Figure 4

Pint is a nuclear long intergenic non-coding RNA (lincRNA) that interacts with PRC2. (A) Pint subcellular localization. Percentage of total RNA found in the nuclear and cytoplasmic fractions of p53-restored doxorubicin-treated p53^LSL/LSL mouse embryonic fibroblasts (MEFs) determined by quantitative real time(RT-qPCR). (B) Single-molecule visualization of Pint. RNA fluorescent in situ hybridization (FISH) of Pint in 3T3 cells untreated (-DOX) or treated (+DOX) with doxorubicin. (C) Quantification of the relative subcellular distribution of Pint FISH foci. (D) Physical association between Pint and PRC2 after chemical crosslinking of cells. Suz12 or Wdr5 were immunoprecipitated from nuclear extracts of formaldehyde-crosslinked p53-restored doxorubicin-treated p53^LSL/LSL MEFs, and associated RNAs were detected by RT-qPCR. The relative enrichment was calculated as the amount of RNA associated with Suz12 or Wdr5 IP relative to the IgG control. Gapdh mRNA was used as control RNA. (E) In vitro interaction of Pint with Polycomb repressive complex 2 (PRC2). Protein associated with biotinylated Pint or the anti-sense (control) RNA incubated with nuclear extracts. The bottom band shows the crossreaction of the antibody with a non-specific binding protein. (F) Direct binding of PRC2 and Pint. Protein bound to Pint or anti-sense (control) RNA when incubated with purified PRC2.