The way organisms respond to radiation exposure is important since induced DNA lesions can lead to mutation, genomic instability, and death, cancer or other deleterious health problems. Previous efforts of ours have focused on fission yeast S. pombe rad9, a gene that promotes gamma-ray resistance, UV-resistance, resistance to the DNA replication inhibitor hydroxyurea, and regulates the associated cell cycle checkpoints. We identified human (HRAD9) and mouse (Mrad9) orthologues, and the corresponding cDNAs were found to partially complement several defects demonstrated by rad9::ura4+ yeast. Furthermore, we found that HRAD9 protein binds the checkpoint proteins HHUS1 and HRAD1 at its C-terminal region, and contains a BH3-like domain at its N-terminal region that can bind the anti-apoptotic proteins BCL-2 and BCL-xL, and can cause apoptosis when overexpressed. We also found that this multifunctional Rad9 protein can bind p53 and co- regulate p21. Recent studies indicate that Rad9 also participates in multiple DNA repair pathways as well. We found that Rad9 physically interacts with Rad51 and functions in homologous recombination repair, and others reported that Rad9 can bind and regulate the activity of several proteins involved in base excision repair and mismatch repair. Interestingly, we identified structurally and functionally similar paralogues of Rad9, which we call HRAD9B (human) and Mrad9B (mouse), indicating that Rad9 is part of a gene family. We constructed Mrad9 and Mrad9B knockout cells and mice and found that both genes are essential for embryogenesis. Moreover, based on several established functions of Rad9, such as roles in maintaining genomic stability and homologous recombination, which are critical for spermatogenesis, we investigated whether Rad9 functions in this process. We now provide novel preliminary data indicating that we constructed mice bearing a targeted deletion of Mrad9 in early lineage spermatogonia, type A, and that these animals in fact demonstrate defects in spermatogenesis. The major focus of this proposal builds on and extends our findings to study Rad9 function. Specifically, we will make use of Mrad9 knockout cells and mice we constructed to address focused hypotheses designed to elucidate the mechanisms by which the gene maintains genomic stability, promotes resistance to DNA damage, and regulates spermatogenesis. These hypotheses include: 1) Mrad9 promotes genomic stability and cellular resistance to DNA damage by regulating specific DNA repair pathways and cell cycle checkpoints;and 2) Mrad9 plays an important role in spermatogenesis and in the meiotic cell cycle by regulating genomic stability, apoptosis and repair in testis. These studies will examine Mrad9 function from molecular to cellular to whole animal levels. In addition, this investigation could impact on a wide array of important issues, including understanding inherent susceptibility to DNA damage, with implications for radiotherapy, as well as the genetic control of sperm development and male infertility.
This proposal focuses on the role of Rad9 in mediating genomic instability, resistance to DNA damage and spermatogenesis. The goal is to characterize mouse cells null for Rad9 and to engineer and analyze a novel mouse model that has Rad9 knocked out in early lineage spermatogonia. The molecular basis for genomic instability, sensitivity to DNA damage and aberrant sperm development when Rad9 is deficient will be assessed. As such, this project is important since the results could impact on human health, both in terms of the treatment and cause of cancer, due to the role of Rad9 in radioresistance and genomic instability, and also in understanding and developing novel approaches to treat male sterility.
|Panigrahi, Sunil K; Hopkins, Kevin M; Lieberman, Howard B (2015) Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair. Nucleic Acids Res 43:4531-46|
|Broustas, Constantinos G; Lieberman, Howard B (2014) RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels. Prostate 74:1359-70|
|Ghandhi, Shanaz A; Ponnaiya, Brian; Panigrahi, Sunil K et al. (2014) RAD9 deficiency enhances radiation induced bystander DNA damage and transcriptomal response. Radiat Oncol 9:206|
|Broustas, Constantinos G; Lieberman, Howard B (2014) DNA damage response genes and the development of cancer metastasis. Radiat Res 181:111-30|
|Lyndaker, Amy M; Vasileva, Ana; Wolgemuth, Debra J et al. (2013) Clamping down on mammalian meiosis. Cell Cycle 12:3135-45|
|Vasileva, Ana; Hopkins, Kevin M; Wang, Xiangyuan et al. (2013) The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse. J Cell Sci 126:3927-38|
|Cheng, Haiying; Zhang, Zhenfeng; Borczuk, Alain et al. (2013) PARP inhibition selectively increases sensitivity to cisplatin in ERCC1-low non-small cell lung cancer cells. Carcinogenesis 34:739-49|
|Broustas, Constantinos G; Zhu, Aiping; Lieberman, Howard B (2012) Rad9 protein contributes to prostate tumor progression by promoting cell migration and anoikis resistance. J Biol Chem 287:41324-33|
|Young, Erik F; Smilenov, Lubomir B; Lieberman, Howard B et al. (2012) Combined haploinsufficiency and genetic control of the G2/M checkpoint in irradiated cells. Radiat Res 177:743-50|
|Broustas, Constantinos G; Lieberman, Howard B (2012) Contributions of Rad9 to tumorigenesis. J Cell Biochem 113:742-51|
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