Figure 7. Implication of the MAX-containing PRC1.6 complex in the blockade of meiosis-related gene expression in ESCs.

(a) Effect of knockdown of Myc superfamily genes on the expression of Stra8 and Ddx4 genes in ESCs. Expression levels of Stra8 and Ddx4 genes were quantitated in Dox-untreated Max-null ESCs subjected to lentivirus-mediated stable knockdown of the indicated genes. Expression levels of Stra8 and Ddx4 genes in cells subjected to knockdown with a scrambled sequence was arbitrarily set to one. Knockdown efficiency of each gene is shown in Supplementary Fig. 10a. (b) Immunocytochemical analyses of STRA8 in Dox-untreated Max-null ESCs in which expression of Max (upper) and Mga (lower) was subjected to lentivirus-mediated stable knockdown. (c) Effect of L3mbtl2 knockdown on meiotic gene expression in ESCs. Lentivirus-mediated knockdown was conducted as described in a. Expression of Stra8, Ddx4, Slc25a31, Sycp3 and Tafl7 was determined by TaqMan-based quantitative PCR. (d) ChIP analyses of meiotic gene promoters with antibodies against MAX and L3MBLT2. Dox-untreated and -treated Max-null ESCs for 4 days were subjected to ChIP with either anti-MAX or L3MBLT2 antibodies. A ChIP reaction was also performed with control IgG. The genomic DNAs recovered by these ChIP reactions were individually subjected to quantitative PCR to quantify the relative amounts of Ddx4, Slc25a31 and Sycp3 gene promoters compared with those used as input samples (n=3, means±s.d.). Unpaired t-test was done to compare immunoprecipitation efficiency statistically between Dox-untreated and -treated Max-null ESCs (*P<0.05; **P<0.01).