Roles and regulation of the RETINOBLASTOMA-RELATED protein in Arabidopsis thaliana

The development of multicellular organisms relies on an accurate coordination of cell division, differentiation and programed cell death. De-regulation of these processes could lead to aberrant development and malformations. For example, uncontrolled cell division and de- differentiation results in tumor formation and cancer development. The RETINOBLASTOMA susceptibility gene (RB) was the first tumor suppressor gene characterized as the causal mutation for eye cancer in children. The clinical importance of RB attracted particular attention to its role as a cell cycle regulator, creating a research bias that masked its properties as a coordinator of distinct cell processes for decades. The study of RETINOBLASTOMA-RELATED (RBR) proteins in other organisms and cellular processes has expanded the picture of the RBR functions as an integrator of environmental and developmental signals in cell division and differentiation. This thesis is framed within an emerging paradigm, and contributes to our understanding of how a single, conserved protein regulates multiple processes that often occur simultaneously, while sometimes being exclusive.

In Chapter 1 we introduce the conserved function of RBR in cell cycle as an example of how RBR activity is regarded as the ability to interact with other proteins such as transcription factors (TF) and chromatin remodelers. RBR activity is regulated by CYCLIN-DEPENDENT KINASE-mediated phosphorylation on multiple sites, which in turn promotes structural changes that interfere with RBR-protein interactions. We also present the current understanding of RBR functions in plant development and the recent description of its role in the plant DNA damage response (DDR), and focusing on the Arabidopsis root meristem, where cell division and differentiation processes are partly coordinated by RBR.

In Chapter 2 we addressed the functional relevance of the 16 putative phosphorylation sites in the Arabidopsis RBR protein. The text book mechanism of RBR regulation by phosphorylation assumes that only an un- or hypo-phosphorylated RBR is active. By analyzing the complementation capacity of a systematic collection of transgenic RBR phospho- variants, both phospho-defective and phospho-mimetic, we show that the pleiotropic effects of RBR can be partially disentangled by its phosphorylation state, supporting the notion of a ‘phosphorylation code’ that governs RBR activity. Moreover, phosphorylation- regulated functions of RBR partially depend on its ability to interact with the LXCXE motif, as revealed by the point mutation N849F (RBRNF).

As an attempt to understand the molecular basis of the observed phenotypic effects in the previous chapter, in Chapter 3 we set out to discover nuclear proteins that interact directly with RBR. In a series of Yeast two-hybrid screenings, we used the most substituted phospho-variants, the RBRNF allele, and the wild type RBR as baits to probe an arrayed library encoding nearly 2000 Arabidopsis nuclear proteins. Our results revealed that the binding properties of RBR are not affected by phosphorylation in some cases. Interestingly, the proteins that bind RBR regardless its phosphorylation state are related to stress- responsive transcriptional programs. Actually, the majority of the interactors participate in stress responses. On the other hand, few interactors are canonical cell cycle regulators, like E2FC and the DREAM complex members, TCX 6 and 7. For these, we demonstrated that an LXCXE motif is responsible for the interaction with RBR, providing an insight on the divergent molecular determinants of the DREAM complex assembly in plants.

From the list of interactors obtained in Chapter 3, we paid special attention to NAC044 protein because it is the closest homolog and direct target of the SUPPRESSOR OF GAMMA RESPONSE 1, a plant specific TF that controls the DDR. NAC044 binds RBR in a LXCXE- dependent manner but independently of RBR phosphorylation, challenging the prevalent paradigm that RBR-LXCXE interactions are regulated by phosphorylation. Since the role of NAC044 is to arrest the cell cycle upon DNA damage and other stresses, in Chapter 4 we set out to elucidate the relevance of its interaction with RBR. RBR has a chromatin-structural role and a transcriptional role in the DDR, and we determined that both are separated by the ability to bind LXCXE-containing proteins. On the one hand, the RBRNF mutation abolishes the recruitment of RBR to nuclear foci right after DNA injury, suggesting that a yet unknown LXCXE-containing protein recruits RBR to repair foci. On the other hand, NAC044 expression is induced by DNA damage but slower than RBR recruitment to foci. Therefore, after RBR has been cleared from repairing foci it binds NAC044. We determined that the RBR-NAC044 interaction contributes to the activation of cell death. Thus, we believe that the structural and transcriptional roles of RBR in the DDR are linked through an integrative feature that informs the transcriptional machinery, that decides between cell death and cell division resumption, about the success of the DNA repair process.

Finally, in Chapter 5 we take an evolutionary perspective to contextualize the possible implications of a conserved RBR-centric network in coordinating eukaryotic cellular processes. For instance, we discuss how RBR activity on E2F TFs and its regulation by CYC- CDKs and CDK-inhibitors couples cell size to cell division both in lower and higher eukaryotes. But in higher eukaryotes, the activity of the RBR network is also important for the overall organ size and shape emergence. Moreover, as the development of plants is particularly sensitive to the environmental conditions, we emphasize the integration of environmental inputs by the RBR network to contribute to a coordinated response. To close the general discussion, we also provide experimental perspectives and open questions to be addressed in the coming future of the RBR research field.