Fine Tuning of Globin Gene Expression by DNA Methylation
Figure 3
Effect of DNA methylation on promoter structure
Mononucleosomes were prepared from either non-erythroid (A) or erythroid (B) cells taken from Cre (methylated) and Mx-cre (unmethylated) founder (64) transgene crosses and subjected to ChIP analysis using anti-Ac-H4.
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 γG or loxP-inserted γA promoter regions.
For each ChIP preparation, the Acta1 gene (green) was assayed as a negative control and the Actb gene (yellow) as a positive control.
The results are summarized in graphic form after normalizing to Acta1 (green) enrichment (set at 1.0) (coefficient of variance = 17%).
The results for the Cre (blue) and Mx-cre (red) mice are shown.
Results for β globin are presented for comparison.
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.
(C) In vivo footprinting of γ promoter.
Erythroblasts (in vivo) or purified erythroblast DNA (in vitro) from mice carrying either a methylated or unmethylated γA promoter were treated with DMS. LMPCR gel.
Maxam-Gilbert lanes (AG and CT) are sequencing controls.
The numbers are according to PubMed accession number NG_000007.
Putative protein factor binding regions (rectangles) [26], DMS footprints, as in vivo protected (open circle) or hyper-reactive (closed circle) nucleotides, are indicated.
The sizes of the circles represent relative differences in footprint intensities.
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.