MGA directly recruits SETDB1/ATF7IP for histone H3K9me3 on meiosis-related genes in mouse embryonic stem cells.

Many of meiosis-related genes in mouse embryonic stem cells (ESCs) bear substantial amounts of trimethylated lysine 9 of histone H3 (H3K9me3) modification. However, how meiosis-related genes in ESCs acquire this histone modification is totally obscure. We hypothesize that the specific functional domain termed FAM of MGA that is the scaffolding component of PRC1.6, one of atypical subtypes of PRC1 is directly involved in the deposition of this histone modification by recruiting SETDB1 via its interaction with ATF7IP. To address the hypothesis, we generated ESCs producing MGA that lacks FAM domain and examined effects of the mutation on global expression profile and H3K9me3 levels by RNA- and ChIP-sequence analyses, respectively. We also generated ESCs producing mutant ATF7IP lacking its FNIII domain that is considered to be crucial for the interaction with MGA in our hypothesis to assess the role of the domain for the deposition of H3K9me3 by ChIP-seq analyses. Mouse ESCs producing MGA that lacks FAM domain or ATF7IP that lacks of FNIII domain were generated by means of lenti virus-mediated CRISPR-Cas9 system. Fragmented chromatins were prepared from these genetically manipulated- and parental-ESCs and used for ChIP-sequence analyses to elucidate the effect of removal of these domains on the magnitude/distribution of H3K9me3 modification in ESCs. Mouse ESCs producing the MGA mutant and their control counterparts were also used for RNA-seq analyses to examine the effect of removal of the FAM domain from MGA on global expression profile.