权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

项目概要已知简单的嘌呤-嘧啶 (Pu/Py) 重复序列 (PPR) 可形成左手、脆弱的 Z-DNA 结构。已知此类重复具有高度诱变性，可诱导大量染色体缺失和重排在高等生物的细胞中。如果 Z-DNA 的诱变作用对一个物种来说是最有害的，如果生殖系中不受控制地发生断裂，并且在未修复的断裂处发生的突变被传递下去给下一代。我们在哺乳动物种系中发现了一种新的生物过程，它控制着 PPR 处的 Z-DNA 结构。使用 Zbtb43 突变小鼠遗传模型和我们的转基因小鼠品系使我们能够分离生殖细胞，我们发现了一种以前未表征的DNA结合蛋白， ZBTB43 重塑 Z-DNA 结构并保护胎儿男性生殖细胞免受双链断裂体内。通过生化检测，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基因组重排。通过体内表观基因组图谱我们在突变生殖细胞中的 ZBTB43 结合位置检测到了 Z-DNA 发生在野生型精原细胞中。因此，我们假设 ZBTB43 保护基因组通过在 PPR 处结合和消除 Z-DNA 来保持种系完整性。此外，我们发现通过消除 Z-DNA、ZBTB43 在整体表观遗传重塑期间促进 PPR 的从头甲基化。我们建议结合遗传学、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 结构的工作假设通过直接与胎儿男性生殖细胞中的 PPR 结合而在体内发挥作用。我们将确定 1) 空间和时间在存在或不存在 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重塑过程依赖于ZBTB43与Z-DNA的直接结合体内结构； 3) ZBTB43 对 Z-DNA 作用的分子要求。在目标 2 中，我们将测试工作假设：ZBTB43 通过间接促进精原细胞中 DNA 从头甲基化消除 Z-DNA，从而揭示作为从头甲基转移酶底物的序列。我们将绘制地图胎儿表观基因组重塑过程中 ZBTB43 存在和不存在下的 DNA 甲基化雄性生殖细胞。我们将在体外测试 DNMT 是否会甲基化 Z-DNA 底物。我们将测试 ZBTB43 是否影响 PPR 上的核小体占据。在目标 3 中，我们将检验 Z-DNA 的假设 ZBTB43 会在种系中产生突变，而 ZBTB43 已经进化到可以控制这种负担。我们将绘制双突变胎儿生殖细胞中的链断裂，测试ZBTB43在细胞培养中的抗突变作用，并搜索用于 Zbtb43 突变雄性精子的基因组重排。到资助期结束时，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的第一个例子，通过控制哺乳动物种系中的 Z-DNA 结构来靶向 DNA 从头甲基化。