Abstract

Histone modifications and DNA methylation together govern promoter availability, thereby influencing gene expression. This study queries the unicellular chlorophyte, Chlamydomonas reinhardtii using a three step “epigenetic assay” design to phenotypically track the variegation of a randomly integrated Paromomycin resistance transgene(s) (Pm^R). Based on its position of integration, the Pm^R gene expression hinged on two epigenetic hallmarks: the spreading of heterochromatin, and the transmissible memory of epigenetic states across generations. Using a spot-dilution analysis, the loss of antibiotic resistance phenotype was scored from 0 to 4, four being maximally silenced. Appropriate construct designs were used to demonstrate that the cis-spread of heterochromatin could be interfered with a stronger euchromatic barrier (TUB2 promoter). When assayed for metal ion stress, a combination of Mn deficiency with excess Cu or Zn stress was shown to induce gene silencing in Chlamydomonas. Cu stress resulted in the accumulation of intracellular ROS, while Zn stress elevated the sensitivity to ROS. As proof of functional conservation, mammalian epigenetic drugs demonstrably interfered with stress-induced gene silencing. Finally, a selected group of transgenic clones responsive to HDACi sodium butyrate, when tested in a gradient plate format showed similarity in phenotype to the plant-derived compound cinnamic acid. This indicated a possible commonality in their mode of action, unlike curcumin which might have a different mechanism. Thus, using binned libraries, based on a common set of responses to known drugs, a cost-effective high-throughput screening strategy for epigenetically active compounds from plants or other sources is described.

摘要

组蛋白修饰和 DNA 甲基化共同控制启动子的可用性，从而影响基因表达。本研究使用三步“表观遗传分析”设计来查询单细胞叶绿体莱茵衣藻，以表型跟踪随机整合的巴龙霉素抗性转基因 ( Pm ^R ) 的变异。根据其整合位置，Pm ^R基因表达取决于两个表观遗传标志：异染色质的扩散和跨代表观遗传状态的可传递记忆。使用点稀释分析，抗生素抗性表型的丧失从 0 到 4 评分，其中四个被最大程度地沉默。适当的构造设计被用来证明异染色质的顺式传播可能会受到更强的常染色质屏障（TUB2启动子）的干扰。当测定金属离子压力时，显示出 Mn 缺乏与过量 Cu 或 Zn 胁迫相结合可诱导衣藻中的基因沉默. Cu胁迫导致细胞内ROS的积累，而Zn胁迫提高了对ROS的敏感性。作为功​​能保护的证据，哺乳动物表观遗传药物明显干扰了应激诱导的基因沉默。最后，一组选定的对 HDACi 丁酸钠有反应的转基因克隆在梯度板形式中测试时显示出与植物衍生化合物肉桂酸的表型相似性。这表明它们的作用方式可能存在共性，这与姜黄素不同，姜黄素可能具有不同的机制。因此，使用分箱库，基于对已知药物的一组常见反应，描述了一种具有成本效益的高通量筛选策略，用于来自植物或其他来源的表观遗传活性化合物。