Potential Role of Trap Clone Accumulation Areas (TCAAs) in Sustaining Pluripotency in Mouse Embryonic Stem Cells

Analysis of gene trap clones (TCs) revealed the existence of regions where TCs accumulate in the absence of genes. These regions were designated as trap clone accumulation areas (TCAAs). To ascertain the physiological function of TCAAs, negative control regions devoid of genes and TCs (NC1 and NC11), two randomly selected known gene sets (G1 and G11), and a set of genes presumed to be involved in maintaining pluripotency in embryonic stem (ES) cells (GP) were generated and compared with TCAAs. The assay for transposase-accessible chromatin with sequencing (ATAC-Seq) results indicated that TCAAs exhibited characteristics comparable to G1, G11, and GP, suggesting an open chromatin structure. Oct4-chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that TCAAs had markedly elevated signals compared to G1 and 11, and a comparable level to that of GP. With regard to H3K4me1 and H3K27ac, which are associated with enhancer activity, TCAAs were observed to exhibit significantly higher levels than G1 and 11 and a comparable level to that of GP. Furthermore, approximately half of the super-enhancers overlapped with TCAAs in an ES cell-specific manner. These findings suggest that TCAAs are involved in maintaining the pluripotency of mouse ES cells.