Figure 6: Max protein associates with histone H3K9 methyltransferases and the promoter regions of PGC-related genes.

(a,b) Immunoprecipitated samples using anti-Max antibody were analysed on western blots. We used the Max-KO ESCs with Max cDNA introduced in ROSA26 gene locus together with tetracycline off system⁶. The Max-null ESCs did not express the Max protein in the presence of 1 μg ml⁻¹ doxycycline (Dox). GLP and G9a were immunoprecipitated with anti-Max antibody only in the absence of Dox (a), but Ezh2 was not (b). Max protein was only present when Dox was absent; therefore, these observations strongly supported the conclusion that Max specifically interacted with GLP and G9a in ESCs. Principally the same result was obtained in two independent experiments (a,b). Asterisk in b indicates a non-specific band. (c) ChIP assays involving anti-Max antibodies and undifferentiated VV3 were used to examine the association of Max protein with promoter region of germ-cell-related genes. Relative ratios of the immunoprecipitated chromatin to input chromatin were determined by real-time PCR. The data shown are representative of data from three independent experiments. (d) ChIP assays involving anti-histone H3K9me2 antibody and Max-KD VV3 were used to examine the amount of methylated histone H3K9 in the promoter region of late PGC marker genes, and of haemoglobin β (Hbb-b1) gene as a negative control. There was a significant reduction in methylated histone H3K9 in germ-cell-related genes in Max-KD VV3 cells compared with control VV3 cells. By contrast, no reduction was observed in Hbb-b1. The amount of methylated histone H3K9 relative to input were calculated and ratios of the amount in Max-KD VV3 to control VV3 were represented. Shown is a representative of two independent experiments. IP, immunoprecipitation; US, upstream region; WB, western blot.