Fig. 1

Analysis of enhancer-blocking activity and histone methylation marks of the Alu elements located flanking the Nanog locus. a Scheme of the enhancer-blocking assay (EBA). b Insulator activity of NANOG x45s and x14s Alu elements using human HEK293 cell line. Constructs (blue bars) were transiently transfected and their activity analyzed by EBA. Data are showed as fold-enhancer blocking activity normalized to the reference pELuc vector. c Chromatin immunoprecipitation (ChIP) and re-ChIP for CTCF binding to the Nanog x45s Alu were done in NTERA2-wt cells left untreated (UT) or 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 CTCF antibody followed by a second immunoprecipitation with AhR antibody. Input DNAs, immunoprecipitation without specific 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 NANOG locus in NTERA2-wt UT, RA for 48 h and NTERA2-sh UT, RA for 48 h. Three biological replicates and three experimental replicates were done for panel B. Three biological replicates and two experimental replicates were done for panels C and D. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD