Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex

Abstract

Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Here, we report that Pleiohomeotic (PHO) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. We identified a conserved sequence motif, named PCC-binding element (PBE), which is required for PcG silencing in vivo. PHO sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive PHO/PCC complex. We termed this nucleoprotein complex silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome.

Figure 1.

DNA templates and purified proteins. (A) Schematic representation of the Ubx locus. The BXD/PBX regulatory regions and bxd PRE and Ubx promoter are indicated. Map positions are according to Bender et al. (1983). The eight PHO sites within the core of the bxd PRE are indicated. Numbering is according to Fritsch et al. (1999) and Mahmoudi et al. (2003). For our binding studies, we used a short 50-bp DNA fragment harboring the PHO4 and PHO5 sites, referred to as PHO4/5-PRE. These strong PHO-binding elements are required for bxd PRE-directed silencing in vivo (Fritsch et al. 1999). (B) Schematic representation of the regulatory region of Abd-B. The parasegment-specific regulatory domains iab-5, iab-6, and iab-7, part of the iab-8, and the iab-7 PRE are indicated (Karch et al. 1985). A minimal iab-7 PRE harboring three PHO sites is used in both reconstituted DNA-binding and in vivo silencing studies (Mihaly et al. 1998). (C) Recombinant PHO, PHO lacking the PBD (ΔPBD: amino acids 1-148/169-520), PC, and PCC were immunopurified from extracts of baculovirus-infected Sf9 cells. The eluted proteins were resolved by SDS-PAGE and visualized by silver staining.

Figure 2.

PHO and PCC bind synergistically to the bxd PRE. (A) PRE binding of PHO, ΔPBD and PCC was studied by bandshift assays. Binding reactions contained ∼5 nM PHO and/or ∼5-15 nM PCC, and radiolabeled PHO4/5-PRE. PRE binding of PHO and PCC was analyzed by primer extension DNaseI footprinting using either naked DNA (B,C) or chromatinized PHO4/5-PRE (D) templates. Binding reactions on naked DNA contained ∼20 nM (+) or 100 nM (‡) PHO and ∼20-60 nM PCC. Chromatin binding reactions contained ∼50 nM PHO and ∼20-60 nM PCC. DNaseI digestion ladders were analyzed by primer extension, resolved on a 6% polyacrylamide gel, and visualized by autoradiography. The positions of the PHO sites and footprinted areas are indicated. Closed arrowheads indicate DNaseI hypersensitive sites induced by protein binding.

Figure 3.

PCC subunits contact DNA. (A) UV cross-linking of PHO and PCC subunits to the PRE. Binding reactions contained a ³²P-labeled, BrdU-substituted PHO4/5-PRE fragment in the absence or presence of the indicated proteins. After UV cross-linking, proteins were analyzed by SDS-PAGE and visualized by autoradiography. The relative positions of PSC, PH, PC, and PHO are indicated. (B) PRE binding of PHO and PC was analyzed by primer extension DNaseI footprinting as described in Figure 2. Reactions contained ∼20 or 80 nM PC. (C) Summary of the DNaseI footprinting patterns on PHO4/5-PRE.

Figure 4.

The PBE is required for PCC recruitment to the PRE. (A) Alignment of a selection of PHO sites and their flanking motifs from Mihaly et al. (1998), including the bxd PRE PHO sites 4 and 5 and iab-7 PHO sites. The PHO/YY1 consensus core (C) sequence GCCAT and the upstream (U) and downstream (D or PBE) flanking motifs are indicated. The U.mt, C.mt, and D.mt are indicated for PHO4 and PHO5. Corresponding D.mts were induced in the iab-7 PBEs (Fig. 5). The effects of mutations on PHO binding were tested by DNaseI footprinting on the PHO4/5 PRE (B) and by bandshift analysis (C). (D) Synergistic binding of PHO and PCC requires both PHO4 and PHO5 sites and both accompanying PBEs. Binding was assayed as described above using probes harboring the indicated mutations. (E) Model illustrating that cooperative DNA binding by PHO and PCC requires (1) at least two PHO sites, (2) their juxtaposed PBEs, and (3) direct protein-protein interactions between PHO and PCC. PHO sites and PBEs form an integrated platform for the synergistic assembly of a repressive PHO/PCC/PRE nucleoprotein complex. Details are discussed in the text.

Figure 5.

The PBE is required for PRE silencing in vivo. (A) Binding of PHO/PCC to the iab-7 PRE is abrogated when the PBEs are mutated (for sequences, see Fig. 4A), as revealed by DNaseI footprinting. (B-D) Mutations in the PBE impair PRE-directed PSS. PSS of mini-white by the minimal 260-bp wild-type iab-7 PRE or a mutant, PBE-less iab-7 PRE (PBE^mt iab-7 PRE) was compared. Multiple independent lines were established harboring the mini-white transgene under control of either the wild-type or PBE^mt iab-7 PRE. For each line, the eye color of homozygotes for the insertion was compared with that of heterozygotes. Representative examples are shown: (B) Homozygotes have a much darker eye color than their heterozygous siblings (P[w⁺]/P[w⁺]P[w⁺]/+), revealing the absence of PSS. (C) Homozygotes have much lighter eyes than heterozygotes ((P[w⁺]/P[w⁺][w⁺]/+), showing strong PSS. (D) The eye color of homozygotes is equal (P[w⁺]/P[w⁺] = P[w⁺]/+) to that of heterozygotes, revealing impaired PSS. (E) The number of lines exhibiting no PSS, impaired PSS, or strong PSS were scored and depicted in a graphical representation with the number of lines displaying a PSS phenotype expressed as percentage of the total number of lines analyzed on the Y-axis and the phenotype on the X-axis. Because the percentages refer to the numbers of independent lines, error bars reflecting the SEM are not applicable. χ² test of statistical significance for bivariate tabular analysis confirmed the high significance of the difference in PSS frequency between lines harboring either the wild-type or PBE^mt iab-7 PREs (χ² = 12.3, p < 0.01).