Fig. 5

PRMT1 and CHAF1B drives the formation of NANOG locus Chromatin loop. a and b Chromosome Conformation capture (3C) assay using coordinate 3 as hook. The relative crosslinking frequency was quantified in NTERA-wt cells untreated (UT, blue), treated with RA for 48 h (red) and in NTERA-wt UT cells transfected with CHAF1B siRNA (a) or PRMT1 (b) (green), RA for 48 h (black). 3 + X primer combination was addressed. c Chromatin immunoprecipitation (ChIP) and re-ChIP for AhR and PRMT1 binding to the Nanog x45s Alu were done in NTERA2-wt cells treated with 1 µM of RA for 48 h. For specificity, one primer for the qPCR reaction to amplify each Alu was located in a unique genomic sequence flanking the transposon (see Additional file 3: Table S2). Re-ChIP involved a first immunoprecipitation with AhR antibody followed by a second immunoprecipitation with PRMT1 antibody. Input DNAs, immunoprecipitation without specifics antibodies and immunoprecipitation with GAPDH antibody were also preformed. d Analysis of the pattern of histone methylation marks in the regions of the Alu elements x45s and x14s flanking the Nanog locus in NTERA2-wt transfected with PRMT1 siRNA in conditions UT, RA for 48 h and NTERA2-sh UT, RA for 48 h. e Expression levels of NANOG mRNAs transfected with PRMT1 siRNA (left) or CHAF1B siRNA (right) were quantified by RT-qPCR in NTERA2 cell line left untreated (UT) or treated with 1 µM RA for 48 h. GAPDH mRNA was used to normalize gene expression (A Ct) and 2^−AACt to calculate variations with respect to control or untreated conditions. Three biological replicates and two experimental replicates were done for a, b, c and d. Three biological replicates and three experimental replicates were done for e. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD