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.
Wang
Yong-Jun
YJ
Department of Zoology, University of British Columbia, 6270 University Boulevard, V6T 1Z4, Vancouver, BC, Canada.
Brock
Hugh W
HW
D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D009698:Nucleoproteins / Q000378:metabolism*; D063150:Polycomb Repressive Complex 1
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D050883:HSC70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C424447:Hsc70-4 protein, Drosophila; D009698:Nucleoproteins; C072820:ph-d protein, Drosophila; D063150:Polycomb Repressive Complex 1