A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

保护哺乳动物生殖系基因组完整性的新工艺

基本信息

批准号：
10445720
负责人：
Piroska Edit Szabó
金额：
$ 51.9万
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

来源：
https://reporter.nih.gov/project-details/10445720
关键词：
Adopted Affect Affinity B-DNA BTB/POZ Domain Binding Binding Sites Biochemical Biochemistry Biological Assay Biological Process Cell Culture Techniques Cells Cellular biology Chromosomal Rearrangement Chromosome Deletion Circular Dichroism DNA DNA Double Strand Break DNA Methylation DNA Modification Methylases DNA Sequence Rearrangement DNA Structure DNA-Binding Proteins Dependence Development EMSA Enzymes Epigenetic Process Generations Genetic Genetic Models Genome Germ Cells Germ Lines Germ-Line Mutation Grant Hand Human Immunohistochemistry In Vitro Left Location Mammalian Cell Maps Measures Methods Methylation Methyltransferase Molecular Monitor Mus Mutant Strains Mice Nucleosomes Organism Population Pregnancy Process Proteins Purines Pyrimidine Refractory Role Short Tandem Repeat Site Spermatocytes Tandem Repeat Sequences Testing Time Transgenic Mice Variant Z-Form DNA Zinc Fingers base epigenome epigenomics fetal genome integrity genome-wide in vivo male mutant mutation assay next generation novel sperm cell

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 1, 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，Z-DNA 的诱变作用对于一个物种来说是最不健康的。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我们在哺乳动物种系中发现了一种控制 Z-的新生物过程。 PPR 的 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允许我们分离生殖细胞，我们发现了一种以前未表征的 DNA 结合蛋白 ZBTB43 重塑 Z-DNA 结构并防止体内胎儿男性生殖细胞发生双链断裂。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。位点形成 Z-DNA 并在哺乳动物细胞中引起大的基因组重排。我们在野生型中 ZBTB43 结合发生的突变生殖细胞中检测到 Z-DNA 因此，我们勇敢地说，ZBTB43 通过以下方式保护生殖系中的基因组完整性。此外，我们发现通过消除 Z-DNA，ZBTB43 促进 de 。我们建议在全球表观遗传重塑期间进行 PPR 的新甲基化。目标，结合使用遗传学、细胞生物学、生物化学和表观基因组方法。在目标 1 中，我们将。测试 ZBTB43 通过直接结合胎儿体内的 PPR 消除体内 Z-DNA 结构的工作假设我们将确定 1) 男性胎儿 Z-DNA 结构的空间和时间变化。体内存在或不存在ZBTB43蛋白的生殖细胞2) Z-DNA重塑的依赖性； ZBTB43在体内与Z-DNA结构直接结合的过程；以及3)分子要求； ZBTB43 对 Z-DNA 的作用在目标 2 中，我们将测试 ZBTB43 促进 DNA 新生的工作假设。通过消除 Z-DNA 间接实现精原细胞中的甲基化，从而揭示作为底物的序列对于从头甲基转移酶，我们将绘制 ZBTB43 存在和不存在情况下的 DNA 甲基化图谱。我们将测试胎儿男性生殖细胞中的表观基因组重塑过程是否从头甲基化 Z-。我们将测试 ZBTB43 是否影响 PPR 的核小体占据。检验以下假设：Z-DNA 在种系中具有诱变性，并且 ZBTB43 已进化到能够管理该突变我们将绘制突变胎儿生殖细胞中的双链断裂图，测试ZBTB43的抗突变作用。在细胞培养物中，并在资助结束时寻找 Zbtb43 突变男性精子中的基因组重排。在此期间，我们将鉴定并描述 DNA 结合蛋白如何保护基因组的第一个例子通过控制哺乳动物种系中的 Z-DNA 结构来实现完整性和从头 DNA 甲基化。