Genomic analysis of histone replacement dynamics in yeast

描述(由申请人提供)：染色质在不同时间尺度下控制的过程中扮演关键角色，以应对环境变化，并需要快速可塑性，而通过多代细胞的长期稳定需要表观遗传的染色质。因此，了解活细胞中动态染色质状态的性质是非常有意义的。我们已经开发了能够在酵母基因组的很大一部分上以高分辨率快速测量染色质结构的工具，并且我们的实验室最近扩展了这些工具的使用，以在G1停滞的酵母中测量整个酵母基因组中的组蛋白H3替换动态。我们的初步数据让我们提出了以下问题。在没有基因组复制的情况下，组蛋白替代的机制是什么？H3/H4和H_2A/H_2B的替代率有何不同？我们能机械地将这两个过程分开吗？H3K56乙酰化是否是组蛋白替代所必需的，它在G1期停滞中的作用是什么？组蛋白替换在细胞周期中如何变化，以及异染色质沉默和染色体凝聚等过程如何影响组蛋白替换？核小体替换是否起到相互隔离染色质结构域的作用？这些问题中的大多数只能通过基因组分析来充分解决，因为在许多情况下，单基因座研究可能具有误导性。我们已经开创了几种染色质结构的基因组分析技术，并处于开展拟议研究的理想位置。综上所述，拟议的研究将为了解染色质的动态性质提供一个非常有价值的窗口。我们将确定组蛋白替代的机制，并将确定细胞周期在调节染色质紧致状态稳定性中的作用。这些实验将极大地加深我们对染色质结构的基础知识，将扩大我们对转录控制的理解，并将限制任何染色质状态稳定遗传的模型。表观遗传是DNA序列以外信息的遗传，是人体不同细胞类型之间差异的基础。在过去的十年里，越来越清楚的是，表观遗传缺陷，除了基因突变之外，在癌症中发挥着重要作用。在这项提案中，我们的目标是研究使细胞能够遗传表观遗传信息的机制，希望我们最终可以利用这些信息来设计更有针对性的抗癌疗法。

DESCRIPTION (provided by applicant): Chromatin plays critical roles in processes governed in different time scales in response to environmental changes and requires rapid plasticity, while long term stability through multiple cell generations requires epigenetically heritable chromatin. It is of great interest, therefore, to understand the nature of dynamic chromatin states in living cells. We have developed tools that enable rapid measurement of chromatin structure at high resolution over a significant fraction of the yeast genome, and our lab has recently extended the use of these tools to measure histone H3 replacement dynamics across the entire yeast genome in G1-arrested yeast. Our preliminary data lead us to ask the following questions. What are the mechanisms for histone replacement in the absence of genomic replication? How do replacement rates differ for H3/H4 and H2A/H2B? Can we mechanistically separate those two processes? Is acetylation of H3K56 required for histone replacement, and what is its role during G1 arrest? How does histone replacement change during the cell cycle, and how do processes such as heterochromatin silencing and chromosome cohesion influence histone replacement? Does nucleosome replacement act to insulate chromatin domains from one another? Most of these questions can only be adequately addressed by genomic analysis, since in many cases single locus studies can be misleading. We have pioneered several techniques for genomic analysis of chromatin structure, and are ideally positioned to carry out the proposed studies. Taken together, the proposed studies will provide an immensely valuable window into the dynamic nature of chromatin. We will identify mechanisms of histone replacement, and will determine the role of cell cycle in regulation of the stability of chromatin compaction states. These experiments will greatly further our basic knowledge of chromatin structure, will expand our understanding of transcriptional control, and will constrain any models for the stable inheritance of chromatin states. Epigenetic inheritance, the inheritance of information beyond DNA sequence, underlies the differences between different cell types in the human body. In the past decade it has become increasingly clear that epigenetic defects, in addition to genetic mutations, play a major role in cancer. In this proposal we aim to investigate the mechanisms that enable cells to inherit epigenetic information, with the hope that we may eventually use this information to design more directed therapies against cancer.