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Molecular and Cellular Biology, July 2001, p. 4528-4543, Vol. 21, No. 14
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.14.4528-4543.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Site-Specific Recognition of a 70-Base-Pair Element Containing d(GA)n Repeats Mediates bithoraxoid Polycomb Group Response Element-Dependent Silencing

Jacob W. Hodgson, Bob Argiropoulos, and Hugh W. Brock*

Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4

Received 5 March 2001/Returned for modification 30 March 2001/Accepted 23 April 2001

Polycomb 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 bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)3 repeats for MHS-70 binding in vitro. Mutation of d(GA)3 repeats within MHS-70 in the context of the 5.1-kb bxd PRE 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)3 repeats 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)3 repeats, the TRL/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, 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 the bxd PRE by d(GA)n repeat binding activities mediates PcG-dependent silencing.

* Corresponding author. Mailing address: Department of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC, Canada V6T 1Z4. Phone: (604) 822-2619. Fax: (604) 822-2416. E-mail: brock{at}zoology.ubc.ca.

Molecular and Cellular Biology, July 2001, p. 4528-4543, Vol. 21, No. 14
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.14.4528-4543.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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