Studies of Spt6, a conserved and essential transcription and chromatin factor

描述(申请人提供)：染色质结构为真核生物基因组提供了必要的紧凑，但它也损害了蛋白质进入DNA的途径，从而对蛋白质-DNA交易施加了额外的调节。这个项目的总体目标是了解染色质是如何控制基因表达的。这一应用主要集中在一种基本和保守的转录延伸因子Spt6上，它在转录延伸中具有关键和广泛的作用：Spt6的突变已被证明会导致包括线虫和斑马鱼在内的高等真核生物的发育缺陷。此外，Spt6还被证明可以调节HIV-1病毒基因和感染免疫抑制患者和新生儿的人类巨细胞病毒基因的转录。综上所述，这些发现突显了Spt6在发育过程中和人类疾病中调节染色质结构和转录的重要性，但Spt6发挥作用的分子机制在很大程度上尚不清楚。这项研究提出了生化和基因组方法来了解Spt6是如何调节转录的。AIM 1中Spt6的TAP-Tag纯化将在体内全面鉴定Spt6相互作用的蛋白。重点将放在Spt6和组蛋白之间的相互作用以及组蛋白代码如何调节转录延伸。目标2中提出的生化实验将阐明Spt6如何与核小体相互作用，在转录延伸过程中改变染色体结构，并在转录后恢复正常结构。主要的焦点将是Spt6在转录依赖的组蛋白交换中的作用。最后，在Aim 3中提出的全基因组ChlP-Chip研究将确定Spt6的结合位置，并将它们与现有的基因组数据相关联，例如已知ORF中的转录活性、spt6表达谱和组蛋白修饰图谱。目标是定义Spt6调控的所有细胞过程，并专注于Spt6的新靶点。拟议的研究将进一步深入了解Spt6在染色质结构调控的DNA代谢活动中的作用。相关性：在将信息从基因转移到蛋白质的过程中，称为转录的紊乱，与许多人类疾病有关，如癌症、心脏病和炎症。本研究的目的是阐明Spt6的功能，Spt6是一种重要的、进化上保守的蛋白质，在转录中具有关键和广泛的作用。

DESCRIPTION (provided by applicant): Chromatin structure provides a necessary compaction to the eukaryotic genome, yet it also impairs protein access to DNA, thus imposing additional regulation on protein-DNA transactions. The overall objective of this project is to understand how chromatin controls gene expression. This application focuses on an essential and conserved transcription elongation factor, Spt6, which has critical and widespread roles in transcription elongation: Mutations in Spt6 has been shown to cause developmental defects in higher eukaryotes including C. elegans and zebrafish. In addition, Spt6 has been shown to regulate transcription of HIV-1 viral genes and genes of the human cytomegalovirus which infects immunosuppressed patients and newborns. Taken together, these findings highlight the importance of Spt6 in regulating chromatin structure and transcription during development and in human diseases, yet the molecular mechanism by which Spt6 functions is largely unknown. This study proposes biochemical and genomic approaches to understand how Spt6 regulates transcription. The TAP-tag purification of Spt6 in Aim 1 will comprehensively identify Spt6-interacting proteins in vivo. The focus will be on the interaction between Spt6 and histones and how the histone code regulates transcription elongation. The biochemical experiments proposed in Aim 2 will elucidate how Spt6 interacts with nucleosomes to alter chromosomal structure during transcription elongation and restore proper structure after transcription. The main focus will be on the role of Spt6 in transcription-dependent histone exchange. Finally the genome-wide ChlP-chip study proposed in Aim 3 will identify the binding locations of Spt6, and correlate them with existing genomic data such as transcription activity in known ORFs, spt6 expression profile, and histone modifications maps. The goal is to define all the cellular processes regulated by Spt6, and focus on novel targets of Spt6. The proposed studies will provide further insights into the role of Spt6 in DNA metabolic activities regulated by chromatin structure. Relevance: Disturbances in the process of transferring information from genes to proteins, known as transcription, are involved in many human diseases such as cancer, heart disease, and inflammation. The goal of this research is to elucidate the function of Spt6, an essential and evolutionarily conserved protein that has critical and widespread roles in transcription.