The Paternal Age Effect - Enhanced Germ Cell Mutagenesis Modulated by the TRP53/APE1/MDM2 Tumor Suppressor Axis

项目摘要 自1980年以来，年长父亲的出生率增加了约60%，而年轻父亲的出生率下降了。的 在2015年近400万的登记出生中，11%（约440，000）是由年长的父亲所生。老年男性的精子 与年轻男性相比，他们有更多的突变，增加了他们的孩子患上 遗传病这种突变的增加，即父亲年龄效应，越来越显著，并直接影响着 与男性生殖年龄和儿童健康有关。随着小鼠年龄的增长，生精细胞中的DNA修复下降， 促进突变的增加。碱基切除修复蛋白水平的降低是修复减少的原因 活性和增加的诱变在生精细胞在老年。我们的初步数据显示 随着年龄的增长，驱动生殖系突变增加的机制发生在减数分裂粗线期精母细胞中， 涉及APEX 1、TRP 53和MDM 2，它们是通常起保护作用以防止诱变的肿瘤抑制因子。 我们假设TRP 53的慢性激活激活了APEX 1的MDM 2泛素化，导致了 APEX 1.在目标1中，我们将测试TRP 53在Ser 18/23处的磷酸化是否触发APEX 1在细胞中的降解。 粗线期精母细胞，但不是减数分裂后的圆形精子细胞，来自老年小鼠。在目标2中。我们将测试MDM 2是否 泛素化APEX 1，导致粗线期精母细胞中大量高度泛素化的APEX 1， 而不是圆形精子细胞，老年小鼠，APEX 1的蛋白酶体降解，以及由此产生的更大的自发性 突变频率在目标3中，基于显示APEX 1独特分布的初步数据，我们将测试 如果APEX 1与减数分裂细胞中的染色质独特地相互作用。方法：富集特定的 将从携带Mdm 2和Trp 53靶向遗传变化的雄性小鼠中制备生精细胞类型 为了测试1）这些肿瘤抑制因子是否在老年小鼠中失调APEX 1丰度，以及2）是否在老年小鼠中变得更小， 衰老期间慢性激活，导致APEX 1丰度降低，碱基切除修复减少， 和增加的诱变。蛋白酶体降解和MDM 2活性的重要性将使用 蛋白酶体和MDM 2抑制剂。邻近标记将在体内用于鉴定APEX 1相互作用蛋白 在减数分裂细胞中。将使用以下方法确定小鼠基因的突变频率和谱 双链测序所提出的研究测试了一种新的模型，其中肿瘤抑制轴（即TRP 53， MDM 2和APEX 1），通常功能是保护基因组的完整性，而不是损害配子遗传 通过降低APEX 1丰度并导致在特定生物学背景下的突变增加， 老化的减数分裂生殖细胞。如果得到证实，这是一个范式转变，从广泛持有的观点，主要驱动力 雄性配子中突变增加的力量是一种被动积累。拟议的研究将针对 基本的生物学机制驱动增加的诱变，导致父亲的年龄效应， 最终揭示潜在的治疗靶点，以防止通过诱变介导的遗传疾病 在生精细胞中

PROJECT SUMMARY Birth rates to older fathers increased approximately 60% since 1980, while births to younger fathers declined. Of the nearly 4 million registered births in 2015, 11% (~440,000) were born to older fathers. Sperm from older men have more mutations compared with young men, increasing the risk that their children will be afflicted with a genetic disease. This increase in mutations, the paternal age effect, is increasingly significant and is directly relevant to male reproductive aging and to child health. DNA repair declines in spermatogenic cells as mice age, promoting an increase in mutations. Reduced levels of a base excision repair protein is causal to reduced repair activity and increased mutagenesis in spermatogenic cells at older age. Our preliminary data indicate the mechanisms driving increased germline mutagenesis with aging occur in meiotic pachytene spermatocytes and involve APEX1, TRP53 and MDM2, tumor suppressors that normally function to protect against mutagenesis. We hypothesize that chronic activation of TRP53 activates MDM2 ubiquitination of APEX1, resulting in reduced APEX1. In Aim 1 we will test if phosphorylation of TRP53 at Ser18/23 triggers degradation of APEX1 in pachytene spermatocytes, but not post-meiotic round spermatids, from older mice. In Aim 2. we will test if MDM2 ubiquitinates APEX1, leading to greater amounts of highly ubiquitinated APEX1 in pachytene spermatocytes, not round spermatids, of older mice, proteasomal degradation of APEX1, and a resulting greater spontaneous mutation frequency. In Aim 3, based upon preliminary data showing a unique distribution of APEX1, we will test if APEX1 uniquely interacts with chromatin in meiotic cells. Methods: Defined populations of enriched specific spermatogenic cell types will be prepared from male mice carrying targeted genetic changes in Mdm2 and Trp53 to test whether 1) these tumor suppressors dysregulate APEX1 abundance in old mice, and 2) become chronically activated during aging resulting in decreased APEX1 abundance, decreased base excision repair, and increased mutagenesis. The importance of proteasome degradation and MDM2 activity will be tested using proteasome and MDM2 inhibitors. Proximity labeling will be used in vivo to identify APEX1 interacting proteins in meiotic cells. Age-associated mutation frequency and spectra will be determined for mouse genes using duplex sequencing. The proposed studies test a novel model in which a tumor suppressor axis (i.e. TRP53, MDM2 and APEX1) that normally functions to safeguard genome integrity, instead compromises gamete genetic quality by reducing APEX1 abundance and resulting in elevated mutagenesis in the unique biological context of aging meiotic germ cells. If confirmed, this is a paradigm shift from the widely held view that the major driving force of increased mutations in male gametes is a passive accumulation. The proposed studies will address a fundamental biological mechanism driving increased mutagenesis that leads to the paternal age effect and may ultimately reveal potential treatment targets to protect against genetic diseases mediated through mutagenesis in spermatogenic cells.