Roles of plant retinoblastoma protein: cell cycle and beyond
2020; Springer Nature; Volume: 39; Issue: 19 Linguagem: Inglês
10.15252/embj.2020105802
ISSN1460-2075
AutoresBénédicte Desvoyes, Crisanto Gutiérrez,
Tópico(s)Plant Molecular Biology Research
ResumoReview31 August 2020Open Access Roles of plant retinoblastoma protein: cell cycle and beyond Bénédicte Desvoyes Bénédicte Desvoyes Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Crisanto Gutierrez Corresponding Author Crisanto Gutierrez [email protected] orcid.org/0000-0001-8905-8222 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Bénédicte Desvoyes Bénédicte Desvoyes Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Crisanto Gutierrez Corresponding Author Crisanto Gutierrez [email protected] orcid.org/0000-0001-8905-8222 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain Search for more papers by this author Author Information Bénédicte Desvoyes1 and Crisanto Gutierrez *,1 1Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain *Corresponding author. Tel: +34 911964638; E-mail: [email protected] The EMBO Journal (2020)39:e105802https://doi.org/10.15252/embj.2020105802 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The human retinoblastoma (RB1) protein is a tumor suppressor that negatively regulates cell cycle progression through its interaction with members of the E2F/DP family of transcription factors. However, RB-related (RBR) proteins are an early acquisition during eukaryote evolution present in plant lineages, including unicellular algae, ancient plants (ferns, lycophytes, liverworts, mosses), gymnosperms, and angiosperms. The main RBR protein domains and interactions with E2Fs are conserved in all eukaryotes and not only regulate the G1/S transition but also the G2/M transition, as part of DREAM complexes. RBR proteins are also important for asymmetric cell division, stem cell maintenance, and the DNA damage response (DDR). RBR proteins play crucial roles at every developmental phase transition, in association with chromatin factors, as well as during the reproductive phase during female and male gametes production and embryo development. Here, we review the processes where plant RBR proteins play a role and discuss possible avenues of research to obtain a full picture of the multifunctional roles of RBR for plant life. Introduction Formation of organs, either during embryogenesis as in animals or post-embryonically as in most plants, relies on a continuous supply of new cells. Failure to properly coordinate cell division, cell cycle exit into cell differentiation, and cell fate acquisition frequently results in abnormal growth, developmental aberrations, or cell transformation and tumorigenesis. Strict control of cell cycle progression is necessary to achieve the goal of producing two daughter cells. A plethora of studies has demonstrated that progression through G1 phase and into S-phase, as well as the G2-to-M and metaphase-to-anaphase transitions, represents key checkpoints during the cell cycle. A crucial role in both transitions is played by cyclin-dependent kinases (CDK), as first demonstrated in the fission yeast Schizosaccharomyces pombe (Nurse & Bissett, 1981). Later, it was shown that human cells contain homologs of the yeast Cdc2 CDK (Lee & Nurse, 1987). Cdc2 homologs were found also in plant cells, with their phosphorylation state being cell cycle-dependent (John et al, 1989). These studies paved the way for identifying Cdc2-like kinases (John et al, 1989; Feiler & Jacobs, 1990; Ferreira et al, 1991), as well as their A- and B-type cyclin partners (Hata et al, 1991; Hemerly et al, 1992; Hirt et al, 1992), in various plant species. Identification of the CDK/cyclin targets controlling the G1/S transition proved to be a difficult task, which was enlightened by research in cancer. It was found that cells of rare human tumors, such as retinoblastoma or sarcoma, harbor inactivating mutations in the retinoblastoma susceptibility gene, RB1 (Friend et al, 1986; Fung et al, 1987; Lee et al, 1987). RB1 suppresses cell proliferation and can bind oncoviral proteins such as SV40 large T-antigen (T-ag; Lee et al, 1987; DeCaprio et al, 1988; Dyson et al, 1989; Ludlow et al, 1989). Moreover, RB1 was found to be phosphorylated in a cell cycle-dependent manner by CDKs (Lees et al, 1991). Together, these results led to the proposal that RB1 is a regulator of cell cycle progression in G1 (Buchkovich et al, 1989). The last piece in the initial puzzle was the identification of E2F (for "adenovirus early gene 2 promoter-binding factor") as a cellular protein that could form complexes with RB1 (Chellappan et al, 1991). These and other findings served to establish that the RB1 complexes are regulators of the G1/S transition, as well as of the switch from quiescence to proliferation (reviewed in Fischer & Müller, 2017). Other RB1 interactors, primarily D-type cyclins, bind RB1 through a LxCxE amino acid motif, an interaction that is mimicked by human oncoviral proteins that inactivate RB1's tumor suppressor function (DeCaprio, 2009); however, although the finding of the LxCxE motif in D-type cyclins proved originally valuable, more recent work deleting this motif in mammalian cells has demonstrated that it is not essential (Landis et al, 2007; Topacio et al, 2019). Retinoblastoma protein in plants Back in the mid-1990s, a couple of apparently unrelated research lines reinforced the hypothesis that plants might share some kind of RB/E2F regulatory module with human cells. D-type cyclin homologs were identified in several plant species (Dahl et al, 1995; Soni et al, 1995), which had—despite limited amino acid sequence homology with human D-type cyclins—two key features in common with them: a similar expression pattern during the cell cycle and a highly conserved LxCxE amino acid motif (Soni et al, 1995; Riou-Khamlichi et al, 1999). These facts strongly suggested that plants might contain proteins that could recognize this motif, as it is the case for human cyclin D and RB1. Independently, geminiviruses, a group of plant DNA viruses that replicate their single-stranded DNA genome in the nucleus of the infected cell, were found to trigger synthesis of the host cell nuclear DNA upon infection (Nagar et al, 1995). Additionally, wheat dwarf virus (WDV) and other monocotyledonous plant-infecting geminiviruses were found to encode an LxCxE motif-containing protein, RepA (Xie et al, 1995). Importantly, this motif required for efficient viral DNA replication was shown to enable interaction of RepA also with human RB1 (Xie et al, 1995), in a manner analogous to human oncoviral proteins. Subsequent studies identified genes encoding retinoblastoma-related (RBR1) proteins in maize (Grafi et al, 1996; Xie et al, 1996; Ach et al, 1997a), and in an immediate follow-up to the cloning of multiple plant E2F and DP factors (Ramirez-Parra et al, 1999; Sekine et al, 1999; Albani et al, 2000; Magyar et al, 2000; Ramirez-Parra & Gutierrez, 2000; Kosugi & Ohashi, 2002). Remarkably, Arabidopsis was found to encode six E2F family members with two types of domain organization. The first group (E2FA, B and C) possesses the same domains as human E2F1-5, including DNA-binding and dimerization domains. The second group (E2FD, E and F, also known as DEL2, 1 and 3, respectively) is unique in that its members contain a duplicated DNA-binding domain but lack the dimerization domain that allows binding to DNA in the absence of DP factors and fails to activate gene expression (Kosugi & Ohashi, 2002; Mariconti et al, 2002). Proteins homologous to these atypical E2F family members were subsequently also identified in mammalian cells (reviewed in Trimarchi & Lees, 2002; Lammens et al, 2009). Genome-wide studies identified putative E2F target genes in the Arabidopsis genome (Ramirez-Parra et al, 2003; Vandepoele et al, 2005; Naouar et al, 2009), a list that—somewhat surprisingly at the time—contained not only bona fide cell cycle control genes, but also genes involved in many other aspects of plant physiology, strongly pointing to a multifunctional role of RBR1. This will be further discussed below. Evolutionary perspective on plant RBR proteins The availability of multiple plant genomes has revealed the presence of RBR-, E2F-, and DP-encoding genes in all species analyzed so far (reviewed in detail in Gutzat et al, 2014; Desvoyes et al, 2014). A first conclusion from the genomic data is that the appearance of the RBR-E2F/DP module preceded the multiple branches of multicellularity that occurred ~800 million years ago, since it is present in unicellular organisms such as Chlamydomonas reinhardtii (Umen & Goodenough, 2001) and Ostreococcus tauri (Robbens et al, 2005), as well as colonial algae such as Volvox carteri (Kianianmomeni et al, 2008; Fig 1). Within the multicellular plant lineages, RBR proteins are present in all of them, and notably, monocotyledonous plants possess several members with different functions (Fig 1; see discussion below); for a more detailed discussion on how the RBR-E2F/DP module has evolved in plants and animals, please refer to (Desvoyes et al, 2014). It is likely that RBR and other family members may have evolved specific functions in the different plant lineages. Thus, contrary to dicotyledonous plants that encode for a single RBR1 protein, monocotyledonous plants such as maize or rice contain two major RBR subfamilies: one whose members are involved in negative regulation of cell cycle, e.g., maize RBR1 and RBR2, and another involved in endosperm development, e.g., maize RBR3 and RBR4 (Sabelli et al, 2005; Sabelli & Larkins, 2006; Lendvai et al, 2007). Figure 1. Phylogenetic relationships of RB family members from representative animal and plant lineagesHomo sapiens (Human, mammal); Drosophila melanogaster (Artropoda); Chlamydomonas reinhardtii (Algae, unicellular); Volvox carteri (Algae, colonial); Azolla filliculoides (Fern); Selaginella moellendorffii (Lycophyte); Marchantia polymorpha (Liverwort); Physcomytrella patents (Moss); Pinus silvestris (Gymnosperm); Arabidopsis thaliana (Angiosperm, dicotyledonous); Zea mays (Angiosperm, monocotyledonous). Download figure Download PowerPoint The current data are consistent with the idea that the RBR-E2F/DP module is an ancient invention likely present already in the last eukaryotic common ancestor (LECA; Desvoyes et al, 2014). Later, the RBR/E2F-DP module has been lost in some plant lineages, e.g., Ulvophyceae (De Clerck et al, 2018), and in other eukaryotes, e.g., S. cerevisiae and other yeast (Desvoyes et al, 2014). It is likely that organisms lacking RBR or related proteins use different pathways to regulate G1 progression. In S. cerevisiae, the Whi5 protein (Jorgensen et al, 2002), which is not homologous to animal RB1 or plant RBR1, seems to play analogous functions, whereby increased Cdk activity associated with the G1 cyclin Cln3 mediates activation of the G1 transcription factor heterodimer Swi4/Swi6, also known as SBF. Like RB proteins, Whi5 negatively regulates SBF, and its phosphorylation by Cdk/Cln3 complexes frees SBF to activate its targets (de Bruin et al, 2004; Costanzo et al, 2004). Together, these evolutionary studies reinforce the usefulness of comparative research on key cellular pathways. Plant RBR1 domains and phosphorylation states As expected from their ancient evolutionary origin, plant RBR proteins share with animal counterparts not only a relatively high degree of amino acid homology but, more importantly, a similar organization of functional domains. This includes an N-terminal domain, the central A/B domain that forms the "pocket," and a C-terminal domain (Fig 2). A groove in the B domain is necessary for binding of LxCxE-containing proteins, and together with the C-terminal domain mediates interaction with E2F (Rubin et al, 2005). In addition, intrinsically disordered regions are present between the A and B domains as well as in the majority of the C-terminal domain. It is remarkable that these regions contain most of the conserved CDK phosphorylation sites, including T373, involved in driving a more globular structure of this region, and S608, whose phosphorylation prevents interaction with E2F, as described for human RB1 (Burke et al, 2010). Figure 2. Domain organization of representative proteins of the RB familyDomain organization of retinoblastoma family proteins comparing the three human proteins (RB1, p107, and p130) with two plant retinoblastoma-related proteins (Arabidopsis thaliana RBR1 and Chlamydomonas reinhardtii MAT3). The two major domains A and B (green) defining the "pocket" together with the amino acid positions are indicated. The CDK/cyclin consensus phosphorylation sites (empty circles) are also indicated. Those experimentally demonstrated to be phosphorylated are shown (closed red circles). Data are taken from the UNIPROT database (Hansen et al, 2001; Farkas et al, 2002; Rubin, 2013; Willems et al, 2020). Download figure Download PowerPoint Plant RBR proteins contain putative CDK phosphorylation sites in similar locations, although the role of individual sites on RBR structure and function is not yet known. In spite of this, there are reports on the overall function of RBR1 phosphorylation in cell cycle control in various plant species. The mechanism mediating RBR1 activity through phosphorylation involves the interaction with CDK/cyclin complexes containing many plant D-type cyclins (Grafi et al, 1996; Nakagami et al, 1999, 2002; Boniotti & Gutierrez, 2001; Gutiérrez et al, 2005; Godínez-Palma et al, 2017) and either CDKA or CDKB types of kinases (Boniotti & Gutierrez, 2001; Kawamura et al, 2006). The CDK/cyclin activity on RBR1 is cell cycle-regulated and highest from mid-G1 phase until the G1/S transition (Boniotti & Gutierrez, 2001; Nakagami et al, 2002; Sanchez et al, 2002; Hirano et al, 2008; Nowack et al, 2012), as well as and in G2 mediated by CDKB1;1 (Kawamura et al, 2006; Nowack et al, 2012). These CDK activities can be suppressed by CDK inhibitors known as Kip-related proteins (KRPs; Pettkó-Szandtner et al, 2006). In unicellular algae, RBR1/CDK/cyclin ternary complexes remain bound to chromatin throughout the cell cycle (Olson et al, 2010), but this has not been corroborated in multicellular plants. A functional connection between members of the RBR-E2F/DP-CDK/cyclin module has been demonstrated by the finding that activation of PCNA expression by E2F/DP is inhibited by co-expression of RBR1, an inhibition counteracted by additional expression of cyclin D (Uemukai et al, 2005; Shimizu-Sato et al, 2008). Antibodies detecting specific phosphorylated residues in human RB1, such as pS807/pS811, proved useful to identify phosphorylated forms of RBR1 that accumulate after the G1/S transition and, in particular, during the G2 phase (Abrahám et al, 2011; Polit et al, 2012). It is interesting that phosphorylated RBR1 can be detected in the nucleus of interphase cells in the form of granules (Abrahám et al, 2011), although the functional relevance of such foci remains to be determined. Proteomic studies have demonstrated RBR1 phosphorylation in vivo at residues T406, S652, and S911 (Reiland et al, 2009; Willems et al, 2020). The identification of other phosphorylated residues, as well as their functions, remains as a future challenge. RBR1 phosphorylation is important not only for cell cycle progression but also for cell fate determination, as exemplified in the stem cell niche of the root apical meristem (RAM; Cruz-Ramirez et al, 2012; see discussion below). Plant RBR1 is also a substrate of other kinases, e.g., PIP5K (Dieck et al, 2012) or S6K (Henriques et al, 2010, 2013), but in these cases the mechanistic implications are not fully understood. RBR in unicellular green organisms The lack of RB1 orthologs in yeasts and their presence in animals and plants had initially suggested that it may have been an evolutionary acquisition linked to multicellularity. This hypothesis was however ruled out by the identification of RBR1 homologs as well as its E2F/DP partners in the unicellular alga Chlamydomonas reinhardtii (Umen & Goodenough, 2001; Fang et al, 2006). This organism operates a peculiar cell division cycle, since a mother cell grows in G1 to much more than twice its size, and upon reaching a critical size then undergoes multiple cycles of S-phases and mitoses to produce multiple daughter cells of the size typical for this organism. However, RBR1 does not regulate the length of G1, but instead acts to counter the number of mitotic divisions, a process that further depends on E2F/DP (Umen & Goodenough, 2001; Fang et al, 2006), a SUMO peptidase (Fang & Umen, 2008), and a unique G-type CDK (Li et al, 2016). Remarkably, periodic expression of cell cycle genes is independent of functional RBR, E2F, and DP in this unicellular alga (Fang et al, 2006), altogether suggesting that an early role in evolution of RBR proteins was likely related to cell size control, rather than cell cycle progression through G1. Non-cell cycle functions of RBR proteins are also found in the colonial alga Volvox carteri, where the RBR1 gene has a gender-specific expression pattern in female cells (Kianianmomeni et al, 2008). In these cells, up to four differentially spliced products of the RBR1 gene have been identified (Kianianmomeni et al, 2008; Hallmann, 2009; Ferris et al, 2010), suggesting a multifunctional role of RBR1 in different processes that still need to be delineated. Transcriptional waves controlled by RBR1 during the cell cycle As in animal cells, two major transcriptional waves can be distinguished in plants during G1 and G2 phases, which regulate expression of gene products required for S-phase and mitotic progression, respectively. RBR1-E2F complexes In the case of G1, several lines of evidence demonstrate that the key role of the RBR1-E2F module includes the negative RBR1 regulation by CDK/cyclin complexes, the counteracting CDK inhibitors (KRPs for Kip-related proteins), and the participation of chromatin remodeling enzymes, such as histone acetyl transferases (HATs), and subsequent recruitment of RNA polymerase II at the target promoters (Fischer & Müller, 2017). High levels of E2FA/DPA or E2FC/DPB expression lead to misregulation of E2F target genes and developmental abnormalities in Arabidopsis (De Veylder et al, 2002; del Pozo et al, 2002, 2006). Modest overexpression of E2FA or CYCD3 directly affects expression of their target genes and demonstrated that E2FC does not counteract E2FA-mediated gene upregulation (see also discussion of E2FC roles in G2, below). Furthermore, cell wall biogenesis depends on E2FA (de Jager et al, 2009) and on E2FF/DEL3 (Ramirez-Parra & Gutierrez, 2007). Direct RBR1 regulation by CDKA;1 was shown using cdka;1 mutants able to rescue the rbr1 mutant phenotype (Nowack et al, 2012). However, RBR1 is not exclusively regulated by CDKA;1, and S6K was found as another RBR1-interacting kinase phosphorylating it and repressing cell proliferation by inhibiting E2FB factors (Henriques et al, 2010, 2013). Interestingly, RBR1 can form a stable repressor complex with E2FA but not with E2FB. In the proliferation area, differentiation genes such as CCS52A1 and CCS52A2 (encoding activators of the anaphase promoting complex/cyclosome (APC/C)), are repressed under conditions of high cyclin D/CDK activity (Magyar et al, 2012). Other upstream regulators included the KRP family of CDK inhibitors, formed by seven members in Arabidopsis, and the F-box-like protein FBL17 (Kim et al, 2008; Zhao et al, 2012; Noir et al, 2015). FBL17 is itself regulated by the RBR1/E2F pathway and at the same time (as subunit of an SCF ubiquitin ligase) mediates degradation of several downstream targets of E2F, e.g., CDT1a (Desvoyes et al, 2019). CDK activity is also inhibited by another family of proteins, SIAMESE (SIM) and SIAMESE-RELATED (SMR), and some SMRs are under control of the TARGET OF RAPAMYCIN (TOR) signaling pathway (Ahmad et al, 2019; Barrada et al, 2019). Therefore, RBR1-mediated gene expression in G1 is controlled by multiple redundant pathways including kinases, inhibitors, and ubiquitin/proteasome-dependent degradation, to finely balance the availability of gene products required for G1/S transition and S-phase progression (Fig 3A and B). Figure 3. Role of Arabidopsis RBR1 complexes in transcriptional control during the cell cycle(A) RBR1-E2F complexes. Genes required for the G1/S transition are bound by E2F-DP heterodimers located at the E2F binding sites in their promoters (grey box). They are repressed by the retinoblastoma-related (RBR1) protein in association with histone deacetylases (HDAC). At this stage, CDKA-cyclin complexes are inactivated by one or more CDK inhibitors (KRPs). In Arabidopsis, E2FA-C, bound to DP partners, participate in regulation of different gene targets. Later in G1, when CYCD levels are sufficiently high, CDKA phosphorylates RBR1 (small red circles) leaving free the E2F-DP complexes to transactivate their target genes, in association with histone acetylases (HAT) after recruitment of RNA polymerase II (Pol II). (B) RBR1-DREAM complexes. RBR1 also participates in other transcriptional regulatory complexes. Briefly, at the repressed state, while CDK are inactive, E2FC is part of the complex together with the repressor MYB3R3 factor and RBR1. E2F and MYB factors bind to different sites in the promoter of target genes (white boxes). The DREAM complex switches to an activator when E2FB and MYB3R4 factors are incorporated (see text for details on composition and function). Download figure Download PowerPoint DREAM complexes RB family proteins form parts of multimeric complexes that coordinate transcriptional waves during the cell cycle, originally identified as dREAM in flies (for Drosophila RBF, E2f2 and Multi-vulval interacting proteins; Lewis et al, 2004), DREAM in mammals (Litovchick et al, 2007), DRM in C. elegans (Harrison et al, 2006), and DREAM-like in plants (Kobayashi et al, 2015). In mammalian cells, DREAM complexes are master regulators of the cell cycle that repress gene expression in quiescent cells and in G1 with the participation of the RB-related pocket proteins p130 and p107 (while RB1 itself is restricted to repressing E2F targets) together with the MuvB core components (LIN-9, LIN-37, LIN-52, LIN-54 and RBBP4). DREAM regulates gene repression in mammalian G1 phase by two different modes, one dependent on repressor E2F4/5 binding to E2F sites and another on the presence of the DREAM core component LIN-54 that recognizes a DNA sequence motif called "cell cycle gene homology region" (CHR), also present in the promoters of many G2/M genes. In S-G2 phase, a different Muv-Myb complex then forms to regulate genes required for mitosis, a complex assembled by subsequent recruitment of B-MYB and the forkhead transcription factor FOXM1 (Sadasivam & DeCaprio, 2013; Fischer & Müller, 2017). In contrast, the Arabidopsis DREAM complex contains both MYB transcription factors and RBR1 alongside orthologues of the core DREAM elements (Fig 3A and B), as shown by mass spectrometry experiments (Kobayashi et al, 2015; Fischer & Müller, 2017; Horvath et al, 2017): ALY2 and ALY3 (ALWAYS EARLY proteins) are orthologues of LIN-9, TCX5 (Tesmin/TSO1-like CXC domain protein) is orthologous to LIN54, and MSI1 (MULTI-COPY SUPRESSOR OF IRA1) is orthologous to RBBP4. While orthologues of LIN-37 and LIN-52 have not been found in Arabidopsis, E2FC/B, DPA/B, CDKA (suggestive of phosphorylation as a potential regulator of DREAM activity), and MYB proteins were all found associated with plant DREAM. Arabidopsis contains multiple MYB3R genes (Kobayashi et al, 2015), with MYB3R1 and MYB3R4 regulating the expression of CYCLIN B or KNOLLE (Haga et al, 2007, 2011). The promoters of these G2/M genes contain the MSA (Mitosis-specific activator) motif, originally identified in tobacco BY-2 cells (Ito et al, 1998). Analysis of Arabidopsis myb3r3, myb3r5 double mutants (myb3R3/5) showed activation of G2/M genes both in proliferative and mature tissues, supporting a role of Arabidopsis DREAM in gene expression control in G2. A triple myb3R1/3/5 mutant exhibits enlarged organs resulting from increased cell proliferation, revealing that MYB3R3/5 are repressors while MYB3R1 has a dual activator and repressor role (Kobayashi et al, 2015). Chromatin immunoprecipitation of MYB3R3 has revealed promoters of early cell cycle and mitotic genes that contain E2F and MSA binding sites, respectively, hinting to the existence of distinct DREAM complexes (Kobayashi et al, 2015). In fact, binding sites of RBR1 and MYB3R3 mostly coincide at promoter regions of S-phase- and G2/M-regulated genes (Bouyer et al, 2018). Furthermore, RBR1, DP, and either repressors E2FC and MYB3R3 or activators E2FB and MYB3R4 are present in the two different complexes acting in G1 and in G2/M, respectively, a situation different from the case of animal cells (Fischer & Müller, 2017). In this regard, it is worth noting that in contrast to animal cells, plant cell G2 phase requires expression of multiple genes regulated by E2F, including CDKB-type kinases (Boudolf et al, 2004). Additional components of the DREAM complex have been identified by genetic interactions. For example, loss-of-function mutations in the TSO1 gene (Andersen et al, 2007), causing overproliferation of meristems and defects in flower development, are suppressed by myb3r1 mutation but not by myb3r4 mutation, although no direct TSO1 interaction with RBR1 was found (Wang et al, 2018). DREAM target genes appear to extend beyond cell cycle genes. In a recent study, TCX5 was found to repress the expression of MET1, CMT3, DDM1, KYP, and VIMs genes involved in maintenance of DNA methylation (Ning et al, 2020). TCX5 is redundant with its paralogue TCX6, and the double mutant tcx5, tcx6 exhibits increased levels of DNA methylation, primarily at CHG sites. Another recent study identified that SOL1/TCX3 and SOL2/TCX2, two SPEECHLESS targets from the TSO1-like family, are important regulators of fate transition in the stomatal lineage (see also discussion on RBR1 role in cell fate acquisition in the stomatal lineage). It was hypothesized that they could compete with DREAM for binding sites on DNA (Simmons et al, 2019), but direct evidence for the participation of TSO1/MYB3R1 and SOL1/SOL2 in the DREAM complex is still lacking. Interplay of RBR1 with chromatin Early studies in plants, parallel to those in animals, already showed a connection of RBR1 with chromatin components and provided insightful information about its potential functional relevance. Plant RBR1 was found to interact with MSI1, a homolog of human RbAp46/48 (Ach et al, 1997b; Lusser et al, 1999; Rossi et al, 2001), and with the histone deacetylase (HDAC) Rpd3 for repression of target gene expression (Rossi et al, 2003). Independent studies of flowering control and response to cold stress identified MSI4 (encoded by the FVE gene), a protein that interacts not only with RBR1 but also with HDACs (Ausin et al, 2004; Kim et al, 2004; Pazhouhandeh et al, 2011). Interestingly, fve mutants show increased histone acetylation levels and abnormal silencing of transposable elements (the latter process also affected by RBR1), through effects on cytosine methylation (mC) at CHH and CHG sites (Gu et al, 2011; Xu et al, 2013). Participation in controlling mC levels implies a role in imprinting, which in plants relies on removing silencing marks. Consistent with this, RBR1 interacts with the mC demethylase DEMETER (DME) and represses MET1 (METHYLTRANSFERASE 1), an E2F target gene (Jullien et al, 2008). Further support for a role of RBR1 in repressing euchromatic genes and in TE silencing comes from more recent genome-wide mapping of RBR1 binding sites, which colocalize largely with previously identified E2F and MYB3R3 target genes (see also discussion on DREAM; Bouyer et al, 2018). It is worth noting that TEs have amplified E2F binding sites, as revealed by the presence of ~85% of all E2F binding sites in the heterochromatin of Arabidopsis and other Brassicaceae (Henaff et al, 2014). In addition to the interaction of RBR1 with factors involved in regulating histone acetylation levels, RBR1 also plays a role in maintenance of the repressed state of polycomb (PcG) chromatin. This relies on the physical interaction of RBR1 with PRC2 (polycomb-repressive complex 2) components such as FIE, CLF, and VNR2 (Mosquna et al, 2004; Guitton & Berger, 2005a; Johnston et al, 2010). Defects in RBR1-PRC2 interaction result in mutant phenotypes during Arabidopsis gametophyte development and during cell fate acquisition (Johnston et al, 2008, 2010), but elucidation of detailed mechanisms involved in the RBR1-PRC2 pathway awaits further research (reviewed in Kuwabara & Gruissem, 2014). RBR1 in the DNA damage response (DDR) Plant and animal DNA damage responses share several general strategies, but they also exhibit several unique features. In addition to the conserved ATM and ATR pathways, the plant DDR depends on transcriptional activation of plant-specific target genes such as SOG1, and on epigenetic modifiers (reviewed in Kim, 2019; Nisa et al, 2019). The first hint of the participation of RBR1 in DDR came from the observation that the typical nuclear foci marked by phosphorylated variant histone H2AX (γH2AX) formed after DNA damage contained E2F and depended on an intact RBR1-binding motif in E2F (Lang et al, 2012). Upon DNA damage, RBR1 and E2FA are recruited to γH2AX foci in an ATM- and ATR-dependent manner (Horvath et al, 2017). This recruitment process stimulated by the plant-sp
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