G9a and Glp1 methylate H1.2K187 in vivo. (a) Reverse transcription PCR analysis of siRNA-transfected cells harvested at indicated time points. HEK293 cells were either transfected with a control siRNA (negative control; NK) or simultaneously with siRNAs against G9a and Glp1 (double knockdown; dkn). mRNA levels were normalised to β-actin expression. The mRNA levels of G9a and Glp1 in the control cells were set as 1 and levels in the double knockdown cells calculated as the ratio of this level (light grey, G9a; dark grey, Glp1). (b) MS analysis of H1.2 after knockdown. Full MS result showing the quantitative comparison of H1 peptides from control and G9a/Glp1 double knockdown samples. We observed an approximate twofold decrease in a peptide (414.750 m/z) from the G9a/Glp1 dkn sample compared with the control. (c) This peptide at 414.750 m/z was sequenced by MS/MS experiments and determined to be the peptide containing the K187 monomethylation mark. The dimethylation of H1.2K187 might have escaped our mass-spectrometric analysis by being below of our current detection threshold.