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.

项目摘要已知简单的纯 - 吡啶胺（PU/PY）重复（PPR）形成左手脆弱的Z-DNA结构。已知这种重复是高度诱导的，诱导的大染色体缺失和重排在较高生物体的细胞中。如果断裂发生在种系中，未经修复时发生的突变通过到下一代。我们在哺乳动物细菌系中发现了一个新型的生物学过程 PPRS的Z-DNA结构。使用ZBTB43突变小鼠遗传模型和我们的转基因小鼠系这使我们能够分离生殖细胞，我们发现先前未表征的DNA结合蛋白， ZBTB43重塑Z-DNA结构，并保护胎儿男性生殖细胞中的双链断裂。体内。通过生化测定，我们发现ZBTB43在体外基因组中与富含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型基因组重排。由invivo 表观基因组映射我们在ZBTB43结合的位置检测到突变生殖细胞中的Z-DNA 发生在野生型Prospermatogonia中。因此，我们假设ZBTB43保障基因组通过结合和消除PPRS的Z-DNA在种植线中的完整性。此外，我们发现通过消除 Z-DNA，ZBTB43在全球表观遗传重塑时期促进PPRS的新甲基化。我们提出以下目标，结合遗传，细胞生物学，生物化学和表观基因组方法。在目标I中，我们将测试ZBTB43消除Z-DNA结构的工作假设在体内通过直接与胎儿男性生殖细胞中的PPR结合。我们将确定1）空间和临时在存在或不存在ZBTB43蛋白的情况下，胎儿男性生殖细胞中Z-DNA结构的变化体内2）Z-DNA重塑过程对ZBTB43与Z-DNA的直接结合的依赖性体内结构； 3）ZBTB43对Z-DNA作用的分子要求。在AIM 2中，我们将测试 ZBTB43的工作假设促进了繁荣中的从头DNA甲基化间接地通过消除Z-DNA，从而揭示序列为从头甲基转移酶的底物。我们将映射在表观基因组重塑过程中，在存在和不存在ZBTB43的情况下，DNA甲基化雄性生殖细胞。我们将在体外测试甲基甲酯Z-DNA底物是否从头开始。我们将测试 ZBTB43是否会影响PPRS的核小体占用率。在AIM 3中，我们将测试Z-DNA的假设种系中的诱变，并且ZBTB43已经进化为管理该伯宁。我们将绘制double- 突变胎胎生殖细胞中的链断裂，测试ZBTB43在细胞培养中的抗氧气作用，然后搜索用于ZBTB43突变雄性精子中的基因组重排。到赠款期结束时，我们将确定并表征了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DNA甲基化。