Figure 2

Release of silencing of the DsRed-LacI gene and loss of DNA methylation from lac operator repeats in a ddm1 mutant. A. DsRed fluorescence in roots of seedlings doubly homozygous for a tagged locus (101, 107, 112) and an epigenetic mutation: drd1 (dr), rdr6 (r), wild type (wt), ddm1 (dd), mom1 (m). Increased fluorescence is observed only in the ddm1 mutant. The bar indicates 2 mm. B. DNA methylation analysis of lac operator repeats. The lac operator repeat array is ~9.2 kb (256 copies of a 24-bp lac O monomer plus a 12-bp linker sequence) [8]. The lac operator repeat arrays can potentially be cut into 315 bp fragments by the restriction enzymes Eco RI (E) and Nar I (N) [15]. Whereas the former is insensitive to cytosine methylation, Nar I will not cleave if the CG in its recognition site (GGCGCC) is methylated (compare N versus E lanes in the three WT panels). Retention of the higher band (mom1, rdr6 and drd1 lanes) after Nar I digestion signifies wild type levels of CG methylation; production of a ladder of smaller bands (ddm1 lanes) indicates loss of CG methylation. Size markers are indicated to the left. Data are shown for three tagged lines (101, 107, 112) into which we successfully introduced all four mutations (drd1, ddm1, sgs2, mom1). For the other two tagged lines (26, 79), we were unable to generate homozygous mutants for all four of the epigenetic factors. However, we did successfully introgress the ddm1 mutation into these lines, which resulted in loss of CG methylation from the lac operator repeats in ddm1 (Additional file 1).