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doi: 10.1242/10.1242/dev.00204

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The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing

¹ Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
² EMBL, Gene Expression Programme, Meyerhofstr. 1, 69117, Heidelberg, Germany
³ Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35/III, 72076 Tübingen, Germany

View larger version (52K): [in a new window]   Fig. 7. Trl is not required for repression or activation of homeotic genes in imaginal disks. (A,B) Wing (W) and haltere (H) imaginal disks with Trl mutant clones, which are marked by the lack of GFP signal, stained with antibodies against GFP (green) and Ubx (red in A) or ßgal (red in B) protein. In all cases, the Minute technique was used and clones were analysed 96 hours after clone induction. (A) No misexpression of Ubx was observed in Trl mutant clones (left). Trl mutant clones induced in pho homozygotes (right) do not show anymore misexpression of Ubx than is seen in pho homozygotes alone (compare with Fig. 3A). (B) Trl mutant clones induced in transgenic larvae that express the PRE1.6 (left) or MCP725 (right) reporter transgenes. Expression of both reporter transgenes is confined to the posterior half (ps 6; marked by asterisk) of the haltere disk in wild-type animals. No misexpression of the PRE1.6 reporter gene is detected in Trl mutant clones; the MCP reporter gene shows patchy expression of ß-gal in 10-20% of the wing disks, independent of whether the cells are wild-type or mutant for Trl, but is not seen in this disk. (C) Requirement for Pho, but not GAGA-binding sites for silencing. (Top) sequence of the 567 bp PRED fragment containing five GAGAG sites (green) and six binding sites for Pho protein (red). All six Pho-binding sites or all five GAGAG motives were mutated to obtain PREDPhomut and PREDGAGAmut, respectively; base substitutions are indicated above the sequence. (Below) X-Gal staining of wing imaginal disks carrying the indicated reporter transgene. In PRED and PREDGAGAmut transformants, the transgene is silenced in wing disks. No silencing is observed in PREDPhomut, transformants (Fritsch et al., 1999). (D) Requirement for trx but not Trl in maintaining homeotic gene expression in imaginal disks. Haltere (H) and third leg (L) imaginal disks with Trl (top) or trx (bottom) mutant clones stained with antibodies against GFP (green) and Ubx (red). In both cases mutant clones are marked by the lack of GFP signal and clones were analysed 96 hours after clone induction, the Minute technique was only used in the case of Trl. Ubx expression is unaffected in Trl mutant clones (white arrowheads), whereas trx mutant clones show a complete loss of Ubx signal (empty arrowheads).

View larger version (30K): [in a new window]   Fig. 1. Comparison of amino acid conservation and DNA-binding properties of Pho and Pho-like. (A) Amino acid identity (boxed) between Pho and Phol over the four zinc fingers. Amino acids from human YY1 that have been shown by X-ray crystallography to interact with DNA are marked (Houbaviy et al., 1996). Black circles represent amino acids that contact the DNA backbone. (+) represents positions that contact the DNA bases. White circles represent amino acids that contact both the DNA backbone and the bases. Cys and His residues of the zinc fingers are in bold. (B) Amino acid conservation within the spacer region between Pho, Phol and human YY1 (hYY1). Bold type indicates amino acids that are conserved between all three proteins. (C) The DNA sequence of the Pho and mutated Pho-binding site oligonucleotides used to test the DNA-binding specificity of the Phol zinc fingers. The mutated bases are denoted by the arrows. The Pho binding site is boxed. (D) Autoradiogram of a gel mobility shift assay showing binding of full-length Pho (lanes 1-3) and Phol zinc-finger protein (lanes 4-6) to the Pho-binding site. Lanes 1 and 4, no competitor DNA; lanes 2 and 5, 100x unlabeled Pho-binding site; lanes 3 and 6, 100x unlabeled mutated Pho-binding site. The specific Pho and Phol complexes are indicated by arrowheads. The broken arrow denotes a faint gel shift due to endogenous YY1 protein in the reticulocyte lysate.

View larger version (31K): [in a new window]   Fig. 2. Characterization of phol mutants. (A) The thick line indicates genomic DNA. The thin line indicates the extent of the phol transcription unit. The arrows indicate the start and direction of transcription of phol and the flanking transcription unit, CG3348. The transcription start sites are the first nucleotides of the ESTs from the Drosophila genome project. The shaded boxes indicate the extent of DNA deleted in the phol mutants. The approximate location of the start of the coding region (ATG), the conserved spacer region (S), and the zinc finger (zf) region are shown. (B,C) Embryos stained with DAPI and for the presence of the sperm tail (Karr, 1991). The embryo in B is from a wild-type mother and the embryo in C is from a phol mutant mother. The sperm tail is evident in both embryos (white arrows), but DAPI staining of nuclear DNA is evident only in the wild-type embryo (bright dots in centre of embryo).

View larger version (47K): [in a new window]   Fig. 3. Essential role for pho and phol in the repression of homeotic genes in embryos and imaginal disks. (A) Wing imaginal disks from larvae stained with Ubx or Abd-B antibody (red in both cases). No Ubx or Abd-B expression is detected in wing imaginal disks of wild-type larvae or in phol homozygotes. In pho homozygotes, Ubx is misexpressed in some cells in the wing pouch; but Abd-B is not misexpressed. Strong misexpression of Ubx and Abd-B is observed in phol, pho double homozygous disks; misexpression of Ubx is confined to the wing pouch, the lack of misexpression in more peripheral parts of the wing disk is possibly the result of downregulation by Abd-B protein, which is expressed at higher levels in these regions. (B) phol function is required throughout development. Wing (W) and haltere (H) imaginal disks with clones of phol mutant cells, marked by the lack of GFP signal (green), were induced in pho homozygous larvae and disks were analysed 96 hours after clone induction by staining with antibody against Ubx (red). The Minute technique was used in this experiment to generate M+ phol—/M+ phol- cells that carry two copies of a wild-type Minute allele; this gives them a growth advantage relative to their M+ phol-/M- phol+ neighbours. Strong misexpression of Ubx is observed in clones in the wing pouch (filled arrowhead). The lack of misexpression in clones in peripheral regions of the wing disk and the reduction of Ubx signal in clones in peripheral regions of the haltere disk (empty arrowhead) might be due to downregulation by Abd-B. (C) Requirement for pho and Pho-binding sites for silencing in embryos. (Top) Embryos carrying the PRED reporter gene (Fritsch et al., 1999) show ß-gal expression restricted to parasegments 6-12 (ps 6-12) in late (stage 13) embryos. In PREDPhomut transformant stage 13 embryos, repression anterior to ps 6 is lost and the reporter gene is active in all segments because of mutation of the six Pho-binding sites in PRED. (Below) Expression of the BP01 reporter gene is restricted to ps 6-12 in wild-type (wt) stage 16 embryos (Müller and Bienz, 1991). pho mutant stage 16 embryos show misexpression of the reporter gene in the nervous system (asterisks) and in the epidermis. In all cases, embryos were stained with anti-ß-gal antibodies and are oriented anterior leftwards, dorsal upwards; the anterior boundary of ps 6 is indicated by an arrowhead.

View larger version (29K): [in a new window]   Fig. 4. Pho and Psc colocalize to many sites on polytene chromosomes. This figure shows a partial spread of polytene chromosomes from a wild-type larva labelled with Pho (Cy2 labelled, green) and Psc (Cy3 labelled, red) antibodies. The individual Pho and Psc patterns are shown, together with an overlay of the two patterns in the final panel. Of the Pho sites, roughly 65% of them are also sites for Psc. Almost all of the Psc sites overlap with the Pho sites.

View larger version (43K): [in a new window]   Fig. 5. Analysis of PcG protein binding sites on the X chromosome. PcG-binding sites on polytene chromosomes were detected by immunofluorescent staining using primary antibodies directed against specific members of the PcG of genes. Secondary antibodies were Cy3-conjugated at 1:100 dilution (red). The DNA was labelled with DAPI (blue). An asterisks represents binding positions on the end of the X chromosome that do not change in the single or double mutants (with the exception of Pho, since all Pho bands are lost in the phol, pho double mutant). The arrow indicates the position of the 2D subdivision. (A) The binding pattern on chromosomes from wild-type (WT), or phol, pho double mutant larvae are shown for Pho, Pc, Psc, Scm and Ph. The Pc, Psc and Scm bands at the 2D position are lost in the phol, pho double mutants. (B) In E(z), a signal at 2D was seen only in about 20% of the wild-type chromosomes, and is not seen here. (C) Binding of Pc to the end of the X chromosome in pho and phol single mutants. The Pc 2D signal is not lost in pho or phol single mutants.

View larger version (27K): [in a new window]   Fig. 6. Analysis of Pc binding sites on 3R. This figure shows the telomere end of 3R. Bands that are present in both wild-type and mutant chromosomes are linked with a white line. Bands that are absent in phol, pho double mutants are marked with an asterisk.

View larger version (39K): [in a new window]   Fig. 8. zeste is not required for repression of homeotic genes in imaginal disks. Wing imaginal disks from z homozygotes (left) or z, pho double homozygotes (right) stained with antibody against Ubx protein. No misexpression is detected in z mutant disks and z, pho double mutants do not show more misexpression (asterisk) than do pho homozygotes alone (compare with Fig. 3A).

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