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The many faces of chromatin assembly factor 1
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The many faces of chromatin assembly factor 1 Elena Ramirez-Parra and Crisanto Gutierrez Centro de Biologia Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Chromatin organization requires that histones associate with DNA in the form of nucleosomes the position and composition of which is crucial for chromatin dynamics. Histone chaperones help to deliver specific histone proteins to the sites where chromatin is being newly formed or remodeled. Association of H3–H4 during DNA replication depends on the chromatin assembly factor 1. The study of Arabidopsis plants carrying loss-of-function alleles in each of the three chromatin assembly factor 1 subunits has highlighted the links between chromatin assembly in proliferating cells and other cellular processes. These are the G2 DNA damage checkpoint, homologous recombination, endoreplication control and transcriptional regulation of specific gene sets, all contributing to the plasticity of plants in dealing with alterations in DNA replication-associated chromatin assembly. Introduction In eukaryotes, genomic DNA associates with histone and non-histone proteins, becoming a highly compact structure – chromatin. The basic structural subunit of chromatin is the nucleosome, which contains 150 base pairs of DNA wrapped around a histone octamer core of two molecules of each of the histones H2A, H2B, H3 and H4. Thus, genome duplication consists not only in DNA replication, but also in the reorganization of new histone octamers onto newly synthesized DNA, a process that is initiated by the incorporation of histones H3 and H4. However, histones do not associate with DNA directly to form mature nucleosomes. Instead, histone chaperones (Box 1) facilitate this process by recruiting histones to the chromosomal sites where chromatin is being reconstituted or reorganized [1]. Maintaining chromatin and genome integrity is crucial for the normal development of eukaryotic organisms. Therefore, the function of histone chaperones, particularly in association with DNA replication, is of primary importance. The chromatin assembly factor 1 (CAF-1) chaperone functions in association with the DNA replication machinery to deposit histone H3 and H4 onto DNA, which is the initial event in DNA replication-associated chromatin assembly (Figure 1). Following the identification of genes encoding CAF-1 subunits in plants, their function has been reviewed [2,3]. More recently, various reports have demonstrated that a loss of function of CAF-1 has a pleiotropic effect. Corresponding author: Gutierrez, C. ([email protected]). Available online 9 November 2007. www.sciencedirect.com
Here, we discuss the various cellular processes for which CAF-1 function is required, with the aim of integrating them in the light of recent findings on chromatin organization of genes particularly affected by CAF-1 function. Plant CAF-1 Subunit organization of CAF-1 CAF-1 is a highly conserved heterotrimeric complex. In yeast, where it is known as chromatin assembly complex (CAC), it consists of Cac1, Cac2 and Cac3 subunits [1,4], whereas in mammalian cells, CAF-1 consists of the p150, p60 and p48 subunits [5–7]. In Arabidopsis, the three subunits are encoded by the FASCIATA1 (FAS1), FASCIATA2 (FAS2) and MULTICOPY SUPPRESSOR OF
Glossary Checkpoint: cell-cycle checkpoints are control mechanisms that ensure the fidelity of cell division in eukaryotic cells, controlling whether each phase of the cell cycle has been accurately completed before progression into the next phase. The main function of checkpoints is to assess DNA damage. Chromocenter: a condensed structure formed by the association of centromeric regions of heterochromatin. Double-strand break (DSB): a DNA lesion that affects both DNA strands. DSBs are the consequence of endogenous or exogenous genotoxic agents (ionizing radiation or chemicals). This lesion is repaired mainly by two different pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). E2F: a family of eukaryotic transcription factors that are essential for the G1–Sphase transition but also for the regulation of other cellular and developmental processes. Arabidopsis thaliana contains six E2F proteins (namely, E2Fa–f). They are regulated by the plant retinoblastoma-related (RBR) protein and can function as transcriptional repressors or activators, depending on the E2F member and the cellular and genomic context. Endoreplication: the process of full-genome duplication without mitosis, resulting in an increase in the nuclear DNA content. Endoreplication is a common process in plants, where it has an essential association with developmental programs. Epigenetic: epigenetic regulation or chromatin modification refers to changes in gene expression that are not caused by DNA sequence. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and modifications of histones, such as methylation, acetylation, ubiquitination, phosphorylation and incorporation of histone variants. Euchromatin: chromosomal regions with a high density of genes, which are often under active transcription. Heterochromatin: chromosomal regions, highly condensed throughout the cell cycle, that have been clearly linked to gene silencing. Constitutive heterochromatin is often associated with telomeres and pericentromeric regions of chromosomes and is rich in repetitive, permanently inactive chromosomal regions. However, facultative heterochromatin can be transcriptionally silenced in specific tissues or developmental stages, and remain active in others. Homologous recombination (HR): a DNA repair pathway that requires extensive DNA sequence homology between the interacting strands. Non-homologous end-joining (NHEJ): a DNA repair pathway that requires limited DNA sequence homology, or even no homology, at the ends of the interacting strands.
1360-1385/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.10.002
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Box 1. Histone chaperones A variety of histone chaperones are specialized in incorporating different histones into DNA. One class of histone chaperones is involved in storage and deposition of histone H2A and H2B. Nucleoplasmin, which is a store of histone H2A and H2B, and nucleolin regulate histone exchange [64,65]. In addition, nucleosome assembly protein 1 (NAP-1) and NAP-related protein (NRP) belong to a family of histone chaperones with a high affinity for H2A and H2B that function as nuclear–cytoplasmic shuttle chaperones and are required to maintain postembryonic growth [66,67]. Other histone chaperones are specific for the exchange and/or incorporation of histones H3 and H4. One of these is CAF-1, which facilitates the deposition of histone H3.1–H4 dimers onto DNA followed by the association of H2A–H2B dimers in close association with DNA replication during the S-phase or DNA repair synthesis [68]. By contrast, HIRA incorporates the histone variant H3.3 at any time during the cell cycle or in differentiated cells [68]. In both cases, these chaperones are assisted by ASF1.
IRA1 (MSI1) genes, which encode the large, middle and small subunits, respectively [8–10] (Figure 2). The p150 subunit of human CAF-1 interacts directly with histones H3–H4 through its acidic domain and contains a PEST domain, rich in S and T residues and involved in protein degradation. The PEST domain is not present in the yeast and plant homologs of the human p150 subunit (Figure 2). This subunit also associates with the proliferating cell nuclear antigen (PCNA), which recruits CAF-1 to replication- and repair-coupled chromatin assembly sites [11]. The middle subunit contains seven WD40 repeats, involved in protein–protein interactions and conserved in human, yeast and plants (Figure 2). The smallest subunit (p48, Cac3 or MSI1), which also contains seven WD40 repeats (Figure 2), can interact with histone deacetylases [12,13], suggesting a role in the acetylation–deacetylation cycle during DNA replication-associated chromatin assembly. In addition, MSI proteins associate, independently of
Figure 1. Histone chaperones involved in H3–H4 dynamics. CAF-1 is a heterotrimeric complex that participates in DNA synthesis-associated processes, such as DNA replication and DNA repair, by incorporating histone H3.1. Histone gene repressor A (HIRA) functions in a DNA synthesis-independent manner, depositing the histone variant H3.3. CAF-1 and HIRA are assisted by the histone chaperone antisilencing factor 1 (ASF1). Dotted red lines in the chromatin indicate newly synthesized DNA. Orange shapes indicate H3–H4 dimers and green shapes indicate H2A–H2B dimers. www.sciencedirect.com
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CAF-1, with other proteins, such as retinoblastoma proteins [14–19], regulators of the E2F pathway [20], Polycomb group histone methyltransferases [21–25] and, in plants, regulators of flowering time [26]. In spite of the evolutionary conservation, the consequences of CAF-1 dysfunction are different in unicellular and multicellular eukaryotes. In yeast, loss of CAF-1 activity leads to underassembly of chromatin, produces defects in telomeric and mating-type locus gene silencing and increases genome instability [27–29], although it does not seem to have a direct role in cell cycle progression. In human cells, defects in CAF-1-mediated nucleosome assembly activate the S-phase checkpoint (see Glossary), eventually leading to S-phase arrest and cell death [30– 32]. In Arabidopsis, plants carrying loss-of-function mutations in genes encoding CAF-1 subunits are viable, although they show developmental defects [33]. Thus, the fas1 and fas2 mutants were isolated in a screening for recessive mutations leading to abnormal meristem structure [8]. The mutant plants are fasciated, having broad and flat stems, disrupted leaf phyllotaxy, dentate and narrow leaves, reduced root growth and altered structure of floral organs [8]. The expression of typical markers of the shoot and root apical meristems, such as WUSCHEL (WUS) and SCARECROW (SCR), respectively, is disturbed [9]. Therefore, one current challenge is to identify the variety of cellular processes in which CAF-1 function is relevant and to understand how they interact with each other to make the defects in DNA replication-associated chromatin assembly compatible with development. It is likely that the integration of these processes contributes significantly to the plasticity of plants to cope with alterations in chromatin organization – for example, failure to incorporate histones H3 and H4 during DNA replication. Genes encoding CAF-1 subunits are cell-cycle regulated The activity of CAF-1 during DNA replication is restricted to the S-phase of the cell cycle. Therefore, it is not surprising that FAS1 expression is cell cycle regulated, showing a peak during S-phase [9]. The promoter region of FAS1 contains binding sites for members of the E2F family of transcription factors: TTTGGCGC (at position –345 from the putative initiation codon, ATG) and TTTCCCGCCAAG (at –290) (Figure 3). Electrophoretic mobility shift assays (EMSA) using E2Ff, one of the Arabidopsis E2F proteins, indicates that it can bind to these two E2F sites in vitro. This was confirmed by chromatin immunoprecipitation (ChIP) studies, revealing that E2F can bind directly to the FAS1 promoter in vivo and regulate FAS1 expression [34]. Furthermore, transgenic Arabidopsis plants overexpressing various E2F proteins (such as E2Fa, E2Fb, E2Fd and E2Ff) or deficient in E2Fe, in all cases, show increased FAS1 expression. In contrast, plants overexpressing the repressor E2Fc or a dominant negative version of DP, the dimerization partner of Arabidopsis E2Fa, E2Fb and E2Fc proteins, and required for E2F activity [34], show decreased FAS1 expression. Therefore, these data demonstrate that members of the E2F family regulate FAS1 gene expression, although the nature of the E2F member (or members) involved remains to be identified.
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Figure 2. The three subunits of the CAF-1 heterotrimeric complex are highly conserved in humans, yeast and plants. The names of the different subunits in humans, yeast and plants are indicated (a). The domain organization of the three CAF-1 subunits in yeast, humans and Arabidopsis is shown in the lower part of the figure (b). Note the presence of the highly repeated WD40 domain in the middle and small subunits. The motifs present in each protein are color coded, as indicated.
The role of the E2F binding sites in the spatial regulation of FAS1 expression has been analyzed using reporter plants expressing the b-glucuronidase (uidA) gene under different FAS1 promoter constructs [34]. These studies have demonstrated that proliferating cells in different plant locations possess high levels of FAS1 expression. Deletion of the distal E2F binding site leads to loss of activity, whereas an increase in activity occurs by deleting the proximal E2F site. This led to the conclusion that the distal E2F binding
site is required for enhanced FAS1 expression and the proximal E2F binding site functions as a repressor [34]. Therefore, as reported for other E2F target genes, such as the ribonucleotide reductase RNR2 gene [35], the minichromosome maintenance 3 (MCM3) gene [36] or the PCNA gene [37], the two E2F sites in the FAS1 promoter seem to have distinct regulatory roles both qualitatively and quantitatively [38]. Note that E2F binding sites are also present in FAS2 and MSI1, which encode the other two plant CAF-1
Figure 3. Location of E2F binding sites (blue boxes) in the promoter regions of genes (orange arrows) encoding the large (FAS1), middle (FAS2) and small (MSI1) CAF-1 subunits in Arabidopsis. The numbers inside the coding regions of the genes are the Arabidopsis gene code numbers. The number above each binding site refers to the position of the first nucleotide of the binding site relative to the putative ATG initiator codon. Note that the distal site, relative to the start of the open reading frame, has a reverse orientation. www.sciencedirect.com
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subunits (Figure 3). The FAS2 promoter contains one site (TTTCGCGCCCAAA at –238 from the putative ATG) and the MSI1 promoter contains another single binding site (TGTGGGCGAAT at –120 from the putative ATG). Based on these observations, it is conceivable that E2F might regulate the expression of the three components of CAF-1 in a coordinated manner. Nevertheless, based on microarray data, neither FAS2 nor MSI1 change their expression dramatically during the cell cycle or in plants overexpressing E2Fa and DPa. It should be noted, however, that other well-characterized E2F target genes, such as MCM3, ORC1a (the large subunit of the origin recognition complex) or PCNA [38], which have a peak of expression at the G1–S transition, do not show significant changes in the available microarray data. Therefore, detailed studies are needed to address individually the possible nature of FAS2 and MSI1 genes as E2F targets using complementary approaches. Role of CAF-1 in gene expression Heterochromatin silencing Saccharomyces cerevisiae cac mutants are defective in maintaining stable gene silencing at telomeres and at the mating-type locus [28,39]. In plants, the situation is not yet clear because conflicting reports have appeared on the relevance of CAF-1 in heterochromatin silencing. In one study, the typical transcriptional silencing of genes located in telomeric and pericentromeric heterochromatic regions was maintained in the absence of CAF-1 function in Arabidopsis [40]. Another study reported that transcriptional silencing of some heterochromatic regions (e.g. the CACTA transposon) disappeared in the fas mutants [41]. However, the extent of transcriptional activation in this case was not large. Cytological studies revealed that Arabidopsis fas1 and fas2 mutants have reduced heterochromatin content and dispersed pericentromeric DNA, indicating that CAF-1 is required for heterochromatin formation [40,42]. MSI1 is also required for maintaining chromatin organization because plants with reduced MSI1 levels contain reduced amounts of heterochromatin [40,42]. Together, these data indicate that the recruitment of chromosomal DNA into chromocenters depends strongly on CAF-1 [10]. Nevertheless, the DNA methylation pattern characteristic of transcriptionally silenced pericentromeric repeats was normal [40,42]. In summary, a correct CAF-1 function seems to be needed for maintaining the correct pattern of heterochromatin silencing, although the molecular links between CAF-1 and the transcriptional silencing mechanisms still remain to be defined. Euchromatic gene expression The transcriptional status of euchromatin genes is also affected by CAF-1. As mentioned earlier, mutations in the FAS1 and FAS2 genes exhibit drastic and pleiotropic phenotypes, suggesting that the expression of a significant number of genes might be dependent, either directly or indirectly, on CAF-1 function. However, somewhat unexpectedly, genome-wide microarray experiments have revealed that a small number of genes (<2.1% of all Arabidopsis genes) are either up- or downregulated in response to mutations in the CAF-1 gene [40]. These genes www.sciencedirect.com
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are not evenly distributed among the different functional categories but, instead, some categories are enriched in genes with altered expression levels. Thus, genes active in late S-phase, such as the genes encoding histones H3.1, H4 and H3.3 and genes belonging to the DNA repair category, such as BRCA1, RAD51, the gene encoding RNA helicase A and PARP1, among others, are upregulated in the fas mutants [40]. These DNA repair genes are characteristic of the G2 DNA damage checkpoint response and some of them also participate directly in homologous recombination (HR). These experiments suggest that CAF-1 activity is required for transcriptional regulation of genes that function during late S-phase. Transcriptional analysis of specific gene sets corroborated the microarray data. Thus, there is a dramatic increase in the expression of the genes encoding histones H3 and H4 and the G2–M cyclin CYCB1;1. In addition, genes involved in double-strand break (DSB) repair are upregulated, whereas other cell-cycle markers or genes that participate in the non-homologous end-joining (NHEJ) pathway did not change their expression level in fas mutants [34,42,43]. Thus, it seems that loss of CAF-1 activity does not cause a generalized misregulation of gene expression but, instead, leads to transcriptional activation of subset of genes – for example, encoding proteins involved in DSB repair. It should be emphasized that misregulation of gene expression in CAF-1 mutants might involve either epigenetic mechanisms that depend directly on CAF-1 activity and rely on local chromatin changes, or global effects on particular genes. Loss of function in the MSI1 gene, encoding the small subunit of CAF-1, strongly affects homeotic gene expression. Thus, genes such as AGAMOUS (AG) and APETALA2 (AP2), which are not expressed in mature leaves, were drastically upregulated in plants lacking MSI1 function [10]. However, it should be emphasized that these effects are probably CAF-1 independent and MSI1 specific because FAS1 and FAS1 are not required for the correct expression of AG and AP2. This reinforces the idea of CAF1-independent functions of MSI1 due to its participation in other complexes [10]. CAF-1 activity and the status of epigenetic histone marks Transcriptional regulation depends on the presence of specific epigenetic modifications [e.g. acetylation and methylation of lysine (K) residues] to histones located in the regulatory region of target genes. Immunolocalization experiments have shown that dimethylation of lysine 9 of histone H3 (H3K9me2), a typical mark of heterochromatin in Arabidopsis [44–46], remains enriched in the heterochromatin of fas1 nuclei [40,42], suggesting that CAF-1 function is not required, at least to maintain this epigenetic mark. In contrast to plants, the trimethylation of the K9 of histone H3 (H3K9me3) is an epigenetic hallmark of heterochromatin in human cells. These marks need to be propagated to daughter DNA strands in coordination with DNA replication. In this context, it has been found that the human methyl-CpG binding protein MBD1 tethers the histone H3K9 methylase SETDB1 to the p150 subunit of CAF-1 in an S-phase-specific manner [47]. Therefore, given
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the conservation in plants of many proteins involved in chromatin dynamics – for example, histone acetylases, deacetylases and methyltransferases, methyl CpG-binding proteins (http://www.chromDB.org) [2] – it is conceivable that a plant MBD-like protein, as yet unidentified, could also be responsible for coordinating DNA replication with chromatin assembly through CAF-1 and the maintenance of appropriate epigenetic marks. Current evidence supports the notion that the chromatin of fas1 mutants has a more ‘open’ conformation compared with wild type, an idea reinforced by the observation that the genomic DNA of fas1 nuclei is significantly more sensitive to DNaseI digestion than the wild type [42]. FAS2 is required for the correct establishment of the root epidermal pattern [48]. In addition, three-dimensional fluorescence in situ hybridization on intact root tissue indicates that most nuclei of root epidermal cells in fas2 mutants contain chromatin in an ‘open’ state covering the entire chromosomal region where the GLABRA2 gene (GL2) is located [48,49]. A recent study demonstrated that histone modifications associated with regulation of GL2 expression, which depends on GEM, a GL2 expression modulator protein, occur within a 300-base pair stretch in the GL2 promoter immediately upstream from the ATG and are sufficient to regulate GL2 gene expression [50]. How this observation relates to that in fas2 mutants needs to be addressed. In line with these observations, the transcriptional activation of several euchromatic genes (see earlier discussion on BRCA1 and RAD51), the expression of which is upregulated in fas1 plants, does not occur as a result of global changes in chromatin organization over large chromosomal regions [34]. ChIP assays show that promoters of genes upregulated in fas1 plants are enriched in acetylated histones H3 and H4 but are deprived of H3K9me2. Therefore, FAS1 loss provokes gene-specific removal of epigenetic silencing marks of transcribed genes preferentially within the regulatory region of the genes [34]. In short, the introduction and maintenance of histone epigenetic marks associated with regulation of gene expression seems to depend on the correct functioning of CAF-1, although whether it participates directly in these processes or is an indirect consequence of CAF-1 loss is not yet fully understood. Associations of the CAF-1, G2 checkpoint and homologous recombination pathways The viability of cac1 and cac2 mutants is not compromised after treatments that produce DNA replication stress but they are hypersensitive to treatment with zeocin, which produces DSBs [51]. These mutations also result in genome instability of the yeast cells [52,53]. In Arabidopsis, fas mutants are hypersensitive to alkylating agents, such as methyl methanesulfonate [54] and to DNA replication stress and DSB-inducing treatments [34]. A slight hypersensitivity to g-ray and UV-C irradiation of fas mutants has also been observed [42,43]. Thus, mutations that lead to abnormal CAF-1 confer hypersensitivity to a variety of DNA-damaging treatments. Direct quantification of DSB production has revealed that even in the absence of external damage, fas1 mutant plants still contain increased numbers of DSBs www.sciencedirect.com
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[43]. Consistent with this, phosphorylation of the histone variant g-H2AX, a typical marker of DSB production, increases in fas mutants compared with wild type [43]. Concomitantly, an increase in the somatic HR frequency in the fas mutants was also detected [42,43]. Enhanced HR rates and DSB production are both associated with increased expression of repair genes, such as RAD51, PARP1 and BRCA1, which are required for the G2 DNA damage checkpoint, but not Ku70, which participates in the G1 checkpoint and NHEJ [34]. Together, these data suggest that lack of CAF-1 activity might lead to genomic instability in fas mutants as a direct consequence of the delayed assembly of histones. The cellular responses to defects in CAF-1-mediated chromatin assembly during S-phase are transduced through the activation of G2 DNA damage checkpoint genes. As discussed earlier, changes in gene expression might be due to a specific role of CAF-1 in their expression control and/or a consequence of more global changes in chromatin organization. The G2 checkpoint phenotype of fas1 mutants might reflect a general response of plants to increased DNA damage. Thus, chronic exposure of wild-type plants to DSB-inducing agents leads to upregulation of H4, CYCB1;1, RAD51 and BRCA1 expression [34], suggesting that the DNA-damaging treatment partially mimics the fas phenotype. Role of CAF-1 in the endoreplication switch and cell differentiation Proliferating cells in a developing organism can respond to developmental cues to abandon the cell cycle and enter a different cycle, a process termed endoreplication, which is not aimed at producing two daughter cells. Instead, several rounds of genome replication occur in the absence of intervening mitosis, leading to an increase in nuclear DNA content. Endoreplication occurs in both animals and plants but it is far more frequent in plants [55–57]. Furthermore, the occurrence of the endocycle is, in many cases, associated with cell length increase and a prerequisite for plant cell differentiation and organogenesis – for example, growth of hypocotyl in the dark, trichome development and the late stages of leaf development [58]. Because rounds of DNA replication occur during both the cell cycle and the endocycle and CAF-1 is required for DNA replication-associated chromatin assembly, it is conceivable that loss of CAF-1 function can have consequences for cell cycle progression, endoreplication and plant development. Budding yeast lacking CAF-1 activity only arrest temporarily, showing an S-phase delay [59], whereas in cultured human cells, a strong S-phase arrest, leading to cell death in the normal way, is produced [30–32]. In Arabidopsis, the fas mutants are viable but exhibit developmental defects [8,9]. Recently, CAF-1 has been implicated not only in the control of meristem structure, but also in the control of genome replication at several stages of development [60]. Thus, CAF-1 mutants had shorter hypocotyls as a result of having fewer cells, although these still underwent an increased number of DNA endoreplication rounds [60]. Likewise, the number of cells in both the adaxial and abaxial leaf epidermal layers and in the mesophyll is reduced several-fold but they undergo extra
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endoreplication rounds and their size increases [34,60]. Trichome differentiation is also altered in CAF-1 mutants. Thus, the number of branches of trichomes, which correlates approximately with nuclear DNA content in trichomes [61,62], increases in CAF-1 mutants, a phenotype that correlates well with the size of trichome nuclei in plants carrying the fas1 mutation [34,60] but not in the fas2 and msi1 mutants [60]. This indicates that trichome branching alteration is not strictly linked to endoreplication in these mutants. In fact, a systemic increase in the endoreplication level in cells of all organs studied (e.g. leaves, cotyledons, roots and flowers) occurs, associated with a premature switch from the proliferating stage to the endocycle early after germination [34,42,43]. Consistent with extended endoreplication potential, markers of early S-phase genes that participate in DNA replication during the cell cycle and the endocycle are upregulated. In addition, transcription of the mitotic cyclin CYCB1;1, although not other cyclins, was increased in fas mutants. This reinforces the idea that defects in CAF-1 probably result in the activation of a G2 checkpoint that, instead of resulting in an irreversible cell proliferation arrest, as occurs in human cells, produces a switch to the endocycle program. Concluding remarks and perspectives Loss of CAF-1 seems to have deleterious consequences in mammals, whereas plants manage to cope with this defect and can grow to form a mature individual with sufficiently well developed vegetative and reproductive organs to confer viability. The ability of plant cells to trigger the endocycle program in response to DNA damage or defects in chromatin assembly as a consequence of mutations in genes encoding CAF-1 subunits (e.g. in the fas mutant) might be advantageous because it enables growth and development instead of an irreversible cell cycle arrest, as occurs in animals. Although we have clear knowledge of the different cellular processes affected by CAF-1 activity and by CAF-1 loss of function, the mechanisms behind them are not yet fully understood. We still need to define how the alterations in DNA replication-associated chromatin assembly are translated into locus-specific changes of gene expression. It is plausible that histone dynamics, in particular that of histones H3.1 and H3.3, might be modified at certain loci. The consequences, if any, of such alterations provide an avenue for future work [63]. This aspect might relate directly to the mechanism of transcriptional silencing and activation, and to the biology of heterochromatin maintenance. Therefore, we envision that future research will concentrate on determining the biochemical activity of CAF-1, defining the consequences of its activity and its malfunction on transcriptional control and heterochromatin dynamics, and understanding its functional relevance during development. Acknowledgements This work has been partially supported by the Spanish Ministry of Science and Technology (grant BFU2006–5662), and by an institutional grant from Fundacio´n Ramon Areces. www.sciencedirect.com
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28 Enomoto, S. and Berman, J. (1998) Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci. Genes Dev. 12, 219–232 29 Adkins, M.W. et al. (2004) Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes. Mol. Cell 14, 657–666 30 Nabatiyan, A. and Krude, T. (2004) Silencing of chromatin assembly factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis. Mol. Cell. Biol. 24, 2853–2862 31 Hoek, M. and Stillman, B. (2003) Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo. Proc. Natl. Acad. Sci. U. S. A. 100, 12183–12188 32 Ye, X. et al. (2003) Defective S-phase chromatin assembly causes DNA damage, activation of the S- phase checkpoint, and S-phase arrest. Mol. Cell 11, 341–351 33 Reinholz, E. (1966) Radiation induced mutants showing charged inflorescence characteristics. Arabid. Inf. Serv. 3, 19–20 34 Ramirez-Parra, E. and Gutierrez, C. (2007) E2F Regulates FASCIATA1, a chromatin assembly gene whose loss switches on the endocycle and activates gene expression by changing the epigenetic status. Plant Physiol. 144, 105–120 35 Chaboute´, M-E. et al. (2000) Cell cycle regulation of the tobacco ribonucleotide reductase small subunit gene is mediated by E2F-like elements. Plant Cell 12, 1987–2000 36 Stevens, R. et al. (2002) Two E2F sites in the Arabidopsis MCM3 promoter have different roles in cell cycle activation and meristematic expression. J. Biol. Chem. 277, 32978–32984 37 Kosugi, S. and Ohashi, Y. (2002) E2F sites that can interact with E2F proteins cloned from rice are required for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters. Plant J. 29, 45–59 38 Ramirez-Parra, E. et al. (2007) E2F-DP transcription factors. Annu. Plant Rev. 32, 138–162 39 Monson, E.K. et al. (1997) The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres. Proc. Natl. Acad. Sci. U. S. A. 94, 13081–13086 40 Scho¨nrock, N. et al. (2006) Functional genomic analysis of CAF-1 mutants in Arabidopsis thaliana. J. Biol. Chem. 281, 9560–9568 41 Ono, T. et al. (2006) Chromatin assembly factor 1 ensures the stable maintenance of silent chromatin states in Arabidopsis. Genes Cells 11, 153–162 42 Kirik, A. et al. (2006) The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants. Plant Cell 18, 2431–2442 43 Endo, M. et al. (2006) Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants. EMBO J. 25, 5579–5590 44 Fischer, A. et al. (2006) Heterochromatin proteins and the control of heterochromatic gene silencing in Arabidopsis. J. Plant Physiol. 163, 358–368 45 Fransz, P. et al. (2006) Composition and formation of heterochromatin in Arabidopsis thaliana. Chromosome Res. 14, 71–82 46 Fuchs, J. et al. (2006) Chromosomal histone modification patterns, from conservation to diversity. Trends Plant Sci. 11, 199–208 47 Sarraf, S.A. and Stancheva, I. (2004) Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol. Cell 15, 595–605
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48 Costa, S. and Shaw, P. (2006) Chromatin organization and cell fate switch respond to positional information in Arabidopsis. Nature 439, 493–496 49 Costa, S. and Shaw, P. (2007) ‘Open minded’ cells: how cells can change fate. Trends Cell Biol. 17, 101–106 50 Caro, E. et al. (2007) A chromatin link that couples cell division to root epidermis patterning in Arabidopsis. Nature 447, 213– 217 51 Linger, J. and Tyler, J.K. (2005) The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNAdamaging agents. Genetics 171, 1513–1522 52 Kolodner, R.D. et al. (2002) Maintenance of genome stability in Saccharomyces cerevisiae. Science 297, 552–557 53 Myung, K. and Kolodner, R.D. (2003) Induction of genome instability by DNA damage in Saccharomyces cerevisiae. DNA Repair (Amst.) 2, 243–258 54 Takeda, S. et al. (2004) BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis. Genes Dev. 18, 782–793 55 Edgar, B.A. and Orr-Weaver, T.L. (2001) Endoreplication cell cycles: more for less. Cell 105, 297–306 56 Kondorosi, E. et al. (2000) Plant cell-size control: growing by ploidy? Curr. Opin. Plant Biol. 3, 488–492 57 Larkins, B.A. et al. (2001) Investigating the hows and whys of DNA endoreduplication. J. Exp. Bot. 52, 183–192 58 Caro, E. et al. Endoreplication control during plant development In Eukaryotic Cell Cycle (Bryant, J. and Francis, D. eds), Taylor and Francis (in press) 59 Prado, F. et al. (2004) The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange. EMBO Rep. 5, 497–502 60 Exner, V. et al. (2006) Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development. Development 133, 4163–4172 61 Hu¨lskamp, M. et al. (1999) Pattern formation and cell differentiation: trichomes in Arabidopsis as a genetic model system. Int. Rev. Cytol. 186, 147–178 62 Castellano, M.M. et al. (2004) DNA replication licensing affects cell proliferation or endoreplication in a cell type-specific manner. Plant Cell 16, 2380–2393 63 Loyola, A. and Almouzni, G. (2007) Marking histone H3 variants: how, when and why? Trends Biochem. Sci. 32, 425–433 64 Loyola, A. and Almouzni, G. (2004) Histone chaperones, a supporting role in the limelight. Biochim. Biophys. Acta 1677, 3–11 65 Angelov, D. et al. (2006) Nucleolin is a histone chaperone with FACTlike activity and assists remodeling of nucleosomes. EMBO J. 25, 1669–1679 66 Zhu, Y. et al. (2006) NRP1 and NRP2 encode histone chaperones and are required for maintaining postembryonic root growth. Plant Cell 18, 2879–2892 67 Galichet, A. and Gruissem, W. (2006) Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development. Plant Physiol. 142, 1412–1426 68 Tagami, H. et al. (2004) Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51–61
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The many faces of chromatin assembly factor 1 Elena Ramirez-Parra and Crisanto Gutierrez Centro de Biologia Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Chromatin organization requires that histones associate with DNA in the form of nucleosomes the position and composition of which is crucial for chromatin dynamics. Histone chaperones help to deliver specific histone proteins to the sites where chromatin is being newly formed or remodeled. Association of H3–H4 during DNA replication depends on the chromatin assembly factor 1. The study of Arabidopsis plants carrying loss-of-function alleles in each of the three chromatin assembly factor 1 subunits has highlighted the links between chromatin assembly in proliferating cells and other cellular processes. These are the G2 DNA damage checkpoint, homologous recombination, endoreplication control and transcriptional regulation of specific gene sets, all contributing to the plasticity of plants in dealing with alterations in DNA replication-associated chromatin assembly. Introduction In eukaryotes, genomic DNA associates with histone and non-histone proteins, becoming a highly compact structure – chromatin. The basic structural subunit of chromatin is the nucleosome, which contains 150 base pairs of DNA wrapped around a histone octamer core of two molecules of each of the histones H2A, H2B, H3 and H4. Thus, genome duplication consists not only in DNA replication, but also in the reorganization of new histone octamers onto newly synthesized DNA, a process that is initiated by the incorporation of histones H3 and H4. However, histones do not associate with DNA directly to form mature nucleosomes. Instead, histone chaperones (Box 1) facilitate this process by recruiting histones to the chromosomal sites where chromatin is being reconstituted or reorganized [1]. Maintaining chromatin and genome integrity is crucial for the normal development of eukaryotic organisms. Therefore, the function of histone chaperones, particularly in association with DNA replication, is of primary importance. The chromatin assembly factor 1 (CAF-1) chaperone functions in association with the DNA replication machinery to deposit histone H3 and H4 onto DNA, which is the initial event in DNA replication-associated chromatin assembly (Figure 1). Following the identification of genes encoding CAF-1 subunits in plants, their function has been reviewed [2,3]. More recently, various reports have demonstrated that a loss of function of CAF-1 has a pleiotropic effect. Corresponding author: Gutierrez, C. ([email protected]). Available online 9 November 2007. www.sciencedirect.com
Here, we discuss the various cellular processes for which CAF-1 function is required, with the aim of integrating them in the light of recent findings on chromatin organization of genes particularly affected by CAF-1 function. Plant CAF-1 Subunit organization of CAF-1 CAF-1 is a highly conserved heterotrimeric complex. In yeast, where it is known as chromatin assembly complex (CAC), it consists of Cac1, Cac2 and Cac3 subunits [1,4], whereas in mammalian cells, CAF-1 consists of the p150, p60 and p48 subunits [5–7]. In Arabidopsis, the three subunits are encoded by the FASCIATA1 (FAS1), FASCIATA2 (FAS2) and MULTICOPY SUPPRESSOR OF
Glossary Checkpoint: cell-cycle checkpoints are control mechanisms that ensure the fidelity of cell division in eukaryotic cells, controlling whether each phase of the cell cycle has been accurately completed before progression into the next phase. The main function of checkpoints is to assess DNA damage. Chromocenter: a condensed structure formed by the association of centromeric regions of heterochromatin. Double-strand break (DSB): a DNA lesion that affects both DNA strands. DSBs are the consequence of endogenous or exogenous genotoxic agents (ionizing radiation or chemicals). This lesion is repaired mainly by two different pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). E2F: a family of eukaryotic transcription factors that are essential for the G1–Sphase transition but also for the regulation of other cellular and developmental processes. Arabidopsis thaliana contains six E2F proteins (namely, E2Fa–f). They are regulated by the plant retinoblastoma-related (RBR) protein and can function as transcriptional repressors or activators, depending on the E2F member and the cellular and genomic context. Endoreplication: the process of full-genome duplication without mitosis, resulting in an increase in the nuclear DNA content. Endoreplication is a common process in plants, where it has an essential association with developmental programs. Epigenetic: epigenetic regulation or chromatin modification refers to changes in gene expression that are not caused by DNA sequence. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and modifications of histones, such as methylation, acetylation, ubiquitination, phosphorylation and incorporation of histone variants. Euchromatin: chromosomal regions with a high density of genes, which are often under active transcription. Heterochromatin: chromosomal regions, highly condensed throughout the cell cycle, that have been clearly linked to gene silencing. Constitutive heterochromatin is often associated with telomeres and pericentromeric regions of chromosomes and is rich in repetitive, permanently inactive chromosomal regions. However, facultative heterochromatin can be transcriptionally silenced in specific tissues or developmental stages, and remain active in others. Homologous recombination (HR): a DNA repair pathway that requires extensive DNA sequence homology between the interacting strands. Non-homologous end-joining (NHEJ): a DNA repair pathway that requires limited DNA sequence homology, or even no homology, at the ends of the interacting strands.
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Box 1. Histone chaperones A variety of histone chaperones are specialized in incorporating different histones into DNA. One class of histone chaperones is involved in storage and deposition of histone H2A and H2B. Nucleoplasmin, which is a store of histone H2A and H2B, and nucleolin regulate histone exchange [64,65]. In addition, nucleosome assembly protein 1 (NAP-1) and NAP-related protein (NRP) belong to a family of histone chaperones with a high affinity for H2A and H2B that function as nuclear–cytoplasmic shuttle chaperones and are required to maintain postembryonic growth [66,67]. Other histone chaperones are specific for the exchange and/or incorporation of histones H3 and H4. One of these is CAF-1, which facilitates the deposition of histone H3.1–H4 dimers onto DNA followed by the association of H2A–H2B dimers in close association with DNA replication during the S-phase or DNA repair synthesis [68]. By contrast, HIRA incorporates the histone variant H3.3 at any time during the cell cycle or in differentiated cells [68]. In both cases, these chaperones are assisted by ASF1.
IRA1 (MSI1) genes, which encode the large, middle and small subunits, respectively [8–10] (Figure 2). The p150 subunit of human CAF-1 interacts directly with histones H3–H4 through its acidic domain and contains a PEST domain, rich in S and T residues and involved in protein degradation. The PEST domain is not present in the yeast and plant homologs of the human p150 subunit (Figure 2). This subunit also associates with the proliferating cell nuclear antigen (PCNA), which recruits CAF-1 to replication- and repair-coupled chromatin assembly sites [11]. The middle subunit contains seven WD40 repeats, involved in protein–protein interactions and conserved in human, yeast and plants (Figure 2). The smallest subunit (p48, Cac3 or MSI1), which also contains seven WD40 repeats (Figure 2), can interact with histone deacetylases [12,13], suggesting a role in the acetylation–deacetylation cycle during DNA replication-associated chromatin assembly. In addition, MSI proteins associate, independently of
Figure 1. Histone chaperones involved in H3–H4 dynamics. CAF-1 is a heterotrimeric complex that participates in DNA synthesis-associated processes, such as DNA replication and DNA repair, by incorporating histone H3.1. Histone gene repressor A (HIRA) functions in a DNA synthesis-independent manner, depositing the histone variant H3.3. CAF-1 and HIRA are assisted by the histone chaperone antisilencing factor 1 (ASF1). Dotted red lines in the chromatin indicate newly synthesized DNA. Orange shapes indicate H3–H4 dimers and green shapes indicate H2A–H2B dimers. www.sciencedirect.com
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CAF-1, with other proteins, such as retinoblastoma proteins [14–19], regulators of the E2F pathway [20], Polycomb group histone methyltransferases [21–25] and, in plants, regulators of flowering time [26]. In spite of the evolutionary conservation, the consequences of CAF-1 dysfunction are different in unicellular and multicellular eukaryotes. In yeast, loss of CAF-1 activity leads to underassembly of chromatin, produces defects in telomeric and mating-type locus gene silencing and increases genome instability [27–29], although it does not seem to have a direct role in cell cycle progression. In human cells, defects in CAF-1-mediated nucleosome assembly activate the S-phase checkpoint (see Glossary), eventually leading to S-phase arrest and cell death [30– 32]. In Arabidopsis, plants carrying loss-of-function mutations in genes encoding CAF-1 subunits are viable, although they show developmental defects [33]. Thus, the fas1 and fas2 mutants were isolated in a screening for recessive mutations leading to abnormal meristem structure [8]. The mutant plants are fasciated, having broad and flat stems, disrupted leaf phyllotaxy, dentate and narrow leaves, reduced root growth and altered structure of floral organs [8]. The expression of typical markers of the shoot and root apical meristems, such as WUSCHEL (WUS) and SCARECROW (SCR), respectively, is disturbed [9]. Therefore, one current challenge is to identify the variety of cellular processes in which CAF-1 function is relevant and to understand how they interact with each other to make the defects in DNA replication-associated chromatin assembly compatible with development. It is likely that the integration of these processes contributes significantly to the plasticity of plants to cope with alterations in chromatin organization – for example, failure to incorporate histones H3 and H4 during DNA replication. Genes encoding CAF-1 subunits are cell-cycle regulated The activity of CAF-1 during DNA replication is restricted to the S-phase of the cell cycle. Therefore, it is not surprising that FAS1 expression is cell cycle regulated, showing a peak during S-phase [9]. The promoter region of FAS1 contains binding sites for members of the E2F family of transcription factors: TTTGGCGC (at position –345 from the putative initiation codon, ATG) and TTTCCCGCCAAG (at –290) (Figure 3). Electrophoretic mobility shift assays (EMSA) using E2Ff, one of the Arabidopsis E2F proteins, indicates that it can bind to these two E2F sites in vitro. This was confirmed by chromatin immunoprecipitation (ChIP) studies, revealing that E2F can bind directly to the FAS1 promoter in vivo and regulate FAS1 expression [34]. Furthermore, transgenic Arabidopsis plants overexpressing various E2F proteins (such as E2Fa, E2Fb, E2Fd and E2Ff) or deficient in E2Fe, in all cases, show increased FAS1 expression. In contrast, plants overexpressing the repressor E2Fc or a dominant negative version of DP, the dimerization partner of Arabidopsis E2Fa, E2Fb and E2Fc proteins, and required for E2F activity [34], show decreased FAS1 expression. Therefore, these data demonstrate that members of the E2F family regulate FAS1 gene expression, although the nature of the E2F member (or members) involved remains to be identified.
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Figure 2. The three subunits of the CAF-1 heterotrimeric complex are highly conserved in humans, yeast and plants. The names of the different subunits in humans, yeast and plants are indicated (a). The domain organization of the three CAF-1 subunits in yeast, humans and Arabidopsis is shown in the lower part of the figure (b). Note the presence of the highly repeated WD40 domain in the middle and small subunits. The motifs present in each protein are color coded, as indicated.
The role of the E2F binding sites in the spatial regulation of FAS1 expression has been analyzed using reporter plants expressing the b-glucuronidase (uidA) gene under different FAS1 promoter constructs [34]. These studies have demonstrated that proliferating cells in different plant locations possess high levels of FAS1 expression. Deletion of the distal E2F binding site leads to loss of activity, whereas an increase in activity occurs by deleting the proximal E2F site. This led to the conclusion that the distal E2F binding
site is required for enhanced FAS1 expression and the proximal E2F binding site functions as a repressor [34]. Therefore, as reported for other E2F target genes, such as the ribonucleotide reductase RNR2 gene [35], the minichromosome maintenance 3 (MCM3) gene [36] or the PCNA gene [37], the two E2F sites in the FAS1 promoter seem to have distinct regulatory roles both qualitatively and quantitatively [38]. Note that E2F binding sites are also present in FAS2 and MSI1, which encode the other two plant CAF-1
Figure 3. Location of E2F binding sites (blue boxes) in the promoter regions of genes (orange arrows) encoding the large (FAS1), middle (FAS2) and small (MSI1) CAF-1 subunits in Arabidopsis. The numbers inside the coding regions of the genes are the Arabidopsis gene code numbers. The number above each binding site refers to the position of the first nucleotide of the binding site relative to the putative ATG initiator codon. Note that the distal site, relative to the start of the open reading frame, has a reverse orientation. www.sciencedirect.com
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subunits (Figure 3). The FAS2 promoter contains one site (TTTCGCGCCCAAA at –238 from the putative ATG) and the MSI1 promoter contains another single binding site (TGTGGGCGAAT at –120 from the putative ATG). Based on these observations, it is conceivable that E2F might regulate the expression of the three components of CAF-1 in a coordinated manner. Nevertheless, based on microarray data, neither FAS2 nor MSI1 change their expression dramatically during the cell cycle or in plants overexpressing E2Fa and DPa. It should be noted, however, that other well-characterized E2F target genes, such as MCM3, ORC1a (the large subunit of the origin recognition complex) or PCNA [38], which have a peak of expression at the G1–S transition, do not show significant changes in the available microarray data. Therefore, detailed studies are needed to address individually the possible nature of FAS2 and MSI1 genes as E2F targets using complementary approaches. Role of CAF-1 in gene expression Heterochromatin silencing Saccharomyces cerevisiae cac mutants are defective in maintaining stable gene silencing at telomeres and at the mating-type locus [28,39]. In plants, the situation is not yet clear because conflicting reports have appeared on the relevance of CAF-1 in heterochromatin silencing. In one study, the typical transcriptional silencing of genes located in telomeric and pericentromeric heterochromatic regions was maintained in the absence of CAF-1 function in Arabidopsis [40]. Another study reported that transcriptional silencing of some heterochromatic regions (e.g. the CACTA transposon) disappeared in the fas mutants [41]. However, the extent of transcriptional activation in this case was not large. Cytological studies revealed that Arabidopsis fas1 and fas2 mutants have reduced heterochromatin content and dispersed pericentromeric DNA, indicating that CAF-1 is required for heterochromatin formation [40,42]. MSI1 is also required for maintaining chromatin organization because plants with reduced MSI1 levels contain reduced amounts of heterochromatin [40,42]. Together, these data indicate that the recruitment of chromosomal DNA into chromocenters depends strongly on CAF-1 [10]. Nevertheless, the DNA methylation pattern characteristic of transcriptionally silenced pericentromeric repeats was normal [40,42]. In summary, a correct CAF-1 function seems to be needed for maintaining the correct pattern of heterochromatin silencing, although the molecular links between CAF-1 and the transcriptional silencing mechanisms still remain to be defined. Euchromatic gene expression The transcriptional status of euchromatin genes is also affected by CAF-1. As mentioned earlier, mutations in the FAS1 and FAS2 genes exhibit drastic and pleiotropic phenotypes, suggesting that the expression of a significant number of genes might be dependent, either directly or indirectly, on CAF-1 function. However, somewhat unexpectedly, genome-wide microarray experiments have revealed that a small number of genes (<2.1% of all Arabidopsis genes) are either up- or downregulated in response to mutations in the CAF-1 gene [40]. These genes www.sciencedirect.com
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are not evenly distributed among the different functional categories but, instead, some categories are enriched in genes with altered expression levels. Thus, genes active in late S-phase, such as the genes encoding histones H3.1, H4 and H3.3 and genes belonging to the DNA repair category, such as BRCA1, RAD51, the gene encoding RNA helicase A and PARP1, among others, are upregulated in the fas mutants [40]. These DNA repair genes are characteristic of the G2 DNA damage checkpoint response and some of them also participate directly in homologous recombination (HR). These experiments suggest that CAF-1 activity is required for transcriptional regulation of genes that function during late S-phase. Transcriptional analysis of specific gene sets corroborated the microarray data. Thus, there is a dramatic increase in the expression of the genes encoding histones H3 and H4 and the G2–M cyclin CYCB1;1. In addition, genes involved in double-strand break (DSB) repair are upregulated, whereas other cell-cycle markers or genes that participate in the non-homologous end-joining (NHEJ) pathway did not change their expression level in fas mutants [34,42,43]. Thus, it seems that loss of CAF-1 activity does not cause a generalized misregulation of gene expression but, instead, leads to transcriptional activation of subset of genes – for example, encoding proteins involved in DSB repair. It should be emphasized that misregulation of gene expression in CAF-1 mutants might involve either epigenetic mechanisms that depend directly on CAF-1 activity and rely on local chromatin changes, or global effects on particular genes. Loss of function in the MSI1 gene, encoding the small subunit of CAF-1, strongly affects homeotic gene expression. Thus, genes such as AGAMOUS (AG) and APETALA2 (AP2), which are not expressed in mature leaves, were drastically upregulated in plants lacking MSI1 function [10]. However, it should be emphasized that these effects are probably CAF-1 independent and MSI1 specific because FAS1 and FAS1 are not required for the correct expression of AG and AP2. This reinforces the idea of CAF1-independent functions of MSI1 due to its participation in other complexes [10]. CAF-1 activity and the status of epigenetic histone marks Transcriptional regulation depends on the presence of specific epigenetic modifications [e.g. acetylation and methylation of lysine (K) residues] to histones located in the regulatory region of target genes. Immunolocalization experiments have shown that dimethylation of lysine 9 of histone H3 (H3K9me2), a typical mark of heterochromatin in Arabidopsis [44–46], remains enriched in the heterochromatin of fas1 nuclei [40,42], suggesting that CAF-1 function is not required, at least to maintain this epigenetic mark. In contrast to plants, the trimethylation of the K9 of histone H3 (H3K9me3) is an epigenetic hallmark of heterochromatin in human cells. These marks need to be propagated to daughter DNA strands in coordination with DNA replication. In this context, it has been found that the human methyl-CpG binding protein MBD1 tethers the histone H3K9 methylase SETDB1 to the p150 subunit of CAF-1 in an S-phase-specific manner [47]. Therefore, given
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the conservation in plants of many proteins involved in chromatin dynamics – for example, histone acetylases, deacetylases and methyltransferases, methyl CpG-binding proteins (http://www.chromDB.org) [2] – it is conceivable that a plant MBD-like protein, as yet unidentified, could also be responsible for coordinating DNA replication with chromatin assembly through CAF-1 and the maintenance of appropriate epigenetic marks. Current evidence supports the notion that the chromatin of fas1 mutants has a more ‘open’ conformation compared with wild type, an idea reinforced by the observation that the genomic DNA of fas1 nuclei is significantly more sensitive to DNaseI digestion than the wild type [42]. FAS2 is required for the correct establishment of the root epidermal pattern [48]. In addition, three-dimensional fluorescence in situ hybridization on intact root tissue indicates that most nuclei of root epidermal cells in fas2 mutants contain chromatin in an ‘open’ state covering the entire chromosomal region where the GLABRA2 gene (GL2) is located [48,49]. A recent study demonstrated that histone modifications associated with regulation of GL2 expression, which depends on GEM, a GL2 expression modulator protein, occur within a 300-base pair stretch in the GL2 promoter immediately upstream from the ATG and are sufficient to regulate GL2 gene expression [50]. How this observation relates to that in fas2 mutants needs to be addressed. In line with these observations, the transcriptional activation of several euchromatic genes (see earlier discussion on BRCA1 and RAD51), the expression of which is upregulated in fas1 plants, does not occur as a result of global changes in chromatin organization over large chromosomal regions [34]. ChIP assays show that promoters of genes upregulated in fas1 plants are enriched in acetylated histones H3 and H4 but are deprived of H3K9me2. Therefore, FAS1 loss provokes gene-specific removal of epigenetic silencing marks of transcribed genes preferentially within the regulatory region of the genes [34]. In short, the introduction and maintenance of histone epigenetic marks associated with regulation of gene expression seems to depend on the correct functioning of CAF-1, although whether it participates directly in these processes or is an indirect consequence of CAF-1 loss is not yet fully understood. Associations of the CAF-1, G2 checkpoint and homologous recombination pathways The viability of cac1 and cac2 mutants is not compromised after treatments that produce DNA replication stress but they are hypersensitive to treatment with zeocin, which produces DSBs [51]. These mutations also result in genome instability of the yeast cells [52,53]. In Arabidopsis, fas mutants are hypersensitive to alkylating agents, such as methyl methanesulfonate [54] and to DNA replication stress and DSB-inducing treatments [34]. A slight hypersensitivity to g-ray and UV-C irradiation of fas mutants has also been observed [42,43]. Thus, mutations that lead to abnormal CAF-1 confer hypersensitivity to a variety of DNA-damaging treatments. Direct quantification of DSB production has revealed that even in the absence of external damage, fas1 mutant plants still contain increased numbers of DSBs www.sciencedirect.com
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[43]. Consistent with this, phosphorylation of the histone variant g-H2AX, a typical marker of DSB production, increases in fas mutants compared with wild type [43]. Concomitantly, an increase in the somatic HR frequency in the fas mutants was also detected [42,43]. Enhanced HR rates and DSB production are both associated with increased expression of repair genes, such as RAD51, PARP1 and BRCA1, which are required for the G2 DNA damage checkpoint, but not Ku70, which participates in the G1 checkpoint and NHEJ [34]. Together, these data suggest that lack of CAF-1 activity might lead to genomic instability in fas mutants as a direct consequence of the delayed assembly of histones. The cellular responses to defects in CAF-1-mediated chromatin assembly during S-phase are transduced through the activation of G2 DNA damage checkpoint genes. As discussed earlier, changes in gene expression might be due to a specific role of CAF-1 in their expression control and/or a consequence of more global changes in chromatin organization. The G2 checkpoint phenotype of fas1 mutants might reflect a general response of plants to increased DNA damage. Thus, chronic exposure of wild-type plants to DSB-inducing agents leads to upregulation of H4, CYCB1;1, RAD51 and BRCA1 expression [34], suggesting that the DNA-damaging treatment partially mimics the fas phenotype. Role of CAF-1 in the endoreplication switch and cell differentiation Proliferating cells in a developing organism can respond to developmental cues to abandon the cell cycle and enter a different cycle, a process termed endoreplication, which is not aimed at producing two daughter cells. Instead, several rounds of genome replication occur in the absence of intervening mitosis, leading to an increase in nuclear DNA content. Endoreplication occurs in both animals and plants but it is far more frequent in plants [55–57]. Furthermore, the occurrence of the endocycle is, in many cases, associated with cell length increase and a prerequisite for plant cell differentiation and organogenesis – for example, growth of hypocotyl in the dark, trichome development and the late stages of leaf development [58]. Because rounds of DNA replication occur during both the cell cycle and the endocycle and CAF-1 is required for DNA replication-associated chromatin assembly, it is conceivable that loss of CAF-1 function can have consequences for cell cycle progression, endoreplication and plant development. Budding yeast lacking CAF-1 activity only arrest temporarily, showing an S-phase delay [59], whereas in cultured human cells, a strong S-phase arrest, leading to cell death in the normal way, is produced [30–32]. In Arabidopsis, the fas mutants are viable but exhibit developmental defects [8,9]. Recently, CAF-1 has been implicated not only in the control of meristem structure, but also in the control of genome replication at several stages of development [60]. Thus, CAF-1 mutants had shorter hypocotyls as a result of having fewer cells, although these still underwent an increased number of DNA endoreplication rounds [60]. Likewise, the number of cells in both the adaxial and abaxial leaf epidermal layers and in the mesophyll is reduced several-fold but they undergo extra
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endoreplication rounds and their size increases [34,60]. Trichome differentiation is also altered in CAF-1 mutants. Thus, the number of branches of trichomes, which correlates approximately with nuclear DNA content in trichomes [61,62], increases in CAF-1 mutants, a phenotype that correlates well with the size of trichome nuclei in plants carrying the fas1 mutation [34,60] but not in the fas2 and msi1 mutants [60]. This indicates that trichome branching alteration is not strictly linked to endoreplication in these mutants. In fact, a systemic increase in the endoreplication level in cells of all organs studied (e.g. leaves, cotyledons, roots and flowers) occurs, associated with a premature switch from the proliferating stage to the endocycle early after germination [34,42,43]. Consistent with extended endoreplication potential, markers of early S-phase genes that participate in DNA replication during the cell cycle and the endocycle are upregulated. In addition, transcription of the mitotic cyclin CYCB1;1, although not other cyclins, was increased in fas mutants. This reinforces the idea that defects in CAF-1 probably result in the activation of a G2 checkpoint that, instead of resulting in an irreversible cell proliferation arrest, as occurs in human cells, produces a switch to the endocycle program. Concluding remarks and perspectives Loss of CAF-1 seems to have deleterious consequences in mammals, whereas plants manage to cope with this defect and can grow to form a mature individual with sufficiently well developed vegetative and reproductive organs to confer viability. The ability of plant cells to trigger the endocycle program in response to DNA damage or defects in chromatin assembly as a consequence of mutations in genes encoding CAF-1 subunits (e.g. in the fas mutant) might be advantageous because it enables growth and development instead of an irreversible cell cycle arrest, as occurs in animals. Although we have clear knowledge of the different cellular processes affected by CAF-1 activity and by CAF-1 loss of function, the mechanisms behind them are not yet fully understood. We still need to define how the alterations in DNA replication-associated chromatin assembly are translated into locus-specific changes of gene expression. It is plausible that histone dynamics, in particular that of histones H3.1 and H3.3, might be modified at certain loci. The consequences, if any, of such alterations provide an avenue for future work [63]. This aspect might relate directly to the mechanism of transcriptional silencing and activation, and to the biology of heterochromatin maintenance. Therefore, we envision that future research will concentrate on determining the biochemical activity of CAF-1, defining the consequences of its activity and its malfunction on transcriptional control and heterochromatin dynamics, and understanding its functional relevance during development. Acknowledgements This work has been partially supported by the Spanish Ministry of Science and Technology (grant BFU2006–5662), and by an institutional grant from Fundacio´n Ramon Areces. www.sciencedirect.com
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