Figure 3. M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting.

(A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated.

(B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown.

(C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T₂AG₃). The graph represents normalized intensities relative to input (mean ± SD).

(D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown.

(E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors.

(F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).