Effect of DNA methylation on promoter structure

<div><p>Mononucleosomes were prepared from either non-erythroid (<b>A</b>) or erythroid (<b>B</b>) cells taken from Cre (methylated) and Mx-cre (unmethylated) founder (64) transgene crosses and subjected to ChIP analysis using anti-Ac-H4.</p> <p>Input (I) and bound (B) DNA fractions were used for semi-quantitative PCR on 1 or 3 µl samples using primer sets specific for the γ^G or loxP-inserted γ^A promoter regions.</p> <p>For each ChIP preparation, the Acta1 gene (green) was assayed as a negative control and the Actb gene (yellow) as a positive control.</p> <p>The results are summarized in graphic form after normalizing to Acta1 (green) enrichment (set at 1.0) (coefficient of variance = 17%).</p> <p>The results for the Cre (blue) and Mx-cre (red) mice are shown.</p> <p>Results for β globin are presented for comparison.</p> <p>The data shown for non-erythroid cells was obtained using mononucleosomes from MEFs, but the graph summarizes results from 3–4 ChIP experiments on both MEFs and lymphocytes.</p> <p>(<b>C</b>) In vivo footprinting of γ promoter.</p> <p>Erythroblasts (in vivo) or purified erythroblast DNA (in vitro) from mice carrying either a methylated or unmethylated γ^A promoter were treated with DMS. LMPCR gel.</p> <p>Maxam-Gilbert lanes (AG and CT) are sequencing controls.</p> <p>The numbers are according to PubMed accession number NG_000007.</p> <p>Putative protein factor binding regions (rectangles) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Ikuta1" target="_blank">[26]</a>, DMS footprints, as in vivo protected (open circle) or hyper-reactive (closed circle) nucleotides, are indicated.</p> <p>The sizes of the circles represent relative differences in footprint intensities.</p> <p>DMS footprinting on spleen lymphocytes did not show any hyperreactive sites on unmethylated DNA, but did reveal some slight reactivity over the distal CCAAT box on methylated DNA.</p>