Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications
Figure 4
Lack of long-term activation of MAGEA1/hph following TSA treatment.
A, Schematic outline of the experiment. MZ2-MEL.TrHM cells were treated or not (control) with 300 nM of TSA for 24h, 80 nM of TSA for 72h, or with 20 nM of 5-azadC for 72h. After three days, 106 cells from each group were transferred into two flasks (75 cm2) and were selected in a medium containing hygromycin (180 µg/mL) during 13 days. B, The level of expression of the MAGEA1/hph transgene was quantified by qRT-PCR at the indicated time point (d1 or d3) in each group of cells. Data represent the mean (± sem) of al least three independent experiments. *** P<0.001. C, The level of DNA demethylation of the MAGEA1/hph 5′-region in the different groups of cells was evaluated by quantitative MS-PCR, using primers that specifically amplify the tagged transgene sequence. The data (fold DNA demethylation) were calculated as in Fig 2C, and correspond to the mean (± sem) of at least three independent experiments. * P<0.05. D, The number of clones that survived hygromycin selection were counted at day 16 in the three groups of cells. Data derive from at least two independent experiments, each in duplicate.