The role of Chd1 chromatin remodelers in the repression of pervasive transcription and dissecting the crosstalk between pervasive transcription, nucleosome positioning and turnover.

真核细胞的染色质结构压缩和保护基因组，但也限制了底层DNA的可及性。染色质修饰活性可以打开染色质并提供对特定基因组基因座的受调控的接近。核小体的位置、占有率和周转率等染色质特征建立了一个复杂的基因组索引机制，负责定义基因组中的功能单位。该过程中的缺陷导致在遗传定义的转录单位之外的转录增加、非编码转录物的毒性积累和基因组不稳定性。然而，基因组索引机制如何准确地定义转录单位及其转录本是染色质生物学中的基本问题之一。该建议的目的是：（i）了解核小体的位置，营业额和普遍的转录抑制之间的关系;和（ii）调查的机制，Chd 1保持核小体阵列在基因编码区的均匀，物种特异性的核小体距离。我们将使用不同的裂殖酵母（S。粟酒裂殖酵母）突变体，这些突变体显示出增加的隐蔽启动子活性，并决定了对抑制启动子区域外的转录起始至关重要的染色质特征。我们计划产生各种Chd 1缺失和点突变体，以剖析核小体定位，营业额和普遍的转录之间的串扰，并进一步了解Chd 1在这些过程中的作用。由于核小体之间的连接DNA的长度在物种之间变化很大，我们将比较Chd 1型酶从粟酒裂殖酵母（典型的连接DNA长度，6 bp），嗜热毛壳菌（典型的连接DNA长度，26 bp）和各种嵌合体蛋白产生的核小体阵列，并测试它们对普遍转录和高阶染色质结构的影响。这些研究将显著增加我们对真核生物基因组组织和染色质在基因组功能单位定义中的作用的理解。扰乱这种高度保守的结构会导致普遍的转录活性增加和基因组不稳定性，这是癌症发展和衰老的主要原因。增加对这些过程背后的分子机制的理解可能具有重要的长期治疗意义。

The eukaryotic chromatin structure compacts and protects the genome but also limits the accessibility of the underlying DNA. Chromatin modifying activities can open the chromatin and provide regulated access to specific genomic loci. Chromatin characteristics, such as position, occupancy and turnover-rate of nucleosomes, establish an elaborate genomic indexing mechanism, which is responsible for defining functional units in the genome. Defects in this process lead to increased transcription outside of genetically defined transcription units, toxic accumulation of non-coding transcripts and genomic instability. However, how genomic indexing mechanisms accurately define transcription units and their transcripts is one of the fundamental questions in chromatin biology. The aims of this proposal are: (i) to understand the relationship between nucleosome position, turnover and repression of pervasive transcription; and (ii) to investigate the mechanism by which Chd1 maintains nucleosome arrays in gene coding regions with uniform, species-specific nucleosome distance. We will use various Schizosaccharomyces pombe (S. pombe) mutants that show increased cryptic promoter activity and determine the chromatin characteristics that are critical for repressing transcription initiation outside of the promoter regions. We plan to generate various Chd1 deletion and point mutants to dissect the crosstalk between nucleosome positioning, turnover and pervasive transcription and to further understand the role of Chd1 in these processes. Since the length of the linker DNA between nucleosomes varies widely between species, we will compare nucleosome arrays generated by Chd1-type enzymes from S.pombe (typical linker DNA length, 6 bp), Chaetomium thermophilum (typical linker DNA length, 26 bp) and various chimera proteins and test their effect on pervasive transcription and higher order chromatin structure. These studies will significantly increase our understanding of the eukaryotic genome-organization and the role of the chromatin in the definition of functional units in the genome. Disturbing this highly conserved structure leads to increased pervasive transcription activity and to genomic instability, which is a major cause of cancer development and aging. Increased understanding of the molecular mechanisms behind these processes may have important long-term therapeutic implications.