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The MCP silencer of the Drosophila Abd-B gene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

¹ Centro de Biología Molecular, Universidad Autónoma de Madrid, CSIC-UAM, Campus de Cantoblanco, Madrid 28049, Spain
² Department of Human Genetics, 5200 Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA

View larger version (21K): [in a new window]   Fig. 1. Pleiohomeotic binding sites are required but not sufficient for MCP silencing function. (A) The basic structure of the reporter construct used to assay the silencing activity of MCP fragments (not drawn to scale). The MCP element was replaced with mutant or deletion fragments discussed in the text. (B) Diagram of the MCP fragments. The top line shows the wild-type MCP822 element which is a SalI-XbaI fragment. The numbers represent nucleotide residues corresponding to those indicated in Fig. 7. Open circles denote locations of the putative PHO binding sites. 5MPHO, 4MPHO, PHO62 and PHO604 contain mutations of PHO binding consensus sequence at sites indicated by X. MCP1 is a fragment containing sequences between nucleotides 573 and 675. In the adjacent table, the ‘# lines’ column shows the total number of independent transformant lines carrying the constructs and examined for lacZ expression in the wing and haltere discs; the ‘silenced’ column indicates the number of lines in which expression of lacZ is silenced in the wing disc (ps4 and ps5) and in the anterior compartment of the haltere disc (ps5) (Fig. 2A); the ‘derepressed’ column indicates the number of lines in which lacZ is derepressed in the wing or haltere discs. (C) In vitro binding of PHO protein to the putative PHO604 binding site in MCP. Radioactively labeled oligonucleotide PHO604 (Fig. 7) was incubated with in vitro-synthesized PHO protein (Materials and Methods). Arrow indicates the PHO-oligo complexes. Unlabeled competitors were PHO604, ADF (non-specific sequence control) and MPHO604 (PHO604 site mutated; see Fig. 7).

View larger version (30K): [in a new window]   Fig. 2. Reporter gene expression patterns in the wing and haltere imaginal discs. (A) MCP822 construct containing the wild-type element. lacZ expression is correctly maintained: it is expressed in the posterior compartment of the haltere disc (ps6) but is silenced in the wing disc (ps4 and ps5) and in the anterior compartment of the haltere disc (ps5). (B) 5MPHO construct in which all the putative PHO binding sites are mutated. The expression of transformant line T4 is shown. lacZ is strongly derepressed in the wing and haltere discs. (C) MCP1 construct carrying the fragment from residues 573 to 675. lacZ is derepressed in the wing and haltere discs. (D) 5MPHO line T8, in which lacZ is weakly derepressed. (E) 5MPHO: a derivative line of 5MPHO line T8 from which 5MPHO had been excised. Expression is strongly derepressed in wing and haltere discs.

View larger version (12K): [in a new window]   Fig. 3. Deletion analysis of MCP. Top line shows a diagram of the wild-type MCP element, with the location of the five PHO consensus sites (circles) and the two putative GAF binding sites (squares) indicated. The deletion constructs MCP12, MCP14, MCP1, MCP2 and MCP7 are shown. The deleted portions are denoted by parentheses and the extent of the deletions are indicated by the nucleotide numbers. MCP7* has mutations in the two putative GAF binding sites, indicated by Xs. In the adjacent table, three columns are shown: ‘# lines’ are the total number of independent transformant lines examined for lacZ expression; ‘silenced’ is the number of lines in which silencing is correctly maintained in the wing disc and the anterior compartment of the haltere disc; ‘derepressed’ is the number of lines in which lacZ is derepressed in the wing or haltere discs.

View larger version (44K): [in a new window]   Fig. 4. Electrophoretic mobility shift assays. Top panel shows the sequences of the oligonucleotides 37/38 and the competitors 37/38 A, B and C used in the assay. The oligo 37/38 contains sequences between residues 543 and 581 including the two putative GAGA binding sites (underlined) at positions 548 and 558 (Fig. 7). The competitor sequences are identical to oligo 37/38 except for the residues replaced with Ts. The lower panel shows the results from EMSA when radioactively labeled 37/38 oligonucleotide was used as probe and incubated with embryonic nuclear extracts in the absence or in the presence of unlabeled competitors (Materials and Methods). Lane 1: oligo 37/38 forms three shifted bands (arrows) in the absence of competitors. Lane 2: 37/38 competitor. The three bands are competed. Lane 3: ADF competitor (non-specific sequence). No competition is observed. Lane 4: oligo 37/38 A as a competitor. The bottom two bands (connected arrows) are competed. Lane 5: 37/38 B competitor. The top band (arrow) is competed. Lane 6: 37/38 C competitor. All three bands are competed. Summary: the top band is competed by sequences included in the left third of the 37/38 oligo and the bottom two bands are competed by sequences in the middle third of the 37/38 oligo. No protein binds to the right third of the oligo 37/38. The results are schematically shown in Fig. 7.

View larger version (66K): [in a new window]   Fig. 5. Binding of in vitro synthesized GAF to two putative GAGA binding sites at MCP residues 548 and 558. Lanes 1-5: EMSA performed in the absence of competitor. Oligonucleotide 55/56 contains the sequence between residues 533 and 572 that includes the putative GAGAG548 and GAGA558 binding sites (Fig. 7). Oligo 55/56M1 has the GAGA548 site mutated, oligo 55/56M2 has the GAGA558 site mutated, and oligo 55/56M3 has mutations in both sites. Radioactively labeled oligonucleotide 55/56 (lane 2) or one of the mutated versions (lanes 3-5) was incubated with in vitro-synthesized GAF factor and run on a 5% acrylamide gel (Materials and Methods). Two shifted bands (arrows) are present when incubated with GAF (lane 2) but not without (lane 1). The two shifted bands presumably correspond to protein-DNA complexes formed by the full-length and a shorter GAF polypeptides synthesized in our in vitro translation system (data not shown). Lanes 6-10: EMSA in the presence of competitors. The binding is competed by excess, unlabeled oligo 55/56 (lane 6) but not by non-specific competitor oligo (lane 10). Lanes 7-9 are competition experiments in which oligo 55/56 was labeled and the mutated oligos were used as excess, unlabeled competitors. The shifted band using 55/56M1 (lane 3) is faint. However, we believe this to be a true shift rather than due to spillover from adjacent lanes, as lane 7 shows shows that oligo 55/56M1 can compete for GAF binding.

View larger version (60K): [in a new window]   Fig. 6. GAGA binding sites are required for the maintenance of silencing. (A) Pattern of lacZ expression in the wing and haltere imaginal discs from larvae containing the MCP7 construct (138 bp MCP minimal sequence that includes the GAGA binding sites, Fig. 3). lacZ is silenced in the wing disc (ps4 and ps5) and in the anterior compartment of the haltere disc (ps5), but it is expressed in the posterior compartment of the haltere disc (ps6). (B) MCP7* construct (138 bp MCP minimal sequence with GAGA binding sites mutated as shown). Both the wing and haltere discs show strong derepression of lacZ.

View larger version (22K): [in a new window]   Fig. 7. The minimal MCP silencer element. The sequence of the 138 bp minimal MCP silencer element is shown. Putative GAGA and PHO binding sites are boxed. Above the sequence, grey ovals show the approximate locations of protein binding sites deduced from DNA binding studies using embryonic nuclear extracts and in vitro-synthesized proteins. The protein in nuclear extracts that interacts with sequences near GAGA558 is unlikely to be GAF since the binding is not competed by the strong GAGA binding sequence at GAGA548 (see text and Fig. 4). Below the sequence, the extent of the oligonucleotides and the mutated residues are indicated.