FIG. 6.

The SET oncoprotein inhibits PP2A-mediated dephosphorylation of phosphohistone H3 and is present at transcriptionally active loci. (A) Purified human core histones containing Ser10-phosphorylated histone H3 (lane 1) were incubated with purified human PP2A (lane 2). The presence of 1 μl of in vitro reticulocyte lysate-translated SET is capable of inhibiting PP2A-mediated dephosphorylation of histone H3 (lane 4). Translation reactions that did not contain SET protein showed minimal inhibition of PP2A activity (lane 3). (B) Antibodies to the SET gene product were generated and used to detect SET in whole Drosophila pupa extracts (lane 1) and the in vitro-translated SET protein expressed via a reticulocyte lysate system and utilized in a PP2A activity assay (lanes 3 and 4). Lane 3 contains 5 μl of reticulocyte lysate that does not contain the expressed SET protein. Lane 4 contains 5 μl of reticulocyte lysate containing expressed SET. Lane 2 shows the Coomassie staining of lane 1. Lanes 5 and 6 show Coomassie staining of lanes 3 and 4, respectively. Molecular mass size standards (in kilodaltons) are indicated at right. (C,D, and E) Genomic distribution of SET protein in polytene chromosomes prepared from wild-type larvae before (C) and after (E) heat shock and immunostained for the presence of SET (SET, red) and the Ser10-phosphorylated histone H3 isoform (phospho-H3, green). Chromosome spreads were also counterstained with DAPI (white) to view the DNA. DAPI counterstain information is represented as blue in merged images. Before heat shock, the SET protein is detected at puffed and H3-phosphorylated (transcriptionally active) loci, including the ecdysone-regulated genes, which are labeled (C). In situ hybridization with a fragment of the hsp70 transcription unit (hsp70, green) reveals that prior to heat shock, the SET protein (SET, red) does not colocalize with the hsp70 genes (D). During heat shock, strong SET staining is also detected at the actively transcribing heat shock puffs (E).