Histone modifications associated with an <i>in vitro</i> methylated <i>MAGEA1</i> transgene.

<p><i>A</i>, Schematic representation of the structure and DNA methylation status of the active unmethylated (empty circles) <i>MAGEA1</i> gene and the inactive methylated (filled circles) <i>MAGEA1/hph</i> transgene in MZ2-MEL.TrHM cells. Black boxes correspond to the <i>MAGEA1</i> exons, the dark gray box within the <i>MAGEA1/hph</i> transgene represents the <i>hph</i> transcription unit, and the asterisk (*) is the site where a 12-bp tag sequence (carrying a <i>Xba</i>I restriction site) was inserted. The lower panel is an enlargement of the amplicon that was amplified in ChIP experiments, and indicates the expected fragment sizes following <i>Xba</i>I digestion. <i>B</i>, ChIP experiments were applied to MZ2-MEL.TrHM. The resulting <i>MAGEA1</i> amplicons were digested with <i>Xba</i>I and separated on agarose gels, thereby revealing relative enrichment of the indicated histone modifications within either the <i>MAGEA1</i> gene (upper band) or the <i>MAGEA1/hph</i> transgene (lower band). <i>C</i>, Relative enrichment of histone marks on the transgene was deduced by quantifying band intensities in gel electrophoresis pictures (ImageJ software), and calculating the lower/upper ratio. Data, which were normalized by the lower/upper ratio in input samples, derive from at least two independent ChIP experiments, with two PCR/XbaI/electrophoresis analyses in each case. * <i>P</i><0.05, *** <i>P</i><0.001.