GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element

Abstract

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Here, using DNaseI footprinting analysis, we provide a high-resolution map of sites for the sequence- specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. We found that while GAGA could efficiently bind to a chromatinized PRE, PHO alone was incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, was strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins.

Figure 1

Schematic representation of the bithorax complex (BX-C) including the pbx/bxd regulatory regions, the Ubx PRE and Ubx promoter. The map coordinates are indicated above in kilobases (kb) and follow the numbering of Bender et al. (70). The Ubx PRE 1.6 is represented schematically as four overlapping fragments termed PRE A, B, C and D, which were separately cloned into pBluescript and used as templates in primer extension DNaseI footprinting experiments. The numbering indicated above each PRE fragment refers to positions (in bp) within the 1.6 kb PRE.

Figure 2

Expression and purification of recombinant GAGA, Zeste, PHO and HB. HA-tagged recombinant GAGA and FLAG-tagged Zeste, PHO and HB were expressed in Sf9 cells and immunopurified from cell extracts using anti-HA or anti-FLAG columns, respectively, and eluted with a peptide corresponding to either the HA or the FLAG epitope. Aliquots of eluted proteins were analysed by SDS–PAGE and visualized with Coomassie Blue staining. The positions and molecular weights (kDa) of the protein standards are indicated on the left.

Figure 3

(Opposite) Identification of binding sites for regulatory proteins within the Ubx PRE and promoter. The binding of GAGA, PHO, Zeste and HB to the PRE A, B, C and D templates were examined by primer extension DNaseI footprinting. The indicated plasmids harboring the distinct PRE fragments were incubated with increasing amounts of recombinant purified GAGA (A), PHO (B), Zeste (C) and HB (D) or in the absence of protein (– lanes), followed by partial DNaseI digestion. (E) Similar DNaseI footprinting analysis of the Ubx promoter region. The concentration of recombinant protein in these reactions ranged from ∼50 to 150 nM as indicated by the triangles above the lanes. Digestion products were analysed by primer extension, resolved on an 8% denaturing polyacrylamide gel followed by autoradiography. The positions within the PRE, as determined by sequencing reactions run in parallel to the DNaseI reactions, are indicated. Vertical bars indicate regions of protection against DNaseI digestion.

Figure 4

Distribution of regulator binding sites on the Ubx PRE and promoter. (A) The various binding sites identified by DNaseI footprinting are indicated in the sequence of the Ubx PRE and highlighted in different colors: GAGA (green), Zeste (blue), PHO (red) and HB (yellow). (B) Schematic representation of protein binding sites within the Ubx PRE and promoter.

Figure 5

GAGA facilitates PHO binding to a chromatinized PRE. (A) GAGA and PHO do not cooperate during binding to naked DNA. The binding of GAGA and PHO to the PRE C template was examined by primer extension DNaseI footprinting. The PRE C plasmid was incubated with increasing amounts of either recombinant purified PHO alone, GAGA alone, both PHO and GAGA or in the absence of protein (– lanes), followed by partial DNaseI digestion. The concentrations of recombinant proteins in these reactions ranged from ∼50 to 150 nM as indicated by the triangles above the lanes. Digestion products were analysed by primer extension, resolved on an 8% denaturing polyacrylamide gel followed by autoradiography. The positions within the PRE, as determined by sequencing reactions run in parallel to the DNaseI reactions, are indicated. Vertical bars indicate regions of protection against DNaseI digestion. (B) The PRE C template was assembled into chromatin and visualized by MNase digestion. After completion of chromatin assembly using the Drosophila embryo S-190 assembly system, the template was digested with increasing amounts of MNase and the digestion patterns were visualized by ethidium bromide staining of agarose gel. (C) GAGA facilitates PHO binding to chromatin. DNaseI footprinting analysis of GAGA and PHO (at higher concentrations) binding to a chromatinized PRE C template. Following completion of assembly, chromatin was incubated for 45 min in either the absence (– lanes) or presence of increasing concentrations of GAGA and PHO (ranging from ∼200 to 400 nM) as indicated by the triangles above the lanes. The reaction shown in lane 18 contains 400 nM of each protein). Arrows indicate bands corresponding to regions bound by PHO. For DNaseI digestion of the chromatin template, a 150-fold higher amount of DNaseI was used than that used for digestion of naked DNA. The DNaseI digestion pattern was visualized by primer extension and separation on an 8% acrylamide gel followed by autoradiography. Footprints are indicated with bars located to the left and right of the autoradiograms.

Figure 6

GAGA and Zeste facilitate PHO binding to a chromatinized PRE. DNaseI footprinting analysis of (A) GAGA and PHO and (B) Zeste and PHO binding to chromatin in the presence of either ∼50 or ∼150 nM of each protein as indicated by triangles above the lanes. The DNaseI digestion pattern was visualized by primer extension and separation on an 8% acrylamide gel followed by autoradiography. Footprints are indicated with bars located to the left and right of the autoradiograms.