Investigating Mitotic Inheritance of Histone Modifications Using Tethering Strategies

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.