Max is a repressor of germ-cell-related gene expression in mouse embryonic stem cells

Embryonic stem cells (ESCs) and primordial germ cells (PGCs) express many pluripotency-associated genes, but ESCs do not normally undergo conversion into PGCs. Thus, we predicted that there is a mechanism that represses PGC-related gene expression in ESCs. Here we identify genes involved in this putative mechanism, by using an ESC line with a Vasa reporter for an RNAi screen of transcription factor genes expressed in ESCs. We identify 5 genes that result in the expression of Vasa when silenced. Of these, Max is the most striking. Transcriptome analysis reveals that Max knockdown (KD) in ESCs results in selective, global derepression of germ-cell-specific genes. Max interacts with histone H3K9 methyltransferases and associates with the germ-cell-specific genes in ESCs. In addition, Max-KD results in a decrease in histone H3K9 dimethylation at their promoter regions. We propose that Max is part of a protein complex that acts as a repressor of germ-cell-related genes in ESCs. Gene expression profile of Vasa-positive cells isolated from Max knockdown ESCs was compared to that of normal ESCs and in vivo PGCs. As an ESCs sample, a mouse embryonic stem cell line that carries a Venus reporter driven by the Vasa gene promoter (VV3) was used for this study. VV3 ESCs were transfected with non-silencing negative control siRNA (AllStars) or siRNA against the Max gene. Vasa-positive cells were purified using fluorescence-activated cell sorting (FACS). As a PGCs sample, a mouse strain that carries a germ-cell-specific GFP reporter (Oct4ΔPE-GFP) was used. PGCs were also purified using FACS. RNA samples were isolated from each sample and labeled and hybridized to Agilent microarrays. Three biological replicates were performed for each group of samples.