Loss of BT2 results in hypermethylation of 35S enhancers in the activation-tagged lines. A, The YUCCA1 locus in the yucca1d line. 35S enhancers are indicated as red arrows. Dotted lines represent regions of DNA subjected to McrBC-PCR analysis. B, McrBC-sensitive PCR analysis of the YUCCA1 locus in WT, yucca1d, and yucca1d bt2-1 plants. (−) and (+) represent mock and McrBC digestion, respectively. With this assay, unmethylated DNA regions are amplified by PCR and can be visualized as a single fragment, while methylated DNA regions are sensitive to digestion by the McrBC endonuclease; hence, their amplification is greatly reduced or eliminated. Note: The lack of amplification of the 35S enhancer (4×)-spanning region in WT samples is because they do not have any 35S enhancers, and as such are not linked to the methylation status. A retrotransposon and ACTIN genomic regions were amplified to serve as positive and negative controls for methylated and unmethylated DNA loci, respectively. C, 35S enhancer methylation status as determined by bisulfite sequencing in the yucca1d bt2-1 line. Methylated CpGs and non-CpGs are highlighted in red and blue font, respectively. The asterisk represents mC also observed in the yucca1d line. D, McrBC-sensitive PCR analysis of 35S enhancers in the pap1d and pap1d bt2-1 lines. ImageJ was used to quantify the intensity of the PCR products in B and D, and the values shown are relative to the WT (set to 1). E and F, Quantitative (q) PCR estimation of McrBC-treated 35S enhancers. In an independent experiment, genomic DNA of WT, yucca1d, yucca1d bt2-1, pap1d, pap1d bt2-1 lines were subjected to McrBC digestion, followed by qPCR-based amplification of 35S enhancers. Unmethylated ACTIN was used to normalize the qPCR data and 35S enhancer amplicon levels were plotted relative to the WT (set to ∼1). The asterisks represent statistically significant differences between the indicated samples and the yucca1d line as determined by two-sample t test (P ≤ 0.01).