Cisplatin-induced epigenomic modifications in male germ cells



 DESCRIPTION (provided by applicant): Testicular cancer is the most common malignancy in men between the ages of 15 and 34. Chemotherapy approaches provide a ~90% cure rate and many of these men will often go on to father children. A critical concern is whether chemotherapy will negatively impact the offspring of these men. In support of this possibility, a number of studies have documented that chemotherapeutic agents used to treat testicular cancer have adverse effects on the subsequent generation. An epigenetic basis for these defects is suggested by chemotherapy-induced changes in the DNA methylation status and expression of specific genes, as well as histone/protamine ratio changes. However, genome-wide analyses of the epigenetic modifications that occur in male germ cells as a result of exposure of the paternal genome to chemotherapy have not yet been performed. This is a critical gap in the field that greatly limits our understanding of the underlying mechanisms by which adverse exposures to the germline lead to disease in the offspring. In this application, we propose to address the epigenetic effects of cisplatin, a drug used in testicular cancer therapy, as well as many other solid tumors. We will determine the effects of cisplatin on the DNA methylome and transcriptome of both transcriptionally active and inactive male germ cells. We will examine round spermatids (RSPs) since these male germ cells are at the last developmental stage that still undergoes active transcription. We will examine mature sperm since they are transcriptionally quiescent but harbor stable RNAs that may be important for fertilization and early embryogenesis. To specifically identify stable alterations that remain afte the affected germ cells are cleared and replaced from the pool of spermatogonial stem cells (SSCs), we will assay gene expression and methylation after 3 cycles of spermatogenesis. In the F1 generation, we will examine male primordial germ cells (PGCs), a unique cell type that undergoes global DNA demethylation. This will allow us to determine whether cisplatin treatment triggers epigenetic changes that allow specific genes to escape this genome-wide epigenetic reprogramming event. We will also define the methyomes and transcriptomes of RSPs and mature sperm from the F1 generation. Given that genome-wide epigenetic effects of cisplatin treatment have not previously been reported, we regard the innovation quotient of this proposal as high. We will also provide new genome-wide epigenetic and transcriptome information on normal germ cell subsets. The experiments proposed here represent a critical first step towards defining the specific mechanisms by which epigenetic marks-deposited as a result of environmental exposure to toxicants-are maintained throughout germ cell development and lead to specific anomalies in future generations. This information may allow physicians to better inform their patients of the reproductive risks associated with chemotherapy treatment.