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

背景：真核生物中的基因沉默是由致密的异染色质介导的，每次经过DNA复制分叉后，异染色质都会重新组装。基因沉默和活跃状态之间的协调转换在后生动物的发育和单细胞生物体的适应中发挥着关键作用。尽管它们意义重大，但人们对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(CAC1、ASF1、RRM3)，它们改变了酿酒酵母端粒的表观遗传转化率。CAC1和ASF1编码组蛋白伴侣蛋白，参与复制叉后面的核小体重组。RRM3促进在暂停的复制分叉处恢复伸长。基于我们和其他人的这些观察，我提出，复制分叉的暂停会导致染色质的异常重组，并易于发生表观遗传学变化。此外，最近我的实验室发现，Cac1p被两种激酶CDK和DDK磷酸化。在这些激酶的靶点携带CAC1突变的细胞在几个基因座上破坏了基因沉默，并显示出细胞周期和生长缺陷。根据我们的数据，我认为染色质重组与CDK和DDK对细胞周期的调控有关。 长期目标：以酿酒酵母为模式生物，我们将探索表观遗传转化和DNA复制暂停之间的联系，并阐明CDK和DDK在这些过程中的作用。 短期目标： 1.了解磷酸化调控CaF-1活性的分子机制。通过结合CDK和DDK靶点的突变，我们将解决以下问题： -Cac1p突变蛋白与染色质相关吗？ -DDK在体内是否使Cac1p磷酸化？ -突变的Cac1p蛋白是否会加剧紧密结合蛋白位置的分叉停顿？ -突变的Cac1p蛋白与体内停滞的复制叉子相关吗？ 2.分析CAC1和ASF1突变体的复制分叉停顿现象。我们将确定这些伴侣蛋白的突变是否会影响体内的叉子加工能力。 3.叉子成分分析。我们将确定在缺乏RRM3的细胞中，Cac1p(包括磷酸化缺陷突变体)和Asf1p与暂停的复制叉的关联是否发生改变。 4.对CAC1、ASF1、RRM3与控制停叉稳定性的基因进行遗传互作分析。使用一种新的测试方法，我们将确定这些基因是否在控制基因沉默和表观遗传转化方面存在遗传交互作用。 原创性和意义：我的研究项目是世界上极少数解决表观遗传转化和DNA复制暂停之间联系的项目之一。因此，它有望在表观遗传学、DNA复制和细胞周期进展等领域做出重大贡献。它还将涉及基因组稳定性以及细胞分化和适应等领域。

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.