Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA.
Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA.
Methods Mol Biol. 2022;2529:419-440. doi: 10.1007/978-1-0716-2481-4_18.
The covalent and reversible modification of histones enables cells to establish heritable gene expression patterns without altering their genetic blueprint. Epigenetic mechanisms regulate gene expression in two separate ways: (1) establishment, which depends on sequence-specific DNA- or RNA-binding proteins that recruit histone-modifying enzymes to unique genomic loci, and (2) maintenance, which is sequence-independent and depends on the autonomous propagation of preexisting chromatin states during DNA replication. Only a subset of the vast repertoire of histone modifications in the genome is heritable. Here, we describe a synthetic biology approach to tether histone-modifying enzymes to engineer chromatin states in living cells and evaluate their potential for mitotic inheritance. In S. pombe, fusing the H3K9 methyltransferase, Clr4, to the tetracycline-inducible TetR DNA-binding domain facilitates rapid and reversible control of heterochromatin assembly. We describe a framework to successfully implement an inducible heterochromatin establishment system and evaluate its molecular properties. We anticipate that our innovative genetic strategy will be broadly applicable to the discovery of protein complexes and separation-of-function alleles of heterochromatin-associated factors with unique roles in epigenetic inheritance.
组蛋白的共价和可逆修饰使细胞能够建立可遗传的基因表达模式，而无需改变其遗传蓝图。表观遗传机制以两种不同的方式调节基因表达：（1）建立，这取决于序列特异性的 DNA 或 RNA 结合蛋白，这些蛋白将组蛋白修饰酶募集到独特的基因组位置，以及（2）维持，这是序列非依赖性的，依赖于在 DNA 复制过程中预先存在的染色质状态的自主传播。基因组中大量组蛋白修饰中只有一部分是可遗传的。在这里，我们描述了一种合成生物学方法，将组蛋白修饰酶固定在活细胞中，以构建染色质状态，并评估它们在有丝分裂遗传中的潜力。在 S. pombe 中，将 H3K9 甲基转移酶 Clr4 与四环素诱导的 TetR DNA 结合域融合，可快速且可逆地控制异染色质的组装。我们描述了一个成功实现诱导性异染色质建立系统的框架，并评估了其分子特性。我们预计，我们的创新遗传策略将广泛适用于发现与表观遗传遗传中具有独特作用的异染色质相关因子的蛋白质复合物和功能分离等位基因。
