913273.fig.004a
(a)
913273.fig.004b
(b)
913273.fig.004c
(c)
Figure 4: Scheme of insertional chromatin immunoprecipitation (iChIP). The system consists of a locus of interest (e.g., a promoter, an enhancer, and an silencer of a gene) linked to LexA-binding elements (LexA BE) (a), and FLAG-tagged, nuclear localization signal (NLS)-fused LexA DNA-binding domain (DB) (3xFNLDD) (b). LexA-binding sites are knocked-in in the genomic locus of interest in cells expressing the tagged LexA DB. Alternatively, cells expressing the tagged LexA DB are transiently or stably transfected with the transgene tagged with LexA BE. These cells are crosslinked with formaldehyde or other crosslinkers, if necessary, and lysed. Then, DNA is fragmented by sonication or other methods. Subsequently, the LexA-tagged genomic region is immunoprecipitated with an anti-FLAG antibody, and crosslink is reversed when a crosslinker is used. Molecules (DNA, RNA, proteins, and others) associated with the LexA-tagged genomic regions are characterized (c).