Inactive uPA loci associate with poised transcription factories rich in the initiating form of RNAP (S5p), prior to activation.
(A, D) MN-ChIP analyses detect the initiating (S5p) form of RNAP bound at the uPA promoter and enhancer before TPA activation (A). Following activation the enhancer is associated with the elongating form of RNAP (S2p), while the promoter is associated with both forms (S5p and S2p) of the enzyme (D). HepG2 cells were grown ±TPA for 3 h, before chromatin preparation and MN-ChIP with antibodies specific for S5p and S2p forms of RNAP. Control (unrelated) antibodies were polyclonal anti-uPA receptor antibodies. (B, E) The position of the uPA locus (red) relative to S5p and S2p sites (green) was determined by immuno-cryoFISH before (B) and after (E) TPA activation for 3 h, using a rhodamine-labelled BAC probe containing the uPA locus and antibodies specific for RNAP phosphorylated on S5 or S2 residues. The association of uPA genes with RNAP (S5p or S2p) was scored as “associated” (signals overlap by at least 1 pixel) or “separated” (signals do not overlap or are adjacent; see Figure S8 for additional examples). Arrowheads indicate the position of uPA loci. Nucleic acids were counterstained with TOTO-3 (blue). Bars: 2 µm. (C, F) Frequency of association of uPA loci with RNAP-S5p or -S2p sites before (C) and after (F) TPA activation. The decrease in association with S5p factories and the increase in association with S2p factories observed after activation were both statistically significant (χ2 tests, p = 0.0006 and p<0.0001, respectively).
