Cellular implications of perturbed post-translational modifications of nuclear RAGE in persistent DNA damage signaling

• Applicant: Kumar Varun, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548178547

This grant is based on the finding that nuclear RAGE (nRAGE), a central orchestrator of DNA repair, can participate in homologous recombination (HR) and DNA interstrand crosslink (ICLs) and replication stress (RS) repair. nRAGE comprises several domains and interaction motifs for its interaction with various factors of the HR-DSBs, ICLs, and RS repair machinery. Each repair step is tightly regulated by various post-translational modifications (PTMs, such as phosphorylation or (de)acetylation) and also by alternative splicing. Perturbation of the RAGE-regulated DNA repair process leads to a cascade that starts with endogenous DNA damage DDR senescence SASP fibrosis loss of organ function. It remains unknown how PTMs of nRAGE are regulated and how PTM adequately controls the different functions of nRAGE to render nRAGE perfectly adapted to the different kinds of DNA repair mechanisms requiring interaction with damage-specific nRAGE binding partners. This grant aims to understand how various PTMs of nRAGE are involved in controlling the different DNA repair factor interactions or functions of nRAGE. These data will help us achieve our long-term goals of studying how this delicately balanced regulation is disturbed in diseases such as diabetic lung fibrosis, aging, and cancer. To this, we plan to study the following two aims: Aim 1 intends to understand the regulation and impact of phosphorylation of nRAGE by asking the following questions: Question 1A: What is the role of nRAGE (de)phosphorylation in HR-DSB response and timely repair? Question 1B: What are the functional implications of the CDK-mediated phosphorylation of the nuclear RAGE in persistent DNA damage signaling and senescence? Question 1C: Which of the different DNA repair properties of nRAGE are affected by each phosphorylation site? Aim 2 intends to understand the regulation and impact of (de)acetylation of nRAGE by asking the following questions: Question 2A: How do acetyltransferases or deacetylases promptly regulate the DNA repair function of nRAGE under genotoxic insults? Question 2B: Which pioneer factor(s) deacetylates nRAGE in response to the genotoxic insults? For each aim, various genotoxic insults known to induce DNA DSBs, ICLs, or RS in conjunction with the various nRAGE mutants generated will be tested in U2OS and Hela cells in parallel. This is essential because U2OS cells have a more stable genome but cannot provide sufficient material for immunoblotting. Hela cells give a better chance to study functional readouts and provide adequate material to study DNA damage and checkpoint signaling, elaborative live cell recruitment kinetics, survival potential or stress resistance, micronuclei frequency, and persistent DNA damage signaling markers together with senescence, senescence-associated secretory phenotype (SASP). In a follow-up grant, the effects seen in these cells will be compared using complex in vivo disease mice models.

• DFG Programme: Research Grants