Effects of Histone H3 depletion on nucleosome occupancy and positioning through the S. cerevisiae genome [single-end MNase-seq]. Saccharomyces cerevisiae

Experiments performed over the past three decades have shown that nucleosomes are transcriptional repressors. In Saccharomyces cerevisiae, depletion of histone H4 results in the genome-wide transcriptional de-repression of hundreds genes. The mechanism of de-repression is hypothesized to be rooted directly in chromatin changes. To test this, we reproduced classical H4 depletion experiments by conditional repression of all histone H3 transcription, which depletes the supply of nucleosomes in vivo. RNA-seq results were consistent with the earlier studies, but much more sensitive, revealing nearly 2500 de-repressed genes. Changes in chromatin organization were determined by MNase-seq. Nucleosomes that were preferentially retained occurred in regions of high DNA-encoded nucleosome affinity, and were marked with H3K36me2, which is linked to transcription elongation. Nucleosomes harboring acetyl marks or that contained the variant histone H2A.z were preferentially lost. Genes that were de-repressed lost or rearranged nucleosomes at their promoter, but not in the gene body. Therefore, a combination of DNA-encoded nucleosome stability and nucleosome composition dictates which nucleosomes will be lost under conditions of limiting histone protein. This, in turn, governs which genes will experience a loss of regulatory fidelity. Overall design: MNase-seq experiments consist of three wildtype (1 single-end and 2 paired-end) and four mutant (DCB200.1/H3 shutoff; 2 single-end, 2 paired-end) replicates. Each replicate contains two timepoints reflecting chromatin immediately after ("O hours") and 3 hours after transition to media containing dextrose. RNA-seq data includes three replicates from wildtype or H3 depleted cells after 3 hours in media containing dextrose.

过去三十年间的实验研究证实，核小体（nucleosomes）是一类转录抑制因子。在酿酒酵母（Saccharomyces cerevisiae）中，组蛋白H4（histone H4）的缺失会引发全基因组范围内数百个基因的转录去抑制现象。学界推测，该去抑制机制直接源于染色质（chromatin）的结构改变。为验证这一假说，我们通过条件性抑制所有组蛋白H3（histone H3）的转录，模拟经典的组蛋白H4缺失实验，以此在活体内耗尽核小体储备。RNA测序（RNA-seq）结果与早期研究一致，但灵敏度显著提升，共检测到近2500个去抑制基因。染色质组织的变化通过微球菌核酸酶测序（MNase-seq）进行分析：优先保留的核小体位于DNA编码的核小体结合亲和力较高的区域，并带有H3K36me2修饰——该修饰与转录延伸过程密切相关。而带有乙酰化修饰或包含变体组蛋白H2A.z（histone H2A.z）的核小体则更易发生丢失。发生去抑制的基因在其启动子区域出现核小体丢失或重排，但在基因本体区域并无此类变化。因此，DNA编码的核小体稳定性与核小体组成的共同作用，决定了在组蛋白供给受限的条件下哪些核小体会发生丢失，这进而决定了哪些基因会出现调控保真度的丧失。实验整体设计：微球菌核酸酶测序（MNase-seq）实验包含3份野生型重复样本（1份单端测序、2份双端测序）与4份突变型重复样本（DCB200.1/H3基因闭合株；2份单端测序、2份双端测序）；每份重复样本包含两个时间点的样本，分别为转移至含葡萄糖培养基后即刻（"O hours"）与3小时后的染色质样本。RNA测序（RNA-seq）数据则包含3份野生型或组蛋白H3缺失细胞的重复样本，均取自含葡萄糖培养基中培养3小时后的细胞。