Regulation of chromatin topology

我的NSERC-DG计划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。改变核小体的动力学和密度是一种简单的染色质修饰形式，可以显著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。在上一个资助期间，我的实验室将注意力集中在芽殖酵母酿酒酵母（Saccharomyces cerevisiae）中两种相关的核质蛋白样组蛋白伴侣Fpr 3和Fpr 4上。使用体外合成染色质纤维，我们表明这些蛋白质具有三种不同的核小体修饰特征，即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酰异构化。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Fpr 3和Fpr 4合作调节磷酸盐和核糖体生物合成基因在细胞核中的转录，但仅需要Fpr 4来维持核仁rDNA的基因组稳定性。 总的来说，核质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。但这些工具何时使用？如何监管？在下一个资助期内，我们将确定核质蛋白的三种染色质修饰工具中的每一种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。如果核质蛋白是染色质的专用调节剂，那么是什么调节它们的活性呢？我们将检验我们的假设，即这些酶是连接磷酸盐和细胞能量状态与核糖体生物合成的主要调节剂。 我们在下一个资助期的短期目标是：1.确定Fpr 3/4组蛋白伴侣如何调节转录沉默。 2.确定Fpr 4组蛋白伴侣如何促进rDNA的基因组稳定性。 3.确定Fpr 4如何调节。影响和意义我的NSERC资助的研究计划的下一阶段将揭示如何一个单一的染色质修饰剂可以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测了染色质控制作为营养素的函数，它也将有助于我们理解代谢状态如何影响表观基因组。我致力于提供第一个研究机会，以本科生的多元化群体，并指导他们实现自己的职业目标。因此，这项建议的一个明显和直接的影响是培训了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.