Regulation of chromatin topology

我的NSERC-DG项目的长期目标是了解细胞如何产生染色质多样性来控制DNA交易。改变核小体的动态和密度是染色质修饰的一种简单形式，可以显着影响染色质区域的拓扑结构。组蛋白伴侣是调节这一过程的酶。在上一个资助期内，我的实验室主要关注出芽酵母酿酒酵母中的两个相关的核纤溶蛋白样组蛋白伴侣，Fpr3和Fpr4。利用体外合成的染色质纤维，我们发现这些蛋白质具有三种不同的核小体修饰特征：i)从游离组蛋白和DNA构建核小体，ii)稳定地结合完整的核小体，以及iii)催化柔性组蛋白尾部的脯氨酸异构化。为了揭示对这些蛋白质敏感的生物过程和基因组位置，我们进行了遗传相互作用筛选和RNA-seq转录组学。这表明，虽然Fpr3和Fpr4协同调节细胞核中磷酸和核糖体生物发生基因的转录，但仅需要Fpr4来维持核仁rDNA上的基因组稳定性。总的来说，核纤溶酶样蛋白就像一把瑞士军刀——它们携带着多种工具来雕刻染色质拓扑结构。但这些工具什么时候使用，如何监管？在下一个资助期内，我们将确定核纤溶蛋白的三种染色质修饰工具在表观基因组中的位置、时间和方式，以及这如何改变局部染色质。如果核浆蛋白是染色质的专门调节剂，那么是什么调节它们的活性呢？我们将验证我们的假设，即这些酶是将磷酸盐和细胞的能量状态与核糖体生物发生联系起来的主要调节剂。我们下一个融资期的短期目标是：确定Fpr3/4组蛋白伴侣如何调节转录沉默。2.确定Fpr4组蛋白伴侣如何促进rDNA上的基因组稳定性。3.确定Fpr4是如何调控的。影响和意义我的nserc资助的研究计划的下一阶段将揭示单个染色质修饰剂如何产生功能不同的染色质拓扑结构。这些细节将对理解染色质如何介导转录过程以及重组事件产生根本性的影响。由于我们的工作还探讨了染色质控制作为营养物质的功能，它也将有助于我们理解代谢状态如何影响表观基因组。我致力于为不同群体的本科生提供首次研究的机会，并指导他们实现自己的职业目标。因此，这项提案的一个明显而直接的影响是培训数十名年轻的高素质人才，他们将在加拿大的科学、研究和知识型经济中发挥至关重要的作用。

The long-term goal of my NSERC-DG program is to understand how cells generate chromatin diversity to control DNA transactions. Altering the dynamics and density of nucleosomes is a simple form of chromatin modification that can dramatically impact the topology of a chromatin region. Histone chaperones are enzymes that regulate this process. In the last funding period my lab focused its attention on two related nucleoplasmin-like histone chaperones in the budding yeast Saccharomyces cerevisiae, Fpr3 and Fpr4. Using synthetic chromatin fibres in vitro we showed that these proteins have three distinct nucleosome modifying features that i) build nucleosomes from free histones and DNA, ii) stably bind to intact nucleosomes, and iii) catalyze prolyl isomerization of the flexible histone tails. To reveal biological processes and genomic locations sensitive to these proteins, we performed genetic interaction screens and RNA-seq transcriptomics. This revealed that while Fpr3 and Fpr4 co-operate to regulate transcription of phosphate and ribosome biogenesis genes in the nucleus, only Fpr4 is required to maintain genomic stability at nucleolar rDNA. Taken together, nucleoplasmin-like proteins are like a swiss army knife- they carry multiple tools for sculpting chromatin topologies. But when are these tools used, and how are they regulated? Objectives In the next funding period we will determine where, when and how each of nucleoplasmin's three chromatin modifying tools is deployed in the epigenome, and how this alters local chromatin. If nucleoplasmins are dedicated regulators of chromatin, what regulates their activity? We will test our hypothesis that these enzymes are master regulators that link phosphate and the energy state of the cell to ribosome biogenesis. Our short-term aims in the next funding period are: 1.Determine how Fpr3/4 histone chaperones regulate transcriptional silencing. 2.Determine how the Fpr4 histone chaperone promotes genome stability at rDNA. 3.Determine how Fpr4 is regulated. Impact and Significance The next phase of my NSERC-funded research program will reveal how a single chromatin modifier can generate functionally distinct chromatin topologies. These details will have a fundamental impact on understanding how chromatin mediates both the process of transcription as well as recombination events. Since our work also probes chromatin control as a function of nutrients, it will also contribute to our understanding of how metabolic state influences the epigenome. I am dedicated to providing first-research opportunities to a diverse group of undergraduates and mentoring them towards their career goals. So, a clear and immediate impact of this proposal is the training of dozens of young highly-qualified personnel that will play a vital role in Canada's science, research and knowledge-based economies.