Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [ChIP-seq]. Schizosaccharomyces pombe

Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the requirements for this critical factor, we have performed genome-wide analyses of transcription, chromatin structure, and histone modifications in an S. pombe spt6 mutant. Our results demonstrate several dramatic changes to transcription and chromatin structure in the spt6 mutant, including an elevation of antisense transcripts at over 70 percent of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for the trimethylation of histone H3 on lysines 4 and 36, marks associated with active transcription. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. Overall design: ChIP-seq experiments were performed on wild type and spt6-1 strains on the following proteins: RNA polymerase II (Rpb1), Paf1 Complex (Ctr9), COMPASS (Swd1), Set2, Spt6, histones H2B and H3, histone modifications H3K4me3 and H3K36me3. Experiments were performed in replicates and matching inputs were also sequenced.

Spt6是一种高度保守的组蛋白伴侣（histone chaperone），可直接结合RNA聚合酶II（RNA polymerase II）与组蛋白，进而调控基因表达。为全面解析这一关键因子的功能必需性，我们对粟酒裂殖酵母（S. pombe）的spt6突变体开展了全基因组范围的转录组、染色质结构及组蛋白修饰分析。本研究结果显示，spt6突变体的转录与染色质结构出现多处显著改变：超过70%的基因的反义转录本水平显著升高，且+1核小体普遍丢失。此外，组蛋白H3第4位赖氨酸（H3K4）与第36位赖氨酸（H3K36）的三甲基化修饰依赖于Spt6，而这两种修饰均与活跃转录过程相关。综上，本研究结果表明，Spt6对于转录的精确性与染色质的完整性至关重要，其功能可能通过与RNA聚合酶II及组蛋白的直接相互作用实现。整体实验设计：针对野生型菌株与spt6-1突变菌株，针对以下蛋白及修饰开展染色质免疫共沉淀测序（ChIP-seq）实验：RNA聚合酶II（Rpb1）、Paf1复合物（Ctr9）、COMPASS复合物（Swd1）、Set2、Spt6、组蛋白H2B与H3，以及组蛋白修饰H3K4me3与H3K36me3。所有实验均设置生物学重复，同时对匹配的输入样本进行了测序。