Epigenetic transmission in S.cerevisiae: links to DNA replication and cell cycle regulation

酿酒酵母的表观遗传传递：与 DNA 复制和细胞周期调控的联系

• 批准号: RGPIN-2020-03874
• 负责人: Yankulov, Krassimir
• 金额: $ 2.62万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744495
• 关键词: Epigenetic; transmission; S.cerevisiae; links; DNA

BACKGROUND: Gene silencing in eukaryotes is mediated by compact heterochromatin, which is reassembled after each passage of the DNA replication forks. Orchestrated conversions between the silenced and active states of genes play key roles in metazoan development and in adaptation of single cell organisms. Despite their significance, the mechanisms of such epigenetic conversions are poorly understood. ONGOING RESEARCH: My lab has identified three genes (CAC1, ASF1, RRM3) that alter the rate of epigenetic conversions at the telomeres of S. cerevisiae. CAC1 and ASF1 encode histone chaperones involved in nucleosome reassembly behind the replication forks. RRM3 promotes the resumption of elongation at paused replication forks. Based on these observations by us and others, I propose that the pausing of replication forks causes aberrant chromatin reassembly and predisposes to epigenetic change. Moreover, recently my lab has shown that Cac1p is phosphorylated by two kinases, CDK and DDK. Cells harboring CAC1 mutations at the target sites of these kinases compromise gene silencing at several loci and show cell cycle and growth defects. Based on our data, I propose that chromatin reassembly is linked to the regulation of the cell cycle via CDK and DDK. LONG-TERM GOAL: Using S. cerevisiae as a model organism, we will explore the link between epigenetic conversions and the pausing of DNA replication and will elucidate the roles of CDK and DDK in these processes. SHORT-TERM OBJECTIVES: 1.To understand the molecular mechanisms of the regulation of CAF-1 activity by phosphorylation. Through combining mutations in the CDK and DDK target sites, we will address the following questions: -Do Cac1p mutant proteins associate with chromatin? -Does DDK phosphorylate Cac1p in vivo? -Do mutant Cac1p proteins exacerbate fork pausing at positions of tightly bound proteins? -Do mutant Cac1p proteins associate with stalled replication forks in vivo? 2.To analyze replication fork pausing in CAC1 and ASF1 mutants. We will determine if mutations in these chaperones affect fork processivity in vivo. 3.To analyze fork composition. We will determine if the association of Cac1p (including phosphorylation-deficient mutants) and Asf1p with paused replication forks is altered in cells lacking RRM3. 4.To perform genetic interaction analysis between CAC1, ASF1, RRM3 and genes that regulate the stability of paused forks. Using a novel assay, we will determine if these genes genetically interact in the control of gene silencing and epigenetic conversions. ORIGINALITY and SIGNIFICANCE: My research program is one of the very few in the world that are addressing the link between epigenetic conversions and the pausing of DNA replication. It is therefore expected to make significant contributions to the fields of epigenetics, DNA replication and cell cycle progression. It will also relate to the fields of genome stability and cell differentiation and adaptation.

背景：真核生物中的基因沉默是由紧密的异染色质介导的，异染色质在DNA复制叉每次传代后重新组装。基因的沉默和激活状态之间的明显转换在后生动物的发育和单细胞生物的适应中起着关键作用。尽管它们的意义，这种表观遗传转换的机制知之甚少。 正在进行的研究：我的实验室已经确定了三个基因（CAC 1，ASF 1，RRM 3），它们改变了S.啤酒。CAC 1和ASF 1编码组蛋白伴侣，参与复制叉后的核小体重组。RRM 3促进在暂停的复制叉处的延长的恢复。基于我们和其他人的这些观察，我认为复制叉的暂停会导致异常的染色质重组，并易于发生表观遗传变化。此外，最近我的实验室已经表明，Cac 1 p是由两种激酶，CDK和DDK磷酸化。在这些激酶的靶位点携带CAC 1突变的细胞损害了几个位点的基因沉默，并显示出细胞周期和生长缺陷。基于我们的数据，我建议，染色质重组是通过CDK和DDK的细胞周期的调节。长期目标：使用S。酿酒酵母作为模式生物，我们将探讨表观遗传转换和DNA复制暂停之间的联系，并将阐明CDK和DDK在这些过程中的作用。短期目标：1.了解CAF-1磷酸化调节活性的分子机制。通过结合CDK和DDK靶位点的突变，我们将解决以下问题：-Cac 1 p突变蛋白与染色质相关吗？- DDK在体内磷酸化Cac 1 p吗？突变的Cac 1 p蛋白会加剧在紧密结合蛋白位置的叉暂停吗？突变的Cac 1 p蛋白与体内停滞的复制叉相关吗？2.分析CAC 1和ASF 1突变体中复制叉停顿现象。我们将确定这些分子伴侣的突变是否会影响叉在体内的持续合成能力。3.分析分叉的构成。我们将确定在缺乏RRM 3的细胞中Cac 1 p（包括磷酸化缺陷突变体）和Asf 1 p与暂停复制叉的关联是否发生改变。4.进行CAC 1、ASF 1、RRM 3与调节暂停叉子稳定性的基因之间的遗传相互作用分析。使用一种新的检测方法，我们将确定这些基因是否在基因沉默和表观遗传转换的控制中发生遗传相互作用。 原创性和意义：我的研究项目是世界上少数几个致力于解决表观遗传转换和DNA复制暂停之间联系的项目之一。因此，它有望为表观遗传学，DNA复制和细胞周期进程领域做出重大贡献。它还将涉及基因组稳定性和细胞分化和适应领域。