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A Novel Process Safeguards Genome Integrity In The Mammalian Germ Line-Administrative Supplement to Support Undergraduate Summer Research Experiences

一种保护哺乳动物种系基因组完整性的新方法——支持本科生暑期研究经历的行政补充

基本信息

批准号：
10807301
负责人：
Piroska Edit Szabó
金额：
$ 1.24万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-20 至 2026-05-31
项目状态：
未结题

项目摘要

PROJECT SUMMARY Simple purine–pyrimidine (Pu/Py) repeats (PPRs) are known to form the left-handed, fragile Z-DNA structure. Such repeats are also known to be highly mutagenic, inducing large chromosomal deletions and rearrangements in the cells of higher organisms. The mutagenic effects of Z-DNA would be the most detrimental to a species if breaks occurred uncontrollably in the germ line and mutations occurring at unrepaired breaks were passed on to the next generation. We discovered a novel biological process in the mammalian germ line that controls Z-DNA structure at PPRs. Using the Zbtb43 mutant mouse genetic model and our transgenic mouse line that allows us to isolate germ cells, we found that a previously uncharacterized DNA binding protein, ZBTB43 remodels Z-DNA structure and protects from double-strand breaks in fetal male germ cells in vivo. By biochemical assays we found that ZBTB43 binds to PPR-rich DNA sites in the genome in vitro. ZBTB43 binding sites form Z-DNA and cause large genomic rearrangements in mammalian cells. By in vivo epigenome mapping we detected Z-DNA in mutant germ cells at the locations where ZBTB43 binding occurs in wild-type prospermatogonia. We hypothesize, therefore, that ZBTB43 safeguards genome integrity in the germ line by binding and eliminating Z-DNA at PPRs. In addition, we found that by eliminating Z-DNA, ZBTB43 promotes de novo methylation at PPRs during the time of global epigenetic remodeling. We propose to pursue the following aims, using a combination of genetic, cell biology, biochemistry, and epigenomic approaches. In Aim I, we will test the working hypothesis that ZBTB43 eliminates Z-DNA structure in vivo by directly binding to PPRs in fetal male germ cells. We will determine 1) the spatial and temporal changes of the Z-DNA structure in fetal male germ cells in the presence or absence of ZBTB43 protein in vivo; 2) the dependence of the Z-DNA remodeling process on the direct binding of ZBTB43 to the Z-DNA structure in vivo; and 3) the molecular requirements of ZBTB43 action on Z-DNA. In Aim 2, we will test the working hypothesis that ZBTB43 facilitates de novo DNA methylation in prospermatogonia indirectly by eliminating Z-DNA, thus revealing the sequences as substrates for de novo methyltransferases. We will map DNA methylation in the presence and absence of ZBTB43 during the epigenome remodeling process in fetal male germ cells. We will test whether de novo DNMTs methylate Z-DNA substrates in vitro. We will test whether ZBTB43 affects nucleosome occupancy at PPRs. In Aim 3, we will test the hypothesis that Z-DNA is mutagenic in the germ line and that ZBTB43 has evolved to manage that burden. We will map double- strand breaks in mutant fetal germ cells, test the anti-mutagenic effect of ZBTB43 in cell culture, and search for genomic rearrangements in sperm of Zbtb43 mutant males. By the end of the grant period, we will have identified and characterized the first example of how a DNA binding protein protects genome integrity and targets de novo DNA methylation by controlling Z-DNA structure in the mammalian germ line.

项目总结简单的嘌呤-嘧啶(Pu/Py)重复序列(Pprs)形成了左手的、脆弱的Z-DNA结构。众所周知，这种重复序列也是高度诱变的，会导致大量的染色体缺失和重排。在高等有机体的细胞中。在以下情况下，Z-DNA的突变效应对物种的危害最大在胚系中发生不可控制的断裂，并且在未修复的断裂处发生的突变被传递献给下一代。我们在哺乳动物生殖系中发现了一种新的生物过程，它控制着 Pprs的Z-DNA结构。利用Zbtb43突变小鼠遗传模型和我们的转基因小鼠系这使得我们能够分离生殖细胞，我们发现一种以前未描述的DNA结合蛋白， ZBTB43重塑胚胎男性生殖细胞Z-DNA结构并保护其免受双链断裂活着。通过生化分析，我们发现ZBTB43在体外与基因组中富含PPR的DNA位点结合。 ZBTB43结合位点形成Z-DNA，并在哺乳动物细胞中引起大规模的基因组重排。通过活体内表观基因组作图我们在ZBTB43结合的突变生殖细胞中检测到Z-DNA 发生在野生型的繁殖原中。因此，我们假设ZBTB43保护基因组通过在PPR上结合和消除Z-DNA来保持胚系的完整性。此外，我们发现，通过消除 Z-DNA，ZBTB43在全球表观遗传重塑期间促进PPR的从头甲基化。我们建议使用遗传学、细胞生物学、生物化学和生物化学相结合的方法来实现以下目标表观基因组学方法。在目标I中，我们将检验ZBTB43消除Z-DNA结构的工作假设在体内，通过与胎儿男性生殖细胞中的Pprs直接结合。我们将决定1)空间和时间 ZBTB_(43)蛋白对小鼠胚胎生殖细胞Z-DNA结构的影响 2)Z-DNA重塑过程依赖于ZBTB43与Z-DNA的直接结合 3)ZBTB43作用于Z-DNA的分子要求。在目标2中，我们将测试 ZBTB43间接促进精原细胞从头DNA甲基化的工作假说消除Z-DNA，从而揭示序列是从头开始甲基转移酶的底物。我们将绘制地图 ZBTB43基因在胎儿表观基因组重塑过程中的DNA甲基化雄性生殖细胞。我们将在体外测试DNMTS是否甲基化Z-DNA底物。我们将测试 ZBTB43是否影响PPR的核小体占有率。在目标3中，我们将检验Z-DNA是在生殖系中具有突变性，而ZBTB43已经进化来处理这一负担。我们将绘制双倍地图- 突变的胎儿生殖细胞的链断裂，在细胞培养中检测ZBTB43的抗突变作用，并寻找用于Zbtb43突变男性精子的基因组重排。到授权期结束时，我们将拥有确定并表征了DNA结合蛋白如何保护基因组完整性和通过控制哺乳动物生殖系中的Z-DNA结构来靶向从头开始的DNA甲基化。