Polyhomeotic stably associates with molecular chaperones Hsc4 and Droj2 in Drosophila Kc1 cells.

Polycomb group (PcG) proteins silence target loci in Drosophila. Although the mechanism of PcG-mediated silencing remains unknown, there is considerable evidence that PcG proteins act via multiple complexes. We have epitope-tagged Polyhomeotic Proximal, PHP, the major isoform of the proximal product of the polyhomeotic locus, at both termini (F-PHP-HA) and generated a stable Kc1 cell line in order to isolate F-PHP-HA-associated proteins. Using either column chromatography followed by immunoaffinity precipitation or a double immunoaffinity precipitation procedure, we observed multiple proteins that stably associate with F-PHP-HA. Sequencing the five major bands identified PHP-170 and PHP-140 isoforms, Polycomb, Heat shock cognate 4 (Hsc4), and a novel Drosophila J class chaperone we term Droj2. Mutations in both chaperone genes enhance homeotic transformations in PcG genes, suggesting that they have a role in silencing. We show by Western blotting that minor components of F-PHP-HA-associated proteins include TBP, TAF(II)42, TAF(II)85, and p55. However, unlike in PRC1, Psc, TAF(II)62, Modulo, dMI-2, or Rpd3/HDAC1 do not associate with F-PHP-HA. We discuss the role of chaperones and F-PHP-HA-associated proteins in PcG-mediated silencing and the evidence for different complexes containing Polyhomeotic in vivo.