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

项目摘要 已知简单嘌呤-嘧啶（Pu/Py）重复序列（PPR）形成左手的、脆弱的Z-DNA结构。 这种重复序列也被认为是高度致突变的，诱导大的染色体缺失和重排 在高等生物的细胞中。Z-DNA的诱变效应对一个物种是最有害的， 在生殖细胞系中不受控制地发生断裂，在未修复的断裂处发生的突变被传递 传给下一代我们在哺乳动物生殖细胞系中发现了一种新的生物学过程， PPR处的Z-DNA结构。使用Zbtb 43突变小鼠遗传模型和我们的转基因小鼠系 我们发现了一种以前未被鉴定的DNA结合蛋白， ZBTB 43重塑Z-DNA结构并保护胎儿男性生殖细胞免受双链断裂的影响， vivo.通过生物化学分析，我们发现ZBTB 43在体外与基因组中富含PPR的DNA位点结合。 ZBTB 43结合位点形成Z-DNA并在哺乳动物细胞中引起大的基因组重排。通过体内 通过表观基因组作图，我们在突变生殖细胞中检测到ZBTB 43结合位点的Z-DNA， 发生在野生型的假丝酵母中。因此，我们假设ZBTB 43保护基因组， 通过结合和消除PPR处的Z-DNA来保持生殖系的完整性。此外，我们发现，通过消除 Z-DNA，ZBTB 43在整体表观遗传重塑期间促进PPR处的从头甲基化。我们 建议采用遗传学、细胞生物学、生物化学和 表观基因组方法。在目的I中，我们将测试ZBTB 43消除Z-DNA结构的工作假设 在体内通过直接结合到胎儿雄性生殖细胞中的PPR。我们将确定1）空间和时间 ZBTB 43蛋白存在或不存在时，胎儿男性生殖细胞中Z-DNA结构的变化， 2）Z-DNA重塑过程依赖于ZBTB 43与Z-DNA的直接结合 ZBTB 43对Z-DNA作用的分子要求。在目标2中，我们将测试 ZBTB 43通过以下方式间接促进了原核细胞中的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 ZBTB 43存在和不存在时胎儿表观基因组重构过程中的DNA甲基化 男性生殖细胞我们将测试从头DNMT是否在体外甲基化Z-DNA底物。我们将测试 ZBTB 43是否影响PPR处的核小体占据。在目标3中，我们将检验Z-DNA是 ZBTB 43在生殖系中具有致突变性，并且ZBTB 43已经进化到管理这种负担。我们将绘制双- 突变胎儿生殖细胞中的链断裂，测试ZBTB 43在细胞培养中的抗突变作用，并搜索 Zbtb 43突变体男性精子中的基因组重排。在补助期结束时，我们将有 鉴定并表征了DNA结合蛋白如何保护基因组完整性的第一个例子， 通过控制哺乳动物生殖系中的Z-DNA结构来靶向从头DNA甲基化。

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.