Systematic dissection of roles for chromatin regulators in a yeast stress response

Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 deletion mutants, on induction/repression dynamics of 200 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We follow up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during diamide stress, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and show that the “activating” mark H3K4me3 functions largely as a repressor in yeast. We set to track how histone modifications change during transcriptional reprogramming. We perfomed a time course experiment following treatment of mid-log growing yeast (BY4741) with diamide. Samples were collected at t=0,4,8,15,30,60 and fixed by crosslinking. We extracted mononucleosomes by MNase digestion and then applied ChIP for selected histone modifications. The IPed material was hybridized to genomic tiling arrays against the input material.

真核基因组组装为染色质的过程，对基因转录具有广泛影响。令人费解的是，学界普遍观察到：敲除某一全局染色质调控因子后，仅会影响该因子结合或修饰的基因中的一小部分的表达。不过，诸多单基因研究已证实，染色质调控因子通常不会影响稳态转录，而是介导环境信号诱导的正常转录重编程过程。为此，我们系统探究了83种组蛋白突变体与119种敲除突变体，对酵母中200个转录本在二酰胺胁迫下的诱导/抑制动力学的影响。值得注意的是，我们发现染色质调控因子在基因诱导/抑制过程中的作用，远强于其在稳态表达中的作用。此外，通过联合分析底物（组蛋白突变体）与酶类（染色质修饰因子敲除突变体），我们鉴定出组蛋白修饰与其调控因子之间的特异性相互作用。我们将这些功能实验结果，与同期开展的全基因组范围内多种组蛋白修饰图谱绘制工作相结合，系统探究了组蛋白修饰的存在与功能之间的对应关系。我们针对一条通路开展了跟进研究，发现二酰胺胁迫下，依赖Set1的H3K4甲基化主要发挥基因抑制作用，且该作用特异性地体现在参与核糖体生物合成的基因上。Set1介导的核糖体基因抑制，在核糖体蛋白基因与核糖体生物发生基因之间采用了不同通路，二者可通过抑制所需的遗传需求，以及基因抑制过程中的染色质变化加以区分。综上，本项动态研究为染色质调控相关研究提供了丰富的资源，同时证实，此前被认为是“激活型”修饰的H3K4三甲基化（H3K4me3）在酵母中大多发挥抑制功能。我们旨在追踪转录重编程过程中组蛋白修饰的变化情况。我们开展了时序实验：将处于对数中期的酵母（BY4741）用二酰胺处理，分别在t=0、4、8、15、30、60分钟时收集样本并通过交联法固定。我们通过微球菌核酸酶（MNase）消化提取单核小体，随后针对目标组蛋白修饰开展染色质免疫共沉淀（ChIP）实验，并将免疫沉淀所得样本与输入样本的基因组平铺芯片进行杂交。