Chromatin-Remodeling and Memory Factors. New Regulators of Plant Development

The establishment and heritable maintenance of specific epigenetic states that lead to differential gene expression are crucial for cell differentiation and development. Over the past few years, it has become apparent that epigenetic control of transcription is mediated through specific states of the chromatin structure. Therefore, changes in the chromatin structure associated with activation and silencing of gene expression are of paramount importance during development. Here, we discuss recent findings on plant proteins involved in modifying, remodeling, or maintenance of chromatin structures. Many of the proteins affect normal development when their function is lost. Cell differentiation and development are controlled through temporal and spatial activation and silencing of specific genes. Once established, cell type-specific pattern of gene expression must be stable over many cell generations and long after inductive developmental signals have disappeared. Although chromatin assembly is still largely unexplored, genetic and biochemical studies in yeast (Saccharomyces cerevisiae), fruitfly (Drosophila melanogaster), and mammals have already revealed that changes in expression patterns require remodeling of the chromatin structure at promoters and other regulatory regions of DNA (for recent reviews, see Kingston and Narlikar, 1999; Aalfs and Kingston, 2000;Urnov and Wolffe, 2001). Thus, packaging of DNA into nucleosomes and higher order structures represents an obstacle to regulatory DNA-binding proteins (e.g. see Niu et al., 1996) and RNA polymerases (Williamson and Felsenfeld, 1978), thereby perhaps imposing a default state in which genetic information is repressed. Chromatin remodeling alters this basal state by promoting either an “open” (activation of transcription) or a “closed” (repression of transcription) chromatin configuration. The propagation of specific transcriptional states to daughter cells through mitosis or even meiosis invokes the stable inheritance of chromatin structures. Chromatin modifiers involved in Arabidopsis development Chromatin modifiers involved in Arabidopsis development Summary of Arabidopsis chromatin components that control development. Time windows of known functions are schematically represented below the protein names (for details, see text). CAF-1 subunit proteins are conserved in different organisms. CAF-1 (CAC) subunits are shown from humans (red), Arabidopsis (green), and yeast (yellow). Numbers represent amino acid positions and percentage of similarity between human and Arabidopsis, and Arabidopsis and yeast, respectively. CAF-1 subunits were termed CAC1, CAC2, and CAC3 in yeast. The corresponding subunits in human and Arabidopsis are p150/FAS1, p60/FAS2, and p48/AtMSI1. CAC1/p150/FAS1 contains a highly charged KER domain, which consists mainly of Lys, Glu, and Arg residues, and an ED region, which consists of Glu and Asp residues. The KER domain is believed to form a coiled-coil structure, and the acidic ED region could interact with basic histones (Kaya et al., 2001). CAC2/p60/FAS2 and CAC3/p48/AtMSI1 contain seven WD40 repeats, which are likely to mediate protein-protein interactions. For MSI-like proteins, direct interaction with histones has been shown (e.g. Rossi et al., 2001). In the Arabidopsis fasciated mutants fas1 andfas2 (Reinholz, 1966; Leyser and Furner, 1992), the corresponding CAC1 and CAC2 genes are disrupted (Kaya et al., 2001). Both mutants show a spectrum of developmental changes caused by the enlarged shoot apical meristem (SAM), including altered phyllotaxy, leaf shape, root growth, and flower organ number. The domain of WUSCHEL expression, usually confined to the SAM quiescent center, is expanded in fas1 andfas2. The expression of SCARECROW, which directs radial patterning in roots and stems, is also affected (Kaya et al., 2001). Thus, CAF-1 appears to be important for the maintenance of plant developmental gene expression patterns. A likely role of CAF-1 in development has also been demonstrated in Xenopus laevisoocytes (Quivy et al., 2001) and mammalian cells (Tchenio et al., 2001). In yeast, CAF-1 is required for stable inheritance of gene silencing (Kaufman et al., 1997; Monson et al., 1997), suggesting that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals. Heredity of histone acetylation patterns during DNA replication. When a genomic region containing deacetylated histones and a condensed chromatin structure is replicated, chromatin structure is transiently altered as a consequence of the incorporation of de novo synthesized histones, which have been acetylated in the cytoplasm by class B HATs and lack any epigenetic information. In contrast, pre-existing histone octamers are segregated equally onto the two daughter DNA molecules, providing information about the degree of acetylation that can be interpreted by HDACs and other protein complexes. How are histone acetylation patterns inherited during development? During DNA replication, histone octamers of the parental DNA molecule segregate approximately equally on the daughter DNA molecules (Fig. 3). New octamers required for chromatin packaging are assembled from de novo synthesized histones. Histones with specific acetylation patterns could therefore be maintained throughout mitosis (Perry et al., 1993) and may serve as templates for HATs and HADCs to modify the newly assembled octamers accordingly. This would explain why alteration of acetylation status induced by trychostatin A, an inhibitor of HDACs, can be epigenetically propagated after the drug has been removed (Ekwall et al., 1997). It should be noted, however that, at present, no evidence exists for epigenetic inheritance of induced changes in acetylation patterns in plants. HATs have been classified into two categories depending on their subcellular distribution (Roth et al., 2001). Type B HATs are cytoplasmic complexes involved in acetylation of histone H4 at positions 5 and 12 before its incorporation into nucleosomes. Maize (Zea mays) HAT type B is a heterodimeric complex (Lusser et al., 1999). The gene for the 50-kD enzymatic subunit is homologous to yeast HAT1. The 45-kD subunit is immunologically related to the mammalian Rb-associated protein (RbAp) and yeast/plant MSI1 proteins (Ach et al., 1997), which are also found in other chromatin-remodeling complexes (see above). HAT type A are nuclear proteins that fall into four classes with different specificities: the GCN5, CBP/p300, TAFII 250, and MYST family of proteins (Marmorstein, 2001). Open reading frames with homology to members of all of these families are present in the Arabidopsis genome. HAT activity has been demonstrated for Arabidopsis p300 and GCN5 homologs, but their functions are still unknown (Bordoli et al., 2001;Stockinger et al., 2001). HATs of the CBP/p300 and GCN5 families are recruited to promoters by specific transcription factors in animals and yeast. Interestingly, Arabidopsis CBF1, a transcription activator involved in cold-regulated gene expression, can interact with GCN5 in vitro (Stockinger et al., 2001), suggesting that similar recruitment of HATs to promoter regions also occurs in plants. The situation for HDACs is equally complex. Four enzyme types have been identified in eukaryotes. Classes I and II compose proteins that are homologous to yeast Rpd3 and HDA1, respectively. Class III proteins share similarity with yeast Sir2 (Khochbin et al., 2001), which unlike class I and II enzymes has NAD+-dependent HDAC activity and ADP-ribosyltransferase activity in vitro. Members of these three classes are present in all eukaryotes including plants. A fourth class of HDACs, whose founding member is the maize HD2 protein (Lusser et al., 1997), has been identified only in plants. At least five class I HDACs are present in the Arabidopsis genome, but functional information is available for only two of them. HDA19 (following the nomenclature of the Chromo database athttp://www.chromdb.org; also termed AtHD1 or AtRPD3A) is expressed at high levels in leaves, stems, flowers, and young siliques. An HDA19 fusion protein can repress transcription when tethered to a promoter through a DNA-binding domain (Wu et al., 2000a), providing direct evidence that HDACs are involved in transcriptional repression in plants. Histone H4 is hyperacetylated in ArabidopsisHDA19 antisense RNA mutants, and plants have developmental abnormalities, including early senescence, suppression of apical dominance, homeotic changes, male and female sterility, and delay of flowering (Tian and Chen, 2001). In contrast, mutations in theHDA6 gene (also called AtRPD3B) increase expression of GUS and HPH transgenes without affecting Arabidopsis development, suggesting that HDA6 plays a more specific role in gene silencing (Murfett et al., 2001). Maize HD2 is a plant-specific HDAC not related to other known HDAC enzymes (Lusser et al., 1997). The maize protein is localized in the nucleolus, suggesting a possible role in the regulation of rRNA genes. Four Arabidopsis gene products (HDA4, HDA3, HDA11, and HDA13) share high sequence similarity with the maize HD2 protein (Wu et al., 2000b). Antisense HDA3 transgenic plants resulted in plants with stunted siliques that contain a high number of aborted seeds, suggesting that HDA3 is involved in embryo development. HDACs are recruited to specific loci by interacting directly or indirectly with DNA-binding proteins that regulate development. For example, Drosophila melanogaster Groucho is a transcriptional corepressor that recruits Rpd3 (Chen et al., 1999) and interacts with transcription factors such as Engrailed or Dorsal. Groucho has three functionally distinct domains: a Gln-rich region, a Gly/Pro-rich region, and six WD40-repeats at the C terminus. Yeast TUP1 repressor (Wu et al., 2001) and LEUNIG (LUG), a repressor of AGAMOUS (AG) expression in Arabidopsis, have striking similarity with Groucho (Conner and Liu, 2000). AG is a class C floral homeotic MADS box gene, which is expressed in whorls 3 and 4 of developing flowers (Yanofsky et al., 1990). AG mRNA is ectopically expressed inlug mutants, resulting in homeotic transformations of floral organ identity. Other mutant phenotypes are independent ofAG, suggesting that LUG is controlling several target genes during Arabidopsis development. Whether LUG interacts with HDACs is currently unknown. In addition to acetylation, methylation is another specific histone posttranslational modification in plants (Waterborg, 1990) and other organisms. But the function of histone methylation was not known until Su(var)3-9, a fruitfly heterochromatin-associated protein involved in position effect variegation, was identified as a histone methyltransferase (Rea et al., 2000). Su(var)3-9 and its yeast and human counterparts contain a SET-domain (first identified inSu(var)3-9, E(z), and trithorax), which is necessary for catalysis and specific methylation of K9 in histone H3. Methylation of this Lys appears to be required during heterochromatin formation, becauseSu(var)3-9 mutants are impaired in heterochromatin-mediated gene silencing (Nakayama et al., 2001). Interestingly, H3 is also methylated at K9 in Arabidopsis heterochromatin (Z. Jasencakova, W. Soppe, P. Fransz, A. Houben, and I. Schubert, unpublished data; see “Note Added in Proof”). Of the more than 30 SET-domain proteins present in the Arabidopsis genome (Baumbusch et al., 2001), nine are homologous to Su(var)3-9. One of them, KRYPTONITE (KYP), is the only plant SET-domain protein for which histone methyltransferase activity has been demonstrated (Jackson et al., 2002). Similar to human SUV39h1, KYP methylates histone H3 at Lys 9. The kyp mutant was isolated as a suppressor of gene silencing at the Arabidopsis SUPERMAN locus. Interestingly,kyp plants show a strong decrease of cytosine methylation at CpNpGp sites, indicating a connection between histone methylation and DNA methylation. The kyp mutants do not show morphological defects in a wild-type background, suggesting that KYP is not directly involved in developmental control. However, given the large number of genes for SET-domain proteins identified in the Arabidopsis genome, it is tempting to speculate that histone methylation will be a regulatory factor in different aspects of gene expression (see below), including control of developmental genes. The role of histone methylation may be better understood through the analysis of proteins that recognize this modification. Heterochromatin protein 1 (HP1), a conserved heterochromatin-associated protein present in animals, fission yeast (Schizosaccharomyces pombe), and plants, contains a chromo domain and chromo-shadow domain. The HP1 chromo domain interacts specifically with K9-methylated H3, which explains the association of these proteins with heterochromatin (Bannister et al., 2001; Lachner et al., 2001). Subsequent oligomerization of HP1 via the chromo-shadow domain may then propagate and maintain heterochromatin structures and gene silencing (Jenuwein, 2001). The Arabidopsis HP1 homolog LHP1 (LIKE HP1) can also interact with histone H3 methylated at Lys 9 (Jackson et al., 2002). Mutations in LHP1 cause early flowering, a general reduction in plant and leaf epidermal cell size, and several other phenotypes (Gaudin et al., 2001). Thus, LHP1 may be required for silencing genes involved in the transition to flowering and other developmental processes. Expression of CONSTANS, a gene regulating time-to-flowering, is increased in lhp1, although it is currently unknown ifCONSTANS is a direct target of LHP1. LHP1 has been shown to interact with CHROMOMETHYLASE3, a DNA methyltransferase specific for CpXpG trinucleotides (Lindroth et al., 2001). cmt3 mutants show no obvious developmental defects but exhibit decreased CpXpG methylation of the SUP gene and other sequences throughout the genome, suggesting that LHP1 may be involved in silencing of methylated loci. The different phenotypes of cmt3 and plants also that LHP1 can function of of will the role of LHP1 for regulation of heterochromatin structure and which could also affect or genomic in the histone of histone H3 is required for during mitosis and in transcription activation in H3 of at mitosis in the chromatin at and at this is with is currently et al., 1999; and 2000). 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During the few years, an number of developmental mutants has been identified in which genes chromatin factors are Thus, in plant development is in a transition from direct transcriptional control to that control chromatin structure and In it is to why developmental are only more genes for functions but in the Mutations in specific transcription factors in phenotypes that can be mutations that affect chromatin factors or more complex and which are more to the other several mutants affected in chromatin factors of and have been isolated in genetic to or suppressor The number of identified target genes directly controlled by chromatin factors is still But even early already that chromatin factors control the expression of specific developmental transcription Thus, chromatin factors can be as the in the regulatory of factors controlling plant development. But and at this many mutants affected in plant chromatin factors are not chromatin factors have perhaps only an role or a high degree of functional The transition to flowering in Arabidopsis is the the SAM has to during development. 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How these and are inherited through meiosis and their are for plant will be a and a and have demonstrated chromatin that and genes localized in the of Arabidopsis are associated with histone H3 methylated at K9 in Interestingly, in mutant not only DNA methylation is but H3 K9 methylation is largely by methylation of the between DNA methylation and histone methylation. This about DNA methylation. The is available in of A. A. Houben, I. Schubert, and for providing information before