Figure 6.
Characterization of Mage-a genes. (A) RNA expression of Mage-a genes. Six micrograms of total RNA prepared from G9a+/+ (TT2), G9a+/− (#36), G9a−/− (2–3 and 22–10), 2–3-expressing exogenous G9a-L (2–3-L), G9a-S (2–3-S), or a drug-selectable molecule alone (2–3+mock1 and 2) were separated, blotted to a nylon membrane, and hybridized with a 32P-labeled Mage-a8 cDNA. Expression of Mage-a2, Mage-a6, and Mage-a8 genes was determined in G9a−/− ES cells by nucleotide sequencing of the RT-PCR products. The lower panel shows 28S rRNA stained with ethidium bromide in the same gel before blotting. (B) ChIP analyses on the Mage-a2 promoter region. Formaldehyde cross-linked chromatin from TT2, 22–10, and 15–3 (2–3-S) ES cells was immunoprecipitated without (−) or with α-dimethyl H3-K9 (K9), α-dimethyl H3-K4 (K4), or α-acetyl-H3-K9+14 (right panel). An equal amount of Mage-a2 promoter sequence in TT2, 22–10, and 15–3 nucleosomal preparations was determined by PCR from 1.4% of the input ChIP/ PCR reaction (lanes 12–14). The linearity of this PCR was confirmed by serial dilution of the TT2 input DNA (lanes 12,15–17). The product size of the Mage-a2 PCR is 242 bp. Similar results were obtained with multiple independent experiments.