Understanding the roles of histone H3 variants in promoting genomic and epigenomic stability during DNA replication and aging

项目总结： 有丝分裂和有丝分裂后都需要维持基因组和表观基因组的稳定性 细胞以防止有害的突变，这些突变可能会导致癌症和 神经退行性疾病。在增殖细胞中，DNA复制需要 与转录和DNA修复相协调，防止发生和遗传 突变的可能性。在像神经元这样的终末分化的非复制细胞中，染色质具有 在很长一段时间内保持稳定以维持既定的转录程序。使用 这一建议，我们试图了解组蛋白H3变体对基因组的贡献 以及DNA复制和老化过程中的表观基因组稳定性。复制级别- 依赖的H3.1/H3.2变体和复制无关的H3.3变体发生变化 在增殖细胞的DNA复制过程中以及在有丝分裂后的一段时间内 细胞。我们之前已经表明，H3.1和H3.3的变种可以有不同的 植物表观基因组的生化功能，这是由可变氨基酸介导的 在这两个高度保守的蛋白质的N端尾部有酸性残基(第31位)。 通过对H3K27甲基转移酶ATXR5和ATXR6(ATXR5/6)的研究，我们 已经证明了新合成的H3.1变异体是由 在DNA复制过程中的ATXR/6，以及H3.1变异体在K27处未甲基化 (H3.1K27me0)，而不是H3.3K27me0导致基因组不稳定。我们将调查 H3.1调控植物基因组稳定性的生化和细胞机制 使用遗传和生化筛选技术筛选染色质相互作用蛋白 区分H3.1和H3.3。我们还将使用组蛋白替换策略来 分析所有组蛋白在DNA过程中维持或破坏基因组稳定性的作用 复制。最后，我们将重复这些实验来研究H3变异体在 以果蝇为模型系统维持老化神经元的异染色质。这个 H3.1、H3.3和染色质修饰蛋白之间的高度相似性 真核生物表明，将通过这一过程揭示的分子机制 工作将在人类中保守，并涉及到由基因组或 表观基因组不稳定性。

Project Summary: Maintaining genome and epigenome stability is required in both mitotic and post-mitotic cells to prevent deleterious mutations that can cause diseases like cancer and neurodegenerative disorders. In proliferative cells, DNA replication needs to be coordinated with transcription and DNA repair to prevent the occurrence and inheritance of mutations. In terminally differentiated, non-replicating cells like neurons, chromatin has to be stable for long periods of time to maintain established transcriptional programs. With this proposal, we seek to understand the contributions of histone H3 variants to genomic and epigenomic stability during DNA replication and aging. Levels of replication- dependent H3.1/H3.2 variants and replication-independent H3.3 variants change drastically during DNA replication in proliferating cells and through time in post-mitotic cells. We have shown previously that H3.1 and H3.3 variants can have different biochemical functions on the epigenome in plants, which are mediated by a variable amino acid residue (position 31) in the N-terminal tail of these two highly conserved proteins. Through the study of the H3K27 methyltransferases ATXR5 and ATXR6 (ATXR5/6), we have demonstrated that newly synthesized H3.1 variants are specifically methylated by ATXR/6 during DNA replication, and that H3.1 variants unmethylated at K27 (H3.1K27me0), but not H3.3K27me0, induce genomic instability. We will investigate the biochemical and cellular mechanisms by which H3.1 regulates genomic stability in plants using genetic and biochemical screens for chromatin-interacting proteins that can discriminate between H3.1 and H3.3. We will also use a histone replacement strategy to analyze the roles of all histones in maintaining or disrupting genomic stability during DNA replication. Finally, we will replicate these experiments to study the role of H3 variants in maintaining heterochromatin in aging neurons using Drosophila as a model system. The high levels of similarity between H3.1, H3.3 and chromatin-modifying proteins across all eukaryotes suggest that the molecular mechanisms that will be uncovered through this work will be conserved in humans and involved in diseases caused by genomic or epigenomic instability.