Site-Specific Recognition of a 70-Base-Pair Element Containing d(GA)nRepeats MediatesbithoraxoidPolycomb Group Response Element-Dependent Silencing

ABSTRACTPolycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd)PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, we identify DNA binding activities that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxdPolycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)3repeats for MHS-70 binding in vitro. Mutation of d(GA)3repeats within MHS-70 in the context of the 5.1-kb bxdPRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)3repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)3repeats, the TRL/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations inTrland psqenhance homeotic phenotypes ofph, indicating that TRL/GAGA factor and Psq are enhancers of Polycomb which have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of thebxdPRE by d(GA)nrepeat binding activities mediates PcG-dependent silencing.