In situ dissection of a Polycomb response element in Drosophila melanogaster

Abstract

Genes of the Polycomb group maintain long-term, segment-specific repression of the homeotic genes in Drosophila. DNA targets of Polycomb group proteins, called Polycomb response elements (PREs), have been defined by several assays, but they have not been dissected in their original chromosomal context. An enhanced method of gene conversion was developed to generate a series of small, targeted deletions encompassing the best-studied PRE, upstream of the Ultrabithorax (Ubx) transcription unit in the bithorax complex. Deletions that removed an essential 185-bp core of the PRE caused anterior misexpression of Ubx and posterior segmental transformations, including the conversion of the third thoracic segment toward a duplicate first abdominal segment. These phenotypes were variable, suggesting some cooperation between this PRE and others in the bithorax complex. Larger deletions up to 3 kb were also created, which removed DNA sites reportedly needed for Ubx activation, including putative trithorax response elements. These deletions resulted in neither loss of Ubx expression nor loss-of-function phenotypes. Thus, the 3-kb region including the PRE is required for repression, but not for activation, of Ubx.

Fig. 1.

Genetic methods for creation of deletions. (A) A plasmid is constructed containing the genomic region of interest, with a pair of FRT recombination sites flanking the segment to be deleted. A selectable marker, such as the rosy (ry) gene, may also be inserted between the FRTs. The plasmid is injected into embryos of a strain with a P-element insertion near the homologous region of interest. Coinjection of a source of the P-element transposase will excise the P element and make possible the integration of the genomic sequences from the plasmid. (B) If the FRT sites in the successful convertant are in the same orientation, they can be induced to recombine in cis by supplying a source of flipase. The rosy selectable marker is also deleted in the recombination event, but a single FRT site remains at the site of the deletion. (C) Any two deletions with residual FRT sites can be induced to recombine in trans, thus creating a larger deletion. Note that the reciprocal recombinant chromosome will contain a smaller deletion, or a duplication, depending on whether the two initial deletions overlap. (D) Some of the conversion constructs carried sites for the homing endonuclease I-Sce I. When I-Sce I is supplied, the chromosome is cut, and some repaired products result from resection, followed by nonhomologous end joining. A series of deletions can be generated extending in either direction from the initial site of the conversion construct.

Fig. 2.

Map of the bxd PRE region dissected with deletions. (A) Fifteen conversion constructs are diagramed, with vertical arrows showing the map positions where the constructs are inserted. The constructs with horizontal bars contained genomic sequences, initially flanked by FRT sites, which were then deleted by flipase-mediated recombination. (B) This DNA sequence map covers 4.5 kb, marked in the coordinates of Seq89E (37). Binding sites for pleiohomeotic protein (GCCAT) and GAGA factor (GAGAG) are indicated by orange and blue balls, respectively. Three vertical arrows mark the insertion sites of P elements used for gene conversion. (C) The white boxes indicate the positions of embryonic enhancers, and blue and violet boxes show subfragments with PRE activity in transgene assays (7, 23). Yellow boxes indicate TREs (17). The TRE1, TRE2, and TRE3 arrows mark short RNA transcripts reported to recruit activators for Ubx (19). (D) The dashed lines indicate the extents of the deletions generated. Orange balls on the deletion lines indicate an FRT site remaining at the position of the deletion. The bottom four deletions also retain a GAL4-VP16 marker gene and an I-SceI cut site. The presence or absence of a posterior transformation phenotype (PS5 to PS6) is indicated for each deletion.

Fig. 3.

Phenotypes of bxd PRE deletion. (A) Transformation of posterior wing to haltere tissue, shown for a Δ1-2 heterozygote. (B) A transformation of anterior wing to haltere tissue, shown in a Δ1-2 hemizygote [heterozygous with Df(3R)Ubx¹⁰⁹]. (C) Transformations of the third thoracic segment toward the first abdominal segment, shown in a Δ1-2 homozygote. (D) Unstained cuticles of third-instar larvae. The denticles of the third thoracic segment are not pigmented in wild type, but are black in the Δ1-2 larva (arrow).

Fig. 4.

Ubx expression in PRE deletion animals. Embryos, ≈16 h after fertilization, were immunostained for the Ubx protein. Overexpression of UBX is most apparent in the developing central nervous system of a homozygous Δ1-2 embryo. Ectopic staining is strong and widespread in the third thoracic segment (large arrow), but occasional cells turn on UBX in more anterior segments (small arrows). UBX in the abdominal segments resembles that in the wild type (WT). A homozygous Δ7-13 embryo also shows no apparent reduction in UBX relative to the wild type, and there is some UBX misexpression in the third thoracic segment (arrow). The enlargement shows the third thoracic through third abdominal segments in the nerve chord of the Δ7-13 embryo.