There are major gaps in our understanding of how DNA damage is sensed and repaired and how DNA repair is coordinated with other dynamic chromatin functions (e.g. transcription, DNA replication) to maintain genome stability. DNA repair prevents environmentally-induced cancer in normal cells and can confer resistance to chemotherapy and radiotherapy in cancer cells. Therefore, knowledge of genome maintenance mechanisms is crucial for us to understand, prevent, and successfully treat cancer. The immediate objective of the proposed exploratory project is to define a new role for the DNA repair protein Rad18 in chromatin modification and regulation of gene expression. Our long-term goal is to determine how Rad18-mediated regulation of gene expression contributes to genome maintenance and tumor suppression (in normal cells) and chemo/radio-resistance (in cancer cells). Previously, Rad18 has been implicated only in regulating DNA repair proteins. Based on strong preliminary data, the central hypothesis is that direct mono-ubiquitination of Histone H2A at K119 by Rad18 is a novel mechanism for transcriptional repression that contributes to genome maintenance. The rationale is that defining mechanisms of Rad18-mediated genome maintenance will provide a better understanding of environmental carcinogenesis and new opportunities for improving cancer therapy.
The Specific Aims (SAs) of this exploratory R21 proposal are: (1) Define novel Rad18 functions in epigenetic control of gene expression. (2) Identify Rad18-responsive histone marks and transcriptional events in vivo. For SA1 we will perform genome-wide analyses to identify Rad18-dependent histone marks associated with altered gene expression following exposure to environmental and therapeutic genotoxic agents (Polycyclic Aryl Hydrocarbons, or PAH, and Ionizing Radiation respectively) in primary human cells. SA2 will determine the extent to which Rad18 influences gene expression and histone modifications in bone marrow (BM) progenitor cells, known targets of genotoxicity from environmental PAH and radiotherapy. To determine the contribution of H2A-ubiquitination to Rad18-mediated genome maintenance we will use 'separation-of-function'Rad18 mutant specifically lacking H2A-directed E3 ligase activity. The proposed ideas and research are innovative because they seek to provide new paradigms where none exist: chromatin modification by Rad18 is novel and indicates that Rad18 may have new genome maintenance functions involving transcriptional repression. The proposed mouse studies are innovative because physiological Rad18 functions in genome maintenance have not previously been studied in BM progenitor cells (or other somatic cells) in vivo. The work is significant because it will define new Rad18 functions that protect normal cells against environmental exposures, and confer resistance to chemotherapy and radiotherapy in cancer cells. This work will identify new biomarkers of environmental and therapeutic genotoxicity. We seek to validate Rad18 and its effectors as druggable targets whose inhibition sensitizes cancer cells to chemotherapy and radiation.
The proposed research is relevant to public health because understanding mechanisms of DNA damage tolerance will further our understanding of the mechanism and etiology of environmentally-induced cancers, may allow early identification of individuals with cancer propensity, and will likely identify druggable targets whose inhibition wil allow better killing of tumor cells by chemotherapies and radiation. The proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help reduce and treat cancer.