De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes

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Abstract
G9a is involved in gene silencing during early embryonic development, catalyzing the methylation of H3K9, which results in heterochromatinization, and also promoting methylation of DNA de novo. These two G9a activities are now dissected, and de novo DNA methylation is shown to occur via recruitment of Dnmt3a/3b and to be necessary and sufficient to prevent reprogramming. The pluripotency-determining gene Oct3/4 (also called Pou5f1) undergoes postimplantation silencing in a process mediated by the histone methyltransferase G9a. Microarray analysis now shows that this enzyme may operate as a master regulator that inactivates numerous early-embryonic genes by bringing about heterochromatinization of methylated histone H3K9 and de novo DNA methylation. Genetic studies in differentiating embryonic stem cells demonstrate that a point mutation in the G9a SET domain prevents heterochromatinization but still allows de novo methylation, whereas biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin domain, by recruiting Dnmt3a and Dnmt3b independently of its histone methyltransferase activity. These modifications seem to be programmed for carrying out two separate biological functions: histone methylation blocks target-gene reactivation in the absence of transcriptional repressors, whereas DNA methylation prevents reprogramming to the undifferentiated state.