A central control hub in DNA damage responses: function and regulation of the RBR module – RHiD

The project has exploited a wide variety of biological technologies, combining:
- genetic approaches, requiring the generation of either, Arabidopsis mutant lines, based on T-DNA insertion and the CRISPR-Cas technologies, or the generation of reporter lines; comparative characterisation upon control or genotoxic-induced stress conditions.
- transcriptional analyses of candidate genes (using RT-qPCR), or at a wide-transcriptome level (RNA sequencing)
- standard protein analyses (western-Blot detection, localisation using reporter lines and/or immunodetection)
- protein interaction tests (Yeast-Two-Hybrid, Immunoprecipitation coupled with Mass Spectrometry)
- protein stability assays (upon drug treatment, ubiquitylation assay)

In this project, the function of the stabilizing deubiquitinase-proteins UBP12 and UBP13, interactors of KNO1 has been elucidated. Excitingly, this research has put the RBR1 target KNO1 at the crossroad of protein stability control via the proteasome and autophagy, and for the first time implicated autophagy in control of DDR in plants, a finding that opened a new line of research and will be followed up in the future.
In addition, the role of the F-box protein FBL17 as a mediator of degradation control via the proteasome has been investigated. While previously identified to control cell cycle progression in a regulatory module with KRPs, CDKA;1, E2FA and RBR1, it was shown that the absence of FBL17 results in transcriptional upregulation of known DDR genes, and that the FBL17 protein colocalise with RBR1 upon DNA-stress conditions. Several new FBL17 functions integrated into the DDR pathways have been elucidated.

This project has provided substantial knowledge in the fundamental aspects of how plant cope with DNA damage. On one hand, the role of the F-box protein FBL17 in DNA damage response signalling, and the role of the FBL17-RBR1 module directly at DNA lesion sites have been revealed and reinforce the implication of the proteasome-dependent protein degradation in the plant DDR. On the other hand, a major breakthrough was the discovery of autophagy as part of the plant DDR including the role of the RBR1 target KNO1 at the crossroad of DNA damage control by proteasome and autophagy. These results have great potential to be included into breeding programs to adjust growth under stress conditions.

- Gentric N, Masoud K, Journot RP, Cognat V, Chabouté M-E, Noir S, Genschik P. The F-box-like protein FBL17 is a regulator of DNA-damage response and co-localizes with RETINOBLASTOMA RELATED 1 at DNA lesion sites. Plant Physiol. 2020 doi.org/10.1104/pp.20.00188
- Gentric N, Genschik P, Noir, S. Connections between the cell cycle and the DNA Damage response in plants. Int. J. Mol. Sci. 2021 doi.org/10.3390/ijms22179558
- Lang L, Pettkó-Szandtner A, Tunçay Elbasi H, Takatsuka H, Nomoto Y, Zaki A, Dorokhov S, De Jaeger G, Eeckhout D, Ito M, Magyar Z, Bögre L, Heese M, Schnittger A. The DREAM complex represses growth in response to DNA damage in Arabidopsis. Life Sci Alliance. 2021 doi.org/10.26508%2Flsa.202101141
- Chen P, De Winne N, De Jaeger G, Ito M, Heese M, Schnittger A. KNO1-mediated autophagic degradation of the Bloom syndrome complex component RMI1 promotes homologous recombination. EMBO J. 2023. doi.org/10.15252/embj.2022111980
- Lang L, Böwer F, Tunçay Elbasi H, Eeckhout D, Marschlich N, de Jaeger G, Heese M, Schnittger A. The two plant-specific DREAM components FLIC and FLAC repress floral transition in Arabidopsis. Development. (in revision)
- The lowdown on breakdown: Open questions in plant proteolysis. Eckardt et al. Plant Cell (2024, in press), in section Proteolysis and cell biology: The cell cycle, DNA damage response, and mitochondrial function:
- Genschik P & Noir S. FBL17: A proteolytic engine for the G1/S phase transition?
- Chen P, Heese M & Schnittger. A Is autophagy a key process in the plant DNA damage response?

DNA damage represents a critical threat to every cell in all organisms. Globally, the DNA Damage Response (DDR) consists of a set of tightly regulated events, from sensing of the inflicted damage, activation of a DDR signalling cascade that also blocks cell cycle progression, accumulation of DNA repair factors at the damaged site, to the physical repair of the lesion and/or the replacement of damaged cells. Remarkably, plants can cope with very high concentrations of harmful agents in comparison to animals. Despite this apparent power and their relevance for agriculture under changing environmental conditions, the plant DNA repair pathways are not very well understood. Moreover, the canonical response pathways of yeast and animals appear to be only partially conserved raising the question of how plants can so efficiently repair DNA damage and which regulators are employed in this process. In this context, the preparatory work of both partners has revealed a DDR network relying on the Arabidopsis pRb homolog, called RETINOBLASTOMA RELATED 1 (RBR1). RBR1 appears to function in at least two ways: first, as a transcriptional regulator of DDR genes and second, as a potential assembly factor of repair complexes at the lesion sites. Thus, RBR1 acts as a central control hub in DDR and hence offers a unique starting point to get mechanistic insights into the DDR of plants. Following up on the genome-wide identification of RBR1 binding sites and the proteome-wide protein-protein interaction network of RBR1 under DNA damaging conditions, an aim of this project is to combine the complementary expertise of both partners to understand the molecular mechanism of how targets of RBR1 are controlled upon DNA damage and explore the function of unknown DDR related RBR1 target genes. At the same time, we will investigate how RBR1 itself is regulated upon DNA damage. Since the preparatory work of both partners indicated a prominent role of proteolytic regulation for both the RBR1 functional complex(es) as well as several of its target genes, special emphasis is put on this aspect. To that end, and supported by extensive preparatory data, we follow the hypothesis that the F-box protein FBL17, previously identified in collaborative effort of the two partners, plays a central role in RBR1 homeostasis and might be also involved in selective degradation of RBR1 targets involved in DDR.