Article Figures & Data
Figures
- FIG. 1.
Purification of tagged Pc-G complex. (A) Structure of the epitope-tagged Pc construct. The chromodomain is indicated by a solid box. The C-terminal FLAG epitope and hexahistidine tags are labeled FLAG and (H)6, respectively. The extra amino acid residues introduced by the linker sequences are shown in single letter code. (B) Silver staining of eluted proteins. Aliquots (3.5 μl) of proteins from the column input and different fractions were resolved on an SDS-7% polyacrylamide gel. The fraction numbers are indicated at the top of each lane and are consistent with those in Fig. 2D and 3B. The position of PC is indicated by the arrows. Note that several discrete protein bands can be resolved in this region on high-resolution gels. (C) Specific association of Pc-G proteins. The eluted fractions prepared from PC-FH or S2 cells were resolved on an SDS-7% polyacrylamide gel. Only a small number of nonspecific proteins were eluted from S2 cell extracts.
- FIG. 2.
CHRASCH binds the (GA) n motif. (A) Map of DNA fragments in B-151 used for binding and competition assays. The locations of the (GA) n motif (solid rectangular box), YY1 (solid circles), and ZESTE (open circles) binding sites are shown. The coordinates of restriction sites and various fragments are indicated. (B) Preferential binding of CHRASCH to the bxd-b fragment. Binding of labeled bxd-a (a) or bxd-b (b) probe was carried out with or without (−) an excess amount of unlabeled bxd-a or bxd-b or a DNA fragment from the polylinker of a pBluescribe vector(ns). Specific binding to bxd-a and bxd-b probes is indicated by the arrowhead and arrow, respectively. (C) Binding of CHRASCH to the (GA) n motif. Binding assays were carried out with the bxd-b probe. The competitors were bxd-a (a), bxd-b (b), bxd-1 to -4 (1 to 4), linker DNA (ns), or fragments containing multiple (GA) n (G) or ZESTE (Z) binding sites. (D) Elution profile of the DNA binding activity. Aliquots (2 μl) of eluted fractions (same fractions as shown in Fig. 1A) were assayed with the bxd-b probe. (E) Antibody supershift assay. Affinity-purified PC antibody (2.5 mg/ml), IgG (2.5 mg/ml), or buffer were preincubated with CHRASCH before the addition of a reaction mixture containing the bxd-b probe. The volume of antibody solution is indicated. The supershifted complex is indicated by the arrowhead. Note that fast-migrating bands appeared after 1 h of preincubation (lanes 2 and 6), suggesting instability of the complex. The EMSA shown here was done with 3.5% polyacrylamide gels. B, bound DNA-protein complex; F, free probe.
- FIG. 3.
Lack of GAF in CHRASCH. (A) Effects of zinc ion on binding activities of GAF and CHRASCH. Different concentrations of ZnSO4 were included in binding reaction mixtures, followed by electrophoresis on 3.5% polyacrylamide gels. The concentrations used are as follows: none (lanes 2 and 10), 10 μM (lanes 3 and 11), 20 μM (lanes 4 and 12), 50 μM (lanes 5 and 13), 100 μM (lanes 6 and 14), 200 μM (lanes 7 and 15), and 500 μM (lanes 8 and 16). Specific bands are indicated by dots. In contrast to stronger binding for GAF at higher ZnSO4 concentrations, CHRASCH is refractory to intermediate concentrations and then becomes completely inactive. (B) Proteins from both column input (1 μl) (lanes 1 and 3) and fraction 8 (5 μl) (lanes 2 and 4) were resolved on an SDS-10% polyacrylamide gel, transferred, and assayed by GAF antiserum before (lanes 1 and 2) or after (lanes 3 and 4) a 1-h preincubation with purified recombinant GAF (∼16 μg/ml). Two major clusters of GAF isoforms, GAGA-519 and GAGA-581, are indicated. Preincubation of recombinant GAF strongly reduces the reactivity of the antiserum to GAF in the input but not to the unknown protein in the purified fraction, indicating that this protein is not related to GAF.
- FIG. 4.
Psq proteins are directly involved in (GA) n binding. (A) Probe used for UV cross-linking of (GA) n binding protein. The DNA sequences of the upper and the lower strands are shown. Two stretches of the (GA) n motif are underlined, which are almost identical to the extended (GA) n motif in the bxd-4 fragment, except that their exact order is reversed. The sequences flanking both sides of the (GA) n motif in the probe bear no similarity to those in the bxd-4 fragment. Note that the incorporation of AB-dUTP and radioactive dCTP are restricted to the (GA) n motif only. (B) UV cross-linking of the binding protein in CHRASCH. Aliquots of recombinant GAF and CHRASCH that gave equivalent DNA binding activities, as judged by EMSA (data not shown), were used for UV cross-linking. After extensive digestion with various amounts of DNase I (10, 20, and 30 U) and 0.5 U of micrococcal nuclease, indirectly labeled proteins were resolved on an SDS-9% polyacrylamide gel. One major protein species of ∼85 kDa was found for purified GAF. Two sets of bands were found for CHRASCH. One set of bands was substantially larger than the 106.5-kDa mass marker, while the other one was ∼70 kDa. (C) Copurification of Psq proteins. The input and peak fractions from extracts containing tagged PC or TBP were resolved on a 7% polyacrylamide gel. After transfer to a nitrocellulose filter, the proteins were probed with an affinity-purified antibody specific for the C-terminal common half of PSQ. The left panel represents the immunoblot for CHRASCH, while the right panel represents that for TBP as a control. Note that PSQ-A and PSQ-B migrate as ∼150- and ∼95-kDa proteins, respectively. Note that the size of PSQ-B is larger than the one (∼80 kDa) reported by Horowitz and Berg (27). (D) Association of PSQ and PC in vivo. Embryonic nuclear extracts were immunoprecipitated by an affinity-purified PSQ antibody (IP) or nonimmune serum (control). The extracts (input) and immunoprecipitates were probed with an affinity-purified PC antibody. The positions for PC and Ig are indicated.
- FIG. 5.
Synergistic effect of psq and Pc mutations on ectopic UBX expression in imaginal discs. First (A and C)- and second (B and D)-leg discs and wing discs (E and F) from wild type (A, B, and E) or double heterozygotes (C, D, and F) of psq 2403 and Pc 4 were stained with a monoclonal UBX antibody. UBX is normally undetectable in the first-leg disk and detectable at a low level in second-leg and wing discs of wild-type larvae. A slight increase of UBX is observed in the wing discs of larvae heterozygous for Pc 4. Strong UBX is observed in the discs of double mutants. The psq mutation alone has no detectable effect on UBX expression. Essentially the same results were observed for psq 0115.
- FIG. 6.
Colocalization of PSC and PSQ on ANTP-C and BX-C. Polytene chromosomes from third-instar larvae were simultaneously stained with an affinity-purified rabbit PSQ antibody and a mouse monoclonal PSC antibody. The merged images are shown with DNA in green (A and E), PSQ staining in red (A, B, E, and F), and PSC staining in blue (A, B, E, and F). The images for single staining of PSQ (C and G) or PSC (D and H) are also shown. The sites for ANTP-C (A to D) and BX-C (E to H) are indicated by arrows.
- FIG. 7.
Relative abundance of Psq proteins in ovaries of psq mutants. (A) A map of the psq locus is redrawn on the basis of the information from Horowitz and Berg (27) and Weber et al. (61). The genomic structure of psq and the positions of class I mutations including three PZ insertion mutants (0115, 2403, and 8109) and a deletion mutant (Δ18) are shown above the map. The structures of two major cDNAs, psq-1 and psq-2, are shown below, with their splicing junctions indicated. The noncoding sequences (blank boxes) and the coding regions (solid boxes), including the N-terminal BTB (stippled boxes) and C-terminal PSQ domains (grey boxes), are also shown. Three class II mutations, F112, E34, and E39, are located around the first exon of psq-2. The approximate sites of insertion (F112) or deletion (E34 and E39) are indicated. psq-1/l(3)S12 represents a fusion product resulting from an aberrant splicing between psq-1 and l(3)S12 adventitiously present in the PZ transposons. The positions of two recombinant proteins, PSQ-N and PSQ-C, used for antibody purification are also indicated. The DNA fragments are drawn to scale except for the ∼40-kb intron. (B) Protein analysis of psq mutants. Ovaries were dissected from homozygous psq or transheterozygous mutant adults, and proteins were analyzed by SDS-polyacrylamide gel electrophoresis. psq proteins were detected with affinity-purified antibody against PSQ-C. Extracts derived from wild-type (WT) or mutants are indicated above the respective lanes. Homozygous psq E39 animals died during pupation. The positions for PSQ-A, PSQ-B, and an internal control (α-tubulin) are marked by arrows. The positions of mass markers (in kilodaltons) are marked. Note that trace amounts of PSQ-A could be detected in psq Δ18/psq 0115 mutants when the film was exposed for a longer amount of time. (C) Relative abundance of psq proteins. The relative amounts of PSQ-A and PSQ-B in these mutants are derived by first calibrating against the internal control (i.e., α-tubulin) in each sample. Each value was then calculated by using wild-type proteins as references.
Tables
- TABLE 1.
Genetic interaction between psq and Pc
Expt a psq allele Avg no. of ectopic sex comb teeth/leg for genotypeb: Bal Pc psq Pc A 0115 4.08 (118) 10.13 (167) 2403 3.44 (148) 8.58 (184) 8109 3.31 (140) 10.02 (131) F112 3.67 (168) 5.17 (216) E34 3.86 (175) 4.41 (216) E39 3.44 (126) 3.81 (108) B 0115 3.77 (102) 7.51 (135) Δ18 2.52 (84) 8.90 (132) 8109 3.00 (66) 8.78 (136) ↵ a Virgin females of various psq mutant alleles were crossed with Pc4 mutant males. Two separate experiments were done to determine the allelic strength in interactions with the Pc4 mutation.
↵ b The average number of ectopic sex comb teeth per leg was calculated by dividing the total number of ectopic sex comb teeth by the number of second and third legs (values in parentheses) for each genotype. The number of sex comb teeth on the first leg of wild-type males usually varies between 10 and 12. A reciprocal cross between Pc4 females and psq2403 males gave a lower number (1.31) of ectopic sex comb teeth in Pc4 progeny but a similar number (8.30 versus 8.58) in double-mutant progeny, indicating a strong maternal effect of psq.
