Analysis of chromatin-RNA interactions during the cell cycle.

DNA由组蛋白包装成染色质，以适应细胞的核。全球，染色质 可以分为开放的、转录活跃的和封闭的、转录沉默的隔室。许多 不同的非组蛋白、染色质结合蛋白对全球染色质结构有贡献。染色质重塑 酶、转录因子和转录相关的RNA对解缩很重要 基因组中转录活跃的部分。异染色质蛋白和相关的非编码RNA是 对基因组转录不活跃部分的浓缩很重要。染色质的变化 组织和紧凑是细胞在发育和染色体过程中状态变化的关键 有丝分裂期间的分离。 染色体结构在有丝分裂开始时发生显著变化，因为染色体紧密并 个人化，为隔离做准备。当细胞进入有丝分裂时，细胞中存在的所有结构 间期核被擦除。相间核结构的擦除与逐步去除相关 粘附素复合体。染色体凝聚和个性化是通过组合完成的 凝集素复合体和拓扑异构酶IIα的作用。有趣的是，染色体的戏剧性变化 结构与转录抑制在时间上是相关的。在有丝分裂染色体的末端 团簇和解聚以改造单一的中间相核。染色体解聚和聚集是 通过重新结合在有丝分裂期间从染色质中移除的许多不同的蛋白质来完成的 在时间上与核转录的恢复有关。 而有丝分裂过程中染色质结构的变化与核的变化有关 在转录活动中，人们对这两个过程是如何联系的知之甚少。我们最近发现 前期去除染色体中的粘附素复合体对于沉默有丝分裂转录至关重要 有丝分裂过程中染色体上染色质结合的RNA的去除是通过磷酸化来实现的 SAF-A。染色体结构变化与染色体结构变化相关的分子通路的鉴定 转录活动提供了一个机会来了解这些事件是如何联系在一起的。在这份提案中，我们 将重建由SAF-A介导的核酸相互作用的生物化学，以提供如何 这种丰富的蛋白质控制着染色体的结构。然后我们将研究如何将SAF-A从 染色质在前期促进染色体凝集。然后我们将研究SAF的重新结合如何- A到有丝分裂末期的染色质促进核重组和转录激活。总而言之， 这项提案中概述的实验将为染色质-RNA相互作用的变化提供新的见解 控制染色质结构以及这些变化如何有助于准确的染色体分离和 转录调控。

DNA is packaged by histone proteins into chromatin in order to fit into the nucleus of a cell. Globally, chromatin can be separated into open, transcriptionally active and closed, transcriptionally silent compartments. Many different nonhistone, chromatin-binding proteins contribute to global chromatin structure. Chromatin remodeling enzymes, transcription factors, and transcription-associated RNAs are important for decondensing transcriptionally active portions of the genome. Heterochromatin proteins and associated noncoding RNAs are important for condensation of the transcriptionally inactive portions of the genome. Changes in chromatin organization and compaction are critical for cell state changes during development and chromosome segregation during mitosis. Chromosome structure changes dramatically at the beginning of mitosis as chromosomes compact and individualize in preparation for segregation. As cells enter into mitosis all of the structure present in the interphase nucleus is erased. Erasure of interphase nuclear structure is correlated with the stepwise removal of the Cohesin complex. Chromosome condensation and individualization are accomplished by the combined action of the Condensin complexes and Topoisomeriase IIα. Interestingly, dramatic changes in chromosome structure are temporally correlated with suppression of transcription. At the end of mitosis chromosomes cluster and decondense to reform a single interphase nucleus. Chromosome decondensation and clustering is accomplished by the rebinding of many different proteins that are removed from chromatin during mitosis and is temporally correlated with the resumption of nuclear transcription. While changes in chromatin structure during mitosis are correlated with changes in nuclear transcriptional activity, little is known about how the two processes are linked. We have recently discovered that prophase removal of the Cohesin complex form chromosomes is critical for silencing mitotic transcription and that removal of chromatin-bound RNAs from chromosomes during mitosis is mediated by phosphorylation of SAF-A. The identification of molecular pathways linking changes in chromosome structure to changes in transcriptional activity presents an opportunity to understand how these events are linked. In this proposal we will reconstitute the biochemistry of nucleic acid interactions mediated by SAF-A to provide a picture of how this abundant protein controls chromosome structure. We will then examine how removal of SAF-A from chromatin promotes chromosome condensation during prophase. We will then examine how rebinding of SAF- A to chromatin at the end of mitosis promotes nuclear reformation and transcriptional activation. Collectively, the experiments outlined in this proposal will provide new insight into changes in chromatin-RNA interactions that control chromatin structure and how these changes contribute to accurate chromosome segregation and transcriptional regulation.