Journal: Molecular and Cellular Biology
Article Title: Mbd1 Is Recruited to both Methylated and Nonmethylated CpGs via Distinct DNA Binding Domains
Figure Lengend Snippet: Localization of Mbd1 in DNA methylation-deficient cells requires the CXXC-3 domain. (A) Schematic representation of the Flag-Mbd1a expression constructs employed. The positions of the MBD (cross-hatched), CXXC-3 (dark gray), and the TRD (hatched) are shown. The CXXC-1 and CXXC-2 positions are shown shaded in light gray, and the Flag tag position is dotted. An asterisk marks the point mutations in the MBD (R22A), and “Δ” marks the sites of deletion of CXXC-3 or the TRD. (B) Diagram illustrating the enrichment for methyl CpG or nonmethyl CpG in the heterochromatic regions of wild-type ( Dnmt1 +/+ ) or methylation-deficient ( Dnmt1 n/n ) cells, respectively. Methyl-CpG is shown with filled circles and nonmethyl-CpGs with open circles. The heterochromatic regions are represented by lightly shaded boxes. (C) Dnmt1 +/+ (panels a to d) or Dnmt1 n/n (panels e to h) mouse embryonic fibroblasts were transfected with constructs expressing Flag-tagged Mbd1a (either full length or the indicated mutants). At 48 h after transfection, the cells were fixed and stained using an anti-Flag antibody (left panels). DNA was counterstained with DAPI (right panels). (D) Schematic of Gal4-DBD-MBD1 fusion constructs analyzed for colocalization with heterochromatic foci in Dnmt1 +/+ or Dnmt1 n/n mouse embryonic fibroblasts. A plus sign indicates colocalization with heterochromatic foci in Dnmt1 n/n cells and diffuse staining in Dnmt1 +/+ cells, whereas a minus sign indicates diffuse staining in cells of either genotype. The fusion constructs encode amino acids 81 to 556, 261 to 556, and 261 to 346 of the MBD1 isoform PCM1. The Gal4-DBD is shown in black. CXXC-2 is shown shaded in light gray, CXXC-3 is shown shaded in dark gray, and the TRD is hatched. (Lower panels) Colocalization with heterochromatic foci of the Gal4-DBD-MBD1 (amino acids 261 to 346) (Gal4-DBD-CXXC-3) in Dnmt1 n/n cells is shown.
Article Snippet: Slides were blocked in 3% bovine serum albumin-PBS before incubation with primary antibody (anti-Flag M2 [Stratagene], anti-Gal4 [Santa Cruz], or anti-Mbd1 [M245] [Santa Cruz] [all at 1:1,000]) for 60 min.
Techniques: DNA Methylation Assay, Expressing, Construct, FLAG-tag, Methylation, Transfection, Staining