Histone Recycling by FACT and Spt6 during Transcription Prevents the Scrambling of Histone Modifications [ChIP-seq]

The genetic information encoded in DNA is framed by additional layers of information, referred to as the epigenome. Epigenetic marks such as DNA methylation, histone modifications and histone variants are concentrated on specific genomic sites as means to instruct, but also sometimes as a consequence of, gene expression. How this information is maintained, notably in the face of transcription, is not understood. Here we show that the histone chaperones FACT and Spt6 are required for maintaining proper localization of several histone modifications including H3K4me1,2,3, H3K36me3, H3K79me3, H3K14ac, H3K18ac and H2Bub in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates massive nucleosome loss which is partially compensated by increased histone assembly by histone chaperones such as Asf1 and HIR. Because re-incorporation of histones by these histone chaperones in not coupled to transcription, the modified histones are randomly incorporated, leading to scrambling of the epigenetic information. Hence, our work highlights the importance of local nucleosome recycling by FACT and Spt6 during transcription in the maintenance of a proper epigenetic landscape. Overall design: Occupancy profiling by ChIP-seq of histone modifications H3K4me3 and H3K56ac as well as histones H4 and H3 in wild-type (WT) and temperature-sensitive mutants for the histone chaperones FACT and Spt6 (spt16-197 and spt6-1004 respectively) in yeast Saccharomyces cerevisiae, in duplicate, for a total of 12 Input DNA and 24 ChIP samples. For all samples, a fixed amount of crosslinked S. pombe cells (representing 10% of the S. cerevisiae cells) was added to the S. cerevisiae cells prior to cell lysis. This spike-in control was used to normalise samples.

DNA所编码的遗传信息，由额外的信息层级所框定，该层级被称为表观基因组（epigenome）。表观遗传标记（epigenetic mark）如DNA甲基化、组蛋白修饰与组蛋白变体，会富集于特定基因组位点，既可以作为调控基因表达的手段，有时也可作为基因表达的结果。目前这类表观遗传信息如何得以维持，尤其是在转录过程中，仍未被阐明。本研究表明，在酿酒酵母（Saccharomyces cerevisiae）中，组蛋白伴侣（histone chaperone）FACT与Spt6对于维持多种组蛋白修饰的正常定位至关重要，这些修饰包括H3K4me1/2/3、H3K36me3、H3K79me3、H3K14ac、H3K18ac以及H2Bub。当功能性FACT或Spt6缺失时，转录会引发大量核小体丢失，而这类丢失可通过Asf1与HIR等组蛋白伴侣介导的组蛋白组装增强得到部分补偿。由于这些组蛋白伴侣介导的组蛋白重新组装并不与转录过程偶联，修饰后的组蛋白会被随机整合至基因组，进而导致表观遗传信息的紊乱。因此，本研究凸显了转录过程中FACT与Spt6介导的局部核小体循环，对于维持正常表观基因组格局的重要性。整体实验设计：针对酿酒酵母中的野生型（WT）以及组蛋白伴侣FACT与Spt6的温度敏感突变体（分别为spt16-197与spt6-1004），通过染色质免疫共沉淀测序（ChIP-seq）对组蛋白修饰H3K4me3、H3K56ac以及组蛋白H4与H3进行结合谱分析，每组设置生物学重复，最终共获得12份Input DNA样本与24份ChIP样本。所有样本在细胞裂解前，均会加入固定数量的交联粟酒裂殖酵母（S. pombe）细胞（其占酿酒酵母细胞总量的10%）作为spike-in内参对照，用于后续样本的归一化处理。