Use of Histone K-M Mutants for the Analysis of Transcriptional Regulation in Mouse Zygotes.

Histone modifications are dramatically altered during the pronuclear (PN) stage of zygotes, and more markedly in paternal than maternal pronuclei. Among various types of histone modifications, lysine methylation exhibits the most dynamic changes in the PN stage . To analyze the physiological functions of histone methylations, it is therefore important to elucidate the mechanism of epigenetic reprogramming. However, loss-of-function approaches using mutant histones whose lysine residues have been substituted with arginine residues are unable to erase histone modifications at all levels, since they are incapable of entirely replacing endogenous histones. To solve this problem, we used an alternative histone mutant whose lysine residues were substituted with methionine (K-M mutants). This mutant cannot be methylated itself but also prevents methylation of endogenous histones. We also developed a simple method for analyzing global transcription levels in early preimplantation embryos, involving using a commercial kit to examine the involvement of histone methylation in zygotic gene activation.