Fig. 2

ICln affects in vivo methylation of histone H3 and binding of PRMT5 to myoD locus, resulting in increased myoD RNA and protein levels. ChIP analysis using chromatin from TIS7 WT or KO MSCs transfected with either YFP or YFP-ICln plasmids. Antisera directed against symmetrically dimethylated histone H3 at arginine 8 (a) or PRMT5 (b) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed in triplicate by qPCR using primers to amplify the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input. ** P ≤ 0.001 and *** P < 0.00005. c TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with anti-MyoD and anti-YFP antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Representative immunoblots are shown. d TIS7 KO MSCs were transfected with the indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 biological replicates and ** P < 0.01). e PRMT5 knockdown significantly increased myoD RNA levels in TIS7 KO MSCs transduced with sh PRMT5-expressing lentivirus. myoD RNA levels were measured by qPCR and PRMT5 protein levels were documented by immunoblots with indicated antibodies