Figure 5.

LEF1 NAT recruits PRC2 to the LEF1 promoter. (A, B) ChIP assays were performed in RWP-1, RWP-1 Snail1 or RWP-1 Snail1 cells transfected with unspliced LEF1 NAT. The immunoprecipitations were performed with the indicated antibodies or with an irrelevant IgG control. The presence of amplicons corresponding to the LEF1 promoter (−931/−750), the NAT promoter (+266/+433) or an irrelevant DNA sequence (+3864/+4068) was determined. The association to these sequences with H3K4me2, an epigenetic mark characteristic of active promoters, is shown in panel A and with H3K27me3, a mark associated to inactive promoters, or of the two members of PRC2 complex, which sets this mark, in panel B. The average ± SD of three experiments performed in triplicate is shown. (C, D) BOPA assays were performed with the biotinylated −1856/+58 LEF1 promoter and cell extracts from RWP-1 Snail1 transfected or not with unspliced NAT (C). In D, when indicated, in vitro synthesized NAT or an irrelevant RNA (YB1X) were added. Samples were incubated with an anti-biotin antibody, the oligonucleotide was pulled-down with protein G-agarose, and the presence of Ezh2 in the precipitate was analyzed by western blot.