Final Report Summary - RBNET (Inference of RBR network and dynamic RBR complexes during leaf development) Summary description of the project objectivesThe RBnet project aimed to map the growth regulatory network connected to the plant Retinoblastoma Related Protein (RBR). RBR is a broadly utilized adaptor protein to dynamically regulate the assembly or disassembly of protein complexes on distinct batteries of genes and it provides a convergence point for signaling pathways for the regulation of cell proliferation, cell differentiation and metabolism. The major aims of the RBnet project were (i) to build an RBR protein-protein interaction network (ii) to computationally identify distinct RB complexes, (iii) perform experiments to verify some of these RBR complexes, (iv) identify RBR target genes and (v) study the dynamic behavior and functions of RBR complexes. The research program and the associated structured training in computational biology provided the fellow a strong basis to become a leader in this field and identify ways to modify the network for optimum growth performance, how the DNA damage response (DDR) pathway is connected to the RBR growth regulatory network.Description of the work performed in the RBnet projectTask1: Inference of RB network and detection of protein complexesList of RBR interacting proteins. In this task we collected all available data on RBR interacting proteins in Arabidopsis including the published cell cycle interactome through directed yeast two hybrid assay, TAP tagging and split YFP fluorescence complementation (Mol Syst Biol. 2010 Aug 10;6:397), our own results of yeast two hybrid (Y2H) interaction screens, mass-spec data of RBR-GFP, E2FA-GFP and E2FB-GFP pull down experiments and a list of LxCxE RBR interaction motive containing proteins. We complemented this with publically available Arabidopsis interactome datasets derived from the INTACT database. Identification of RBR complexes using microarray data, coexpression analysis (guilt by association). A genome-wide transcriptome analysis was performed comparing RBR silenced root tips using the rRBr line with wild type Col0 root tips of 4, 6 and 10 days old seedlings. We compared our microarray with the published microarrays of inducible silenced RBR in seedling and of those using CycD3;1 overexpression, E2FA/DPA overexpression lines and of those carried out with DDRs in Arabidopsis.Task2: Verification of RBR complexes experimentallyWe focused this part of the work on the novel interaction that we have discovered in task 1 between RBR and components of the DDR pathways involving BRCA1 and the novel DDR genes we discovered, an E3 ligase that we named LIFERING (LFR). We established tools and techniques to study these interactions with multiple complementary approaches; 1) interaction screen between in vitro wheat germ translated proteins, 2) transient expression in transformed protoplasts, 3) plants where we transformed GFP tagged complementing genomic constructs.Task3: Discovery of dynamic RBR complexesThis work was focused on the dynamic recruitment of RBR complexes to the sites of DNA damage. To do this we constructed complementing GFP-tagged genomic constructs in the corresponding mutant backgrounds for E2Fs, RBR, BRCA1, PARP2, LFR. We also established the labeling of double strand break (DSB) foci using γH2AX antibody.Task4: RBR targets, functions of specific RBR complexesTo determine whether genes within the BRCA1 cluster are direct targets of RBR complexes we performed chromatin immuno-precipitation (ChIP) with RBR antibody and E2FA-GFP in root tissues followed by qRT-PCR. We used the PCNA1 (At1g07370) promoter as a positive control. To reveal the functional relevance of RBR interaction with DDR components we analyzed mutants for DDR including cell death labeled by PI, DSBs labeled by γH2AX, root meristem sizes, stem cell functions, root growth kinetics upon genotoxic stress conditions.Description of the main results achievedIn the RBnet project we set out to identify novel interacting proteins to plant RBR, dynamics and the functional relevance of this interaction. After collecting publically available data-sets and amalgamating those with our own results we have discovered a novel function for RBR in DNA damage response. This led to the discovery of a novel DDR pathway that is independent to the currently known central DDR transcription factor in plants, SOG1. We suggest that this novel SOG1-independent DDR pathway is organized around the RBR protein based on the following evidence; 1) silencing of RBR leads to DNA damage, recognized by the γH2AX labeling, 2) and to cell death which is SOG1 independent but fully dependent on E2FA (Perelli and Scheres), 3) silencing of RBR results in the up-regulation of DDR genes also in a SOG1 independent-manner, 4) in the absence of SOG1 and RBR silencing, in the amiRBR,sog1-1, DDR genes can still be induced by genotoxic stress, 5) RBR and the downstream transcription factor, E2FA directly binds to the promoter of the DDR gene, AtBRCA1,6) RBR and E2FA are both required for the repression of DDR genes, while E2FA is required for DDR gene activation upon genotoxic stress, 7) the genotoxic stress-induced cell death and DDR gene expression are fully dependent on the combined activities of ATM and ATR while the RBR-regulated cell death and DDR gene expression is only partially dependent on ATR but not on ATM 8) ATM through SOG1 provide an inhibitory cross-regulation to the RBR-dependent pathway, 9) RBR and AtBRCA1 are independently co-recruited to the γH2AX labeled DNA damage foci and can interact, 10) using the amiRBR;brca1-1 cross we show that RBR maintains genome integrity and regulates cell death partially through AtBRCA1. Based on these findings, we propose a model for a SOG1 independent DDR pathway where RBR through a repressive function on E2FA provides novel inputs to DDR downstream of ATM and ATR.Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).In plants, protection against DNA damaging environmental factors and internal metabolic activities is particularly important to maintain genome integrity where germ lines are not specified early during embryogenesis, but arise during the entire life span that potentially can be as long as 1000 years. For this reason, plant cells are equipped with efficient DDR pathways built around evolutionary conserved and plant specific components. Central in the initial signaling of the DNA damage response are the ATM/ATR protein kinases that provide signals around the damaged sites through the phosphorylation of H2AX (γH2AX) to recruit repair. ATM also phosphorylates the plant-specific transcription factor, SOG1, and thus directly initiates the transcriptional response to DNA damage. In Arabidopsis, we discovered that the RETINOBLATOMA RELATED (RBR) is connected to DNA damage and response. RBR independently functions to SOG1, a plant specific DNA damage response (DDR) transcription factor with orthologous functions to the animal p53. We show that RBR directly represses DDR genes through E2FA, a downstream transcription factor required for DDR gene expression and cell death. RBR performs these functions downstream of ATM and ATR, independently recruited to the γH2AX-labelled DNA damage foci with BRCA1 and they interact. Using the amiRBR;brca1-1 cross we show that RBR functions in the maintenance of genome integrity and regulation of cell death through BRCA1. We suggest that RBR, in parallel with imposing quiescence, protects the genome directly as part of DDR signaling with BRCA1 but in parallel to the canonical SOG1 pathway. This might have important implications in crop performance and stress tolerance as well as in plant evolution.
