GENOME WIDE IMPACT OF SSL2 MUTANTS ON TRANSCRIPTION START SITE USAGE. Saccharomyces cerevisiae

We used next generation sequencing (NGS) approach to evaluate Ss2's effect on TSS usage on promoters genome-wide (Chapter IV). We have defined two potential classes of ssl2 alleles in terms of their functions in Pol II scanning and propose they are TFIIH processivity alleles that control TSS distributions by narrowing or widening scanning windows at promoters. Representatives of these two classes and their TSS effects have been tested at the model genes of IMD2 and ADH1 in S. cerevisiae. To gain an insight into the impact of TFIIH's activity on TSS usage genome-wide, we have examined 5' ends of RNA transcripts for these two classes of ssl2 allele in S. cerevisiae. We first investigated TSS distribution in two classes of ssl2 alleles by examining the distribution of the reads at 5979 yeast promoters. We also compared the shift of TSS distribution between WT and ssl2 mutants. As observed in model gene promoters, ssl2 alleles that shift TSS distribution upstream or downstream at IMD2 or ADH1 promoters also shift TSS distribution at most of the other examined gene promoters. We, therefore, measured the degree of TSS shift in ssl2 alleles by comparing the median TSS positions of the mutant and the WT. We also asked if the degree of TSS shift correlated with the architecture of the promoter, for example, the distance between the PIC and the dominant TSS (PIC-TSS distance), so we also examined the relationship between the degree of the TSS shift and the distance between PIC-TSS distance. Our Shooting Gallery model predicts that the hypothetical ssl2 processivity GOF alleles have the potential to expand the scanning window and the ssl2 processivity LOF alleles would reduce the scanning window. To test these predictions, we examined the width of the scanning window by measuring the distribution of 80% TSS spanning region in candidate ssl2 processivity alleles genome-wide.

本研究采用下一代测序（next generation sequencing, NGS）技术，评估Ss2对全基因组启动子区域转录起始位点（transcription start site, TSS）使用情况的影响（第四章）。我们基于Ssl2在RNA聚合酶II（RNA polymerase II, Pol II）扫描过程中的功能，将ssl2等位基因划分为两类潜在亚型，并提出它们属于转录因子IIH（transcription factor IIH, TFIIH）持续合成能力等位基因，可通过缩小或扩大启动子处的扫描窗口来调控TSS分布。我们在酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）的IMD2与ADH1模式基因中，对这两类等位基因的代表株及其对TSS的影响进行了验证。为深入解析TFIIH活性对全基因组范围内TSS使用情况的影响，我们对酿酒酵母中这两类ssl2等位基因的RNA转录本5'端进行了检测。我们首先通过分析5979个酵母启动子的测序读段分布情况，探究了两类ssl2等位基因的TSS分布特征。同时，我们还比较了野生型（wild type, WT）与ssl2突变体之间的TSS分布偏移情况。正如模式基因启动子中观察到的结果，在IMD2或ADH1启动子处可使TSS分布向上游或下游偏移的ssl2等位基因，在绝大多数其他检测过的基因启动子中也能引发类似的TSS分布偏移。为此，我们通过比较突变体与野生型的TSS中位位置，量化了ssl2等位基因的TSS偏移程度。我们还探究了TSS偏移程度是否与启动子结构存在关联，例如预起始复合物（pre-initiation complex, PIC）与优势TSS之间的距离（PIC-TSS距离），因此我们分析了TSS偏移程度与PIC-TSS距离之间的相关性。本研究的"Shooting Gallery"模型预测，假设的ssl2持续合成能力功能获得型（gain-of-function, GOF）等位基因可扩大扫描窗口，而ssl2持续合成能力功能缺失型（loss-of-function, LOF）等位基因则会缩小扫描窗口。为验证上述预测，我们通过检测候选ssl2持续合成能力等位基因中覆盖80% TSS的区域的分布情况，分析了扫描窗口的宽度。