found in chromatin at the 5’-end of genes (Deal et al., 2007; Zilberman et al., 2008). In the absence of machinery for H2A.Z incorporation, canonical H2A-containing nucleosomes cause constitutive expression of many genes that otherwise are expressed only under warm temperature treatments (Kumar and Wigge, 2010). It is well established that H2A.Z incorporation requires the ATP-dependent chromatin remodeling complex SWR1 (Swi2/Snf2-Related 1) in Arabidopsis as in other organisms. In yeast, Chz1 (Chaperone for H2A.Z) has been identified as an H2A.Z-preferential histone chaperone and shown to deliver H2A.Z to SWR1 for H2A replacement in chromatin (Luk et al., 2007). Interestingly, although without preference for H2A.Z, NAP1 is also an important H2A.Z-binding chaperone. Chz1 and NAP1 have overlapping functions and can reciprocally substitute for the binding to H2A.Z-H2B (Luk et al., 2007). Moreover, NAP1 but not Chz1 is involved in maintaining a soluble pool of H2A.Z in the cytoplasm (Straube et al., 2010). As expected, depletion of Chz1 reduces H2A.Z incorporation in yeast genome chromatin;


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however, depletion of NAP1 increases H2A.Z levels, presumably due to a compensatory mechanism using H2A.Z to replace a more drastically reduced H2A incorporation in the genome chromatin

(Liu

et al., 2014a). Chz1 homologues can be found in the green lineage as early as from moss, and Arabidopsis contains two genes encoding Chz1 homologues (Table 1). Future characterization of plant Chz1 homologues likely will provide important insight into mechanisms of nucleosomal H2A.Z replacement and its roles in chromatin regulation and function in gene transcription during plant growth and development. In a study of tRNA gene transcription by RNA Pol III in yeast, the SPT16 of FACT has been shown to play a role in both deposition and removal of H2A.Z in the gene flanking nucleosomes (Mahapatra et al., 2011). A more recent genome-wide study of RNA Pol II transcription shows that yeast SPT16 disfavors the H2A.Z-occupied genes, which is Chz1-dependent, reflecting an in vivo competition between the two different chaperones (Liu et al., 2014b). FACT also plays an important role in H2A.X exchange during DNA damage repair in human cells (Heo et al., 2008). It appears to act as both a chaperone and an exchange factor for this reaction, since FACT is co-purified with soluble H2A.X and FACT promotes both the removal of H2A.X from nucleosomes and H2A.X incorporation into canonical nucleosomes (Du et al., 2006; Heo et al., 2008). H2A.X phosphorylation facilitates the exchange of nucleosomal H2A.X mediated by FACT (Heo et al., 2008). In plants, H2A.X and its phosphorylation have been well established in relation with DNA damage response (Amiard et al., 2011; Lang et al., 2012; and references therein). FACT is essential for Arabidopsis viability and its down-regulation affects many aspects of plant growth and development (Lolas et al., 2010). Yet, FACT function in plant DNA damage repair remains unexamined and whether FACT can act as a plant H2A.X chaperone and/or exchange factor needs to be investigated.

Histone H2A/H2B chaperones in transcription regulation Nucleosome compaction is inhibitory both to the recruitment of transcription factors and RNA polymerases at transcription initiation and to the elongation of RNA polymerases through transcribed chromatin regions. Efficient transcription through chromatin in vivo implicates diverse chromatin structure modifiers, including histone modification enzymes, ATP-dependent remodeling factors, and histone chaperones (Avvakumov et al., 2011; Berr et al., 2011; Kwak and Lis, 2013; Van Lijsebettens


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and Grasser, 2014; Zhu et al., 2012). Histone H2A/H2B N-terminal tails are remarkably inhibitory to the nucleosome traversal of RNA Pol II elongation (Ujvári et al., 2008). The NAP1 and FACT histone chaperones could interfere with transcription via nucleosome disassembly/reassembly and/or nucleosome loosening/compaction (Figure 5).

Roles of NAP1-family proteins in transcription In an in vitro biochemical system recapitulating the transcription process, NAP1 forms complex with H2A and H2B, facilitates nucleosome dissociation in the presence of the ATP-dependent chromatin-remodeling factor RSC (Remodels Structure of Chromatin), and facilitates RSC-dependent RNA Pol II transcription (Levchenko and Jackson, 2004; Lorch et al., 2006). Nucleosome disassembly occurs in a stepwise manner, with the removal of H2A-H2B dimers, followed by the rest of the histones and the exposure of naked DNA. A more recent study demonstrates that in the presence of NAP1, RSC generates a subnucleosomal hexasome; and that despite the propensity of RSC to evict histones, NAP1 retains the hexasome on the DNA template during multiple rounds of RNA Pol II passage during transcription (Kuryan et al., 2012). The complete eviction of nucleosomes in vivo had been first reported to occur at promoter regions genome widely in yeast (Lee et al., 2004). Deletion of NAP1 affects expression of approximately 10% of the yeast genome (Ohkuni et al., 2003) and increases the H2A/H2B density across genes (Andrews et al., 2010). Yeast NAP1 is recruited to sites of active transcription and cooperates with the ATP-dependent chromatin-remodeling factor CHD1 (Chromo-ATPase/Helicase-DNA-binding protein 1) in nucleosome disassembly at promoters as well as in coding regions (Walfridsson et al., 2007). In Arabidopsis, the three ubiquitously expressed genes encoding canonical NAP1 (AtNAP1;1, AtNAP1;2 and AtNAP1;3) have redundant functions, and their simultaneous loss-of-function perturbs expression of a few hundreds of genes, representing roughly 2.5% of the total number of Arabidopsis genes (Liu et al., 2009a). Interestingly, the perturbed genes include many responsive genes such as DNA repair genes, and AtNAP1;3 has been shown to bind chromatin at both promoter and coding regions of several DNA repair genes, including AtCEN1, AtCEN2 and AtXPB1/2 involved in nucleotide excision repair (Liu et al., 2009a). In spite of their sequence homologies and similar domain structure organizations, NRPs and AtNAP1s likely regulate different sets of Arabidopsis genes. The misregulated genes found in the loss-of-function double mutant nrp1 nrp2 (Zhu et al., 2006) do not overlap with the misregulated


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genes found in the Atnap1;1 Atnap1;2 Atnap1;3 (m123-1) mutant (Liu et al., 2009a). Chromatin binding of NRP1 has been detected at some genes involved in root development, e.g. the homeodomain transcription factor gene GLABRA 2 (GL2) and the AP2-type transcription factor gene PLETHORA 2 (PLT2), as well as at some heterochromatin regions (Zhu et al., 2006). The yeast Vps75 together with ASF1 plays an important role as a chaperone for the histone acetyltransferase Rtt109 catalyzing H3K56 acetylation, which is important during transcription (D'Arcy and Luger, 2011). H3K56 acetylation is recognized as mark of newly synthesized H3, and Vps75 and ASF1 presumably supply new histones for nucleosome assembly/reassembly after RNA Pol II passage. Yet, NAP1 likely can play a similar role such that both Vps75 and NAP1 promote H3K56 acetylation and suppress the cryptic transcription within gene body (Xue et al., 2013). Notably, Vps75 but not NAP1 shows a synergistic genetic interaction with the H3K36-methyltrasferase SET2 in suppression of cryptic transcription, suggesting that Vsp75 and NAP1 may also play separate roles during transcription elongation (Xue et al., 2013). In Arabidopsis, the SET2 homologue SDG8 (SET DOMAIN GROUP 8) catalyzes H3K36 methylation and plays a crucial role in gene transcription (Berr et al., 2011; Zhao et al., 2005; and references therein). Future studies are necessary to examine nucleosome occupation and histone modifications at perturbed genes in the Atnap1 and nrp mutants to unravel more precisely mechanisms of AtNAP1 and NRP in transcription regulation.

Roles of FACT in transcription As its name originated from, FACT was found to play important role in transcription elongation in vitro (Orphanides et al., 1998). FACT binds H2A-H2B dimer, destabilizes nucleosomes in the path of RNA Pol II by facilitating the removal of H2A/H2B dimers (Belotserkovskaya et al., 2003). In yeast, FACT can be recruited onto target chromatin during transcription (Takahata et al., 2009a,b). It is involved together with RSC in the disassembly of H2A-H2B dimers selectively at the PHO5 promoter during transcriptional induction (Takahata et al., 2009b; Lorch et al., 2011). Moreover, FACT is required for the transient nucleosome eviction at the promoters of the G1-cyclin genes CLN1 and CLN2 for their expression during a normal cell cycle (Ransom et al., 2009; Takahata et al., 2009a). Interestingly, FACT can stimulate transcription elongation on nucleosomal templates regardless of histone tails, suggesting that FACT can overcome the intrinsic barrier from histone tails (Ujvári et al., 2008). RNA Pol II pauses principally at two sites, about 15 and 45 nucleotides into the nucleosome,


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which mark the contact between H2A-H2B and DNA and the transition to (H3-H4)2 and DNA contacts, respectively. FACT could alleviate RNA Pol II pausing by altering the stability of (H3-H4)2 and DNA interaction or by altering the internal dynamics of nucleosomes by removing H2A-H2B dimers (Formosa, 2012; Figure 5). Consistently, mutations in the yeast FACT can lead to significantly decreased histone levels and a concomitant reduction of genome-wide nucleosome occupancy (Hennig et al., 2012). In Arabidopsis, both AtSPT16 and AtSSRP1 are broadly localized to the cytologically defined and decondensed euchromatin, which is in agreement with a general role of FACT in transcription regulation (Duroux et al., 2004). The Arabidopsis FACT binds at actively transcribed genes and enhances expression levels of some of these target genes, e.g. the flowering repressor gene FLOWERING LOCUS C (FLC), the heat shock-inducible gene HSP70 and the salicylic acid (SA)-inducible gene PR-1 (Duroux et al., 2004; Lolas et al., 2010). It is noteworthy that both FLC and HSP70 are regulated by H2A.Z incorporation (Deal et al., 2007; Kumar and Wigge, 2010), and FLC is also regulated by SDG8-mediated H3K36 methylation (H3K36me2/3) and HUB1/HUB2-mediated H2Bub1 deposition (for a review, see Berr et al., 2011). FACT likely acts in concern with H2A.Z, H2Bub1 and H3K36me2/3, and in cooperation with a variety of other transcript elongation factors in RNA Pol II transcription (Van Lijsebettens and Grasser, 2014). A specific role of AtSSRP1 has uncovered in DNA demethylation and control of imprinted gene expression in the Arabidopsis central cell before fertilization (Ikeda et al., 2011). However, this AtSSRP1 function has been considered as FACT-independent because a similar function could not be found for AtSPT16 (Ikeda et al., 2011).

Histone H2A/H2B chaperones in genome replication and repair NAP1 and FACT in genome replication Histone incorporation into chromatin is critical for the genome replication, because the random transfer of parental histones is not sufficient to meet the requirement of package of the duplicated nascent daughter DNA strands. Histone chaperones that escort histones from the cytoplasm to the nucleus and further assist nucleosome assembly are considered to play important roles in completion of genome duplication during the S-phase of cell cycle (Avvakumov et al., 2011; Desvoyes et al., 2014; Ransom et al., 2010; Raynaud et al., 2014). The in vitro formation of regularly spaced


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nucleosomes on DNA requires the synergistic action of NAP1 with other chromatin factors such as CAF-1 (Ito et al., 1996). Inhibition of human NAP1L1 led to decreased cell proliferation (Simon et al., 1994). Heterologous NAP1 expression in slow-growing pulmonary artery endothelial cells increases their growth potential, whereas the inhibition of NAP1 expression in fast growing pulmonary microvascular endothelial cells decreases their growth potential (Clark et al., 2008). Thus, these studies in mammals are in line with NAP1 function in cell proliferation. Strikingly, the Arabidopsis triple mutant Atnap1;1 Atnap1;2 Atnap1;3 does not display any obvious defect in cell division/proliferation (Liu et al., 2009a). And the nrp1 nrp2 mutant only shows mild delay of G2/M transition in Arabidopsis root tip cells (Zhu et al., 2006). It will be interesting to examine whether or not AtNAP1s and NRPs have redundant function in control of cell division/proliferation. Yeast FACT interacts with the DNA polymerase alpha catalytic subunit (Wittmeyer and Formosa, 1997), and the SPT16 subunit is ubiquitylated by the cullin-E3 ligase Rtt101 in vitro and in vivo, which is specifically correlated with the interaction of FACT with replicative helicase MCM at replication origins (Han et al., 2010). S-phase onset and progression were delayed when Pob3 was mutated, and genetic interactions between pob3 mutants and the genes encoding several DNA replication factors including POL1 caused sensitivity to DNA replication inhibitor (Schlesinger and Formosa, 2000). Although immunolocalization did not allow detection of more AtSPT16 or AtSSRP1 in dividing than in nondividing interphase cells (Duroux et al., 2004), downregulation of FACT in Atspt16 and Atssrp1 mutants clearly reduced cell division in leaf and likely also in other organs (Lolas et al., 2010). Future investigations are necessary to distinguish histone chaperone functions between transcription and replication in the regulation of cell division/proliferation.

NAP1 and FACT in maintenance of genome integrity Deficiencies in nucleosome assembly/reassembly as well as more loosen nucleosomal configuration expose DNA to instability and damage. In line with this idea, increased level of DNA lesions had been observed in the Arabidopsis mutant nrp1 nrp2 (Zhu et al., 2006). Among various types of DNA damage, double strand break (DSB) is the most severe/toxic type that challenges eukaryotic cell viability. Histone chaperones may participate in DSB repair via histone exchange and nucleosome disassembly/reassembly to mark the lesion site, to help DNA repair machinery to overcome the nucleosome obstacle, and to restore chromatin structure after DNA repair (Figure 6).


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Upon DNA damage, the H2A.X is incorporated and quickly phosphorylated in chromatin regions surrounding the damage site, which can extend to a large region of up to mega-base pairs (Avvakumov et al., 2011). In human cells, FACT can catalyze the integration of H2A.X-H2B but not H2A.Z-H2B into nucleosome by replacing H2A-H2B during DNA damage repair (Heo et al., 2008). In contrast, NAP1 can play chaperone function for H2A.Z-H2B exchange (Luk et al., 2007), but not for H2A.X-H2B (Heo et al., 2008). Thus, it appears that FACT and NAP1 may be both involved in histone variant exchange, nucleosome disassembly and reassembly during DNA repair. While SPT16 poly(ADP-ribosyl)ation by PARP1 inhibits FACT activity in H2A.X exchange, H2A.X phosphorylation destabilizes nucleosome structure and favors the exchange of nucleosomal H2A.X by FACT (Huang et al., 2006; Heo et al., 2008). MRN (MRE11-RAD50-NBS1) complex is required for H2A.X phosphorylation (named γ-H2A.X) by the ATM (ATAXIA TELANGIECTASIA MUTATED) and ATR (ATM AND RAD3 RELATED) kinases in response to irradiation-induced DSB in Arabidopsis (Amiard et al., 2010). The γ-H2A.X foci are rarely detectable in Arabidopsis plants under normal growth conditions, but the foci number drastically increases under genotoxic stress such as irradiation. Nevertheless, the mechanism of plant H2A.X incorporation into nucleosome and FACT function in plant DNA damage repair remain to be investigated.

Two major pathways are involved in DSB repair: homologous recombination (HR) and non-homologous end joint (NHEJ) pathways (Knoll et al., 2014). Remarkably, a crucial role of NAP1 and NRP in HR has been demonstrated in Arabidopsis (Gao et al., 2012). Somatic HR frequency is reduced in both the triple mutant Atnap1;1 Atnap1;2 Atnap1;3 and the double mutant nrp1 nrp2 as compared to wild-type, in plants grown under normal growth conditions as well as under a wide range of genotoxic or abiotic stress conditions (Gao et al., 2012). This study suggests that AtNAP1/NRP-mediated H2A-H2B exchange, eviction and/or nucleosome disassembly might be crucial for important steps during HR. Exchange or removal of nucleosomal H2A-H2B nearby DSB site might be essential for the establishment of a favorable chromatin platform to recruit HR repair machinery proteins. Alternatively or in addition, formation and maintenance of nucleoprotein filaments, D-loop and heteroduplex extension during the HR process might require NAP1/NRP to interact with chromatin structure. More recently it has been demonstrated that NAP1 positively regulates HR also in mammalian cells and that release of higher order chromatin structure involving


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histone H1 may be facilitated by NAP1 during HR (Machida et al., 2014). In contrast to AtNAP1/NRP, the depletion of CAF-1 (e.g. in fas1 and fas2 mutants) resulted in increase of HR frequency in Arabidopsis (Endo et al., 2006; Kirik et al., 2006; Gao et al., 2012). Strikingly, the combined higher order mutant nrp1 nrp2 fas2 displays a HR phenotype similar to nrp1 nrp2, indicating that NRPs override CAF-1 in HR determinacy (Gao et al., 2012). This finding highlights the unique role of H2A/H2B-type chaperone in the progression of HR repair. Human FACT subunit SPT16 can interact with the E3 ubiquitin ligase RNF20 in vivo, RNF20 regulates chromatin structure via H2B ubiquitination to facilitate DNA access to HR repair proteins, and depletion of SPT16 impairs the progression of HR repair (Oliveira et al., 2014). It will be interesting to examine whether Arabidopsis FACT is also involved in HR.

In addition to HR, H2A/H2B-type histone chaperones are also involved in other DNA damage

repair

pathways.

Depletion

of

either

AtNAP1;2

or

AtNAP1;3

can

reduce

Agrobacterium-mediated T-DNA transformation efficiency (Crane and Gelvin, 2007), indicating a possible role of AtNAP1 in NHEJ because T-DNA integration occurs about randomly in plant genome by illegitimate recombination. The triple mutant Atnap1;1 Atnap1;2 Atnap1;3 exhibits a hypersensitivity to UV-C radiation, and consistently protein extracts from the mutant plants compared to the wild-type plants show lower efficiency in in vitro nucleotide excision repair (NER) assay (Liu et al., 2009a). The protein kinase CK2 phosphorylates the maize SSRP1, inducing SSRP1-binding to UV-damaged plasmid DNA, suggesting a role of FACT in UV damage response (Krohn et al., 2003).

Roles of histone H2A/H2B chaperones in plant growth and development The single, double and triple gene mutants of loss-of-function of the three ubiquitously expressed canonical NAP1 genes AtNAP1;1, AtNAP1;2 and AtNAP1;3 show plant growth and development similar to wild-type under the standard laboratory growth conditions (Liu et al., 2009a). As predicted by its low level of expression and absence of a canonical NAP1 structure, AtNAP1;4 has no detectable function in either the single Atnap1;4 mutant or the quadruple Atnap1;1 Atnap1;2 Atnap1;3 Atnap1;4 mutant analyses (Liu et al., 2009a; She et al., 2013). This absence of nap1 mutant phenotype in Arabidopsis is in striking contrast to the crucial roles of NAP1 described in animals.


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Deletion of the NAP1 gene in Drosophila results in lethality, either embryonic lethal or poorly viable but adult lethal (Lankenau et al., 2003). Depletion of zygotic xenopus NAP1-like protein from embryos unravels its specific function in hematopoiesis (Abu-Daya et al., 2005). In spite of multiple NAP1-like genes, deletion of the mouse NAP1L2 gene alone causes a lethal phenotype, which is associated with severe neural tube defects and the overproduction of neural stem cells during mouse embryonic development (Rogner et al., 2000; Attia et al., 2011). Nevertheless, similar to result in Arabidopsis, deletion of yeast NAP1 barely affects normal cell growth (Ohkuni et al., 2003). Remarkably, the Arabidopsis triple mutant Atnap1;1 Atnap1;2 Atnap1;3 exhibits hypersensitivities to various treatments, including genotoxic stresses (Liu et al., 2009a; Gao et al., 2012), abscisic acid response and salt stress (Liu et al., 2009b; Gao et al., 2012), and phosphate starvation (Iglesias et al., 2013). Thus, although AtNAP1s are dispensable for normal plant growth and development under standard laboratory growth conditions, they do play important roles in Arabidopsis plant growth fitness to varied environmental changes.

Simultaneous knockout of both NRP1 and NRP2 in Arabidopsis leads to plant short-root phenotype (Zhu et al., 2006). Arrest of cell cycle progression at G2/M and disordered cellular organization has been observed in the primary root of nrp1 nrp2 seedling. In contrast, the nrp1 nrp2 mutant plant can produce sufficient lateral roots, and in vitro root callus regeneration as well as the embryo and the aerial organs (leaf, rossette, inflorescence, flower and fruit) all develop normally in the mutant (Zhu et al., 2006; Zhu et al., 2007). The root-limited growth phenotype of the nrp1 nrp2 mutant may be associated with down-regulation of PLT2 and GL2 transcription factor genes caused by the absence of NRP1/NRP2 histone chaperone activity (Zhu et al., 2006). In yeast, deletion of Vps75 results in a detectable inhibitory effect on cell growth/proliferation only under drug treatment or in some combined mutant genetic backgrounds (Selth et al., 2009). Similarly, the Arabidopsis NRP1/NRP2 play important roles in plant tolerance to various types of genotoxic stresses, in maintenance of plant genome integrity, and in plant DNA damage repair (Zhu et al., 2006; Gao et al., 2012). It is noteworthy that overlapping/redundant roles between the NAP1-subfamily and the NRP/Vps75-subfamily genes had not been directly examined in plant or yeast. Thus, the conclusion about the NAP1-family histone chaperones in organism growth and development remains incomplete so far.


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In contrast to the mild mutant phenotype ascribed for NAP1 and NRP/Vps75, FACT is essential for both yeast and Arabidopsis viability (Lola et al., 2010; Formosa, 2012). Reduction of AtSSRP1 or AtSPT16 causes pleiotropic defects of Arabidopsis plant growth and development, including increased number but drastically reduced size of leaves, early bolting/flowering, 'bushy' with increased number of primary and secondary inflorescences, smaller flower and deformed floral organs, reduced fertility, abnormal embryogenesis, and smaller fruits (Lola et al., 2010; Van Lijsebettens and Grasser, 2010; Ikeda et al., 2011). The FACT effect on flowering time has been shown to be independent from photoperiods (long day or short day), and from vernalization or gibberellin treatment (Lola et al., 2010). Both AtSSRP1 and AtSPT16 bind chromatin at the flowering repressor FLC locus and are involved in activation of FLC expression (Lola et al., 2010), providing a molecular mechanism of FACT function in inhibition of plant flowering. As a comparison, much remains unknown regarding molecular mechanism of FACT in regulation of cell division and differentiation during plant growth and development.

Concluding remarks We have reviewed and discussed current research findings that help to understand molecular basis of function and regulation of the histone H2A/H2B chaperones NAP1 and FACT, and to appreciate their critical roles in transcription, DNA replication and repair, and in plant growth and development or in plant response to environmental stresses. Linking with covalent histone modifications and histone variants, NAP1/FACT-mediated dynamic exchange of H2A-H2B within the nucleosome has great implication with regard to chromatin structure and function, and to epigenetic reprogramming and inheritance. Future studies are necessary to characterize into more detail the plant NAP1 and FACT in these various processes. Most strikingly, neither the Arabidopsis NAP1-subfamily nor the NRP-subfamily genes are essential for plant life; yet overlapping/redundant function of the two subfamilies remains to be examined. Although binding of Arabidopsis NAP1, NRP or FACT has been detected at chromatin of some selected genes, their genome-wide distribution/association with chromatin remains unexamined. In line with mechanisms of their chromatin association with specific genes/genome regions, identification and characterization of functional and/or physical partners of plant NAP1/NRP and FACT will likely have great importance. Novel types of plant histone


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chaperones need to be identified and characterized; it is particularly interesting to note the presence of H2A.Z-specific chaperone Chz1 homologues in plants (Table 1) and the requirement to identify H2A.W chaperones. Future studies are expected to reveal the unique and/or overlap roles of these different families of histone chaperones in plant response to developmental and environmental cues, which is important to understand the epigenetic mechanism for the adaptive growth of multi-cellular organisms.

Acknowledgments Current work in authors' laboratories was supported by National Basic Research Program of China (973 Program, grants no. 2012CB910500 and 2011CB944600), National Natural Science Foundation of China (grant no. 31271374), Science and Technology Commission of Shanghai Municipality (grant no. 13JC1401000), and the French Agence Nationale de la Recherche (ANR-12-BSV2-0013-02). The research was conducted within the context of the International Associated Laboratory Plant Epigenome Research, LIA PER.

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Table 1 . Evolutionarily conserved histone H2A/H2B chaperones found in green alga, moss and higer plants. O. tauri NAP1 family NAP1;1 (NFA3601)

FACT family Spt16 (GTC3601) SSRP1 (SSRP3601)

P. patens

O. sativa

A. thaliana

NAP1;1 (NFA1501)*

NAP1;1 (LOC_Os06g05660)

NAP1;1 (At4g26110)

NAP1;2 (NFA1502)

NAP1;2 (LOC_Os05g46230)

NAP1;2 (At5g56950)

NRP1 (NFA1503)*

NAP1;3 (LOC_Os01g51450)

NAP1;3 (At2g19480)

NRP2 (NFA1504)

NAP1;4 (LOC_Os06g40920)*

NAP1;4 (At3g13782)

NRP1 (LOC_Os04g38620)

NRP1 (At1g74560)

NRP2 (LOC_Os02g36710)

NRP2 (At1g18800)

Spt16 (GTC1501)

Spt16;1 (LOC_Os04g25550)

Spt16 (At4g10710)

SSRP1 (SSRP1501)*

Spt16;2 (LOC_Os08g31240)

SSRP1 (At3g28730)

Spt16;3 (LOC_Os12g26030) SSRP1;1 (LOC_Os01g08970) SSRP1;2 (LOC_Os05g08970) Chz1 family

None

Chz1 (XP_001773750.1)

Chz1 (LOC_Os11g34190)

Chz1a (At4g08310) Chz1b (At1g44780)

GenBank accession or ChromDB ID numbers are indicated in the parentheses. * indicates that no cDNA is available.

FIGURE LEGENDS Figure 1. Domain organization and complex formation of NAP1-subfamily and NRP-subfamily proteins. (a) Schematic representation of domain organization of the yeast and Arabidopsis NAP1-family proteins. Evolutionary conserved domains are indicated and highlighted with different colors. DH, AC, β/α, and AT are referred respectively to Dimerization Helix, Accessory Domain, β-sheets/α-helices region, and Acidic C-Tail. Both the yeast Vps75 and the Arabidopsis NRPs lack the accessory domain. (b) Diagrams representing NAP1 dimer and NRP dimer formation in Arabidopsis. The NAP1-subfamily or NRP-subfamily members can form homo- or hetero-dimers, but they cannot form inter-subfamily complexes.

Figure 2. Different subcellular localization patterns of plant NAP1-family proteins. Subcellular localization patterns of plant NAP1-family proteins can be grouped into three categories: cytoplasm, cytoplasm & nucleus, and nucleus. Double-head arrow indicates for nucleocytoplasmic shuttling.


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Figure 3. Domain organization of the FACT subunits and FACT complex formation in human, yeast and Arabidopsis. (a) Schematic representation of domain organization of human, yeast and Arabidopsis FACT subunits. Evolutionary conserved domains are indicated and highlighted with different colors. NTD, DD, MD, CTD, IDD, HMG and CID are referred respectively to N-Terminal Domain, Dimerization Domain, Middle Domain, C-Terminal Domain, Intrinsically Disordered Domain, High Mobility Group box, C-terminal Intrinsically Disordered. (b) Diagrams representing composition of the human/plant FACT complex as compared to the yeast FACT complex.

Figure 4. Diagram depicting histone exchanges that could affect nucleosome composition, structure configuration and functionality. The canonical H2B-H2A, and the variant H2B-H2A.Z, -H2A.X and -H2A.W dimers are indicated using different colors. Possible examples of post-translational modifications on nucleosomal histones are indicated for ubiquitination (ub) and methylation (me). Double-head arrows indicate exchange activity mediated by possible histone chaperones with known names or by unknown with question mark.

Figure 5. Models of the H2A/H2B-chaperone NAP1 and FACT functioning in Pol II transcription. (a) The ‘dimer eviction’ model. NAP1 and FACT could facilitate RNA Polymerase II (Pol II) to access DNA by H2A-H2B dimer eviction and nucleosome restor by H2A-H2B incorporation. (b) The ‘global access’ model. FACT could reorganize the nucleosome configuration into a more open structure without obliged H2A-H2B eviction. Different players are indicated.

Figure 6. Model depicting possible functions of the H2A/H2B-chaperone NAP1 and FACT in DNA repair. NAP1 and FACT escort H2A-H2B, H2A.X-H2B and H2A.Z-H2B dimers and facilitate dimer exchange, eviction, and incorporation at different steps during DNA double strand break (DSB) repair. Different events and players are indicated.


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