Core promoter sequences contribute to ovo-B regulation in the Drosophila melanogaster germline

Abstract

Utilization of tightly linked ovo-A vs. ovo-B germline promoters results in the expression of OVO-A and OVO-B, C(2)H(2) transcription factors with different N -termini, and different effects on target gene transcription and on female germline development. We show that two sex-determination signals, the X chromosome number within the germ cells and a female soma, differentially regulate ovo-B and ovo-A. We have previously shown that OVO regulates ovarian tumor transcription by binding the transcription start site. We have explored the regulation of the ovo-B promoter using an extensive series of transgenic reporter gene constructs to delimit cis-regulatory sequences as assayed in wild-type and sex-transformed flies and flies with altered ovo dose. Minimum regulated expression of ovo-B requires a short region flanking the transcription start site, suggesting that the ovo-B core promoter bears regulatory information in addition to a "basal" activity. In support of this idea, the core promoter region binds distinct factors in ovary and testis extracts, but not in soma extracts, suggesting that regulatory complexes form at the start site. This idea is further supported by the evolutionarily conserved organization of OVO binding sites at or near the start sites of ovo loci in other flies.

Figure 1.—

Expression of ovo-B- and ovo-A-specific reporters. The organization of the ovo locus and germline transcripts (top) and an expanded view outlining the structure of the base ovo-A- and ovo-B-specific reporters (bottom) are shown above the gonad photos. Shown are: ovo promoters (solid bent arrows), exons (bars), introns (bent lines), open reading frames (solid bars) encoding repressive (red) and activating (green) OVO isoforms, and lacZ encoding fragments (blue bars). The alternative exons defining the ovo-A and ovo-B mRNA are spliced to a common exon 2. Photomicrographs show expression of ovo-B and ovo-A reporters in wild-type and sex-transformed gonads. The sex chromosome karyotype and somatic differentiation phenotype are shown at the top and the reporter genotype is shown at the left. (A and E) Wild-type females. (B and F) Wild-type males. (C and G) Males transformed into females with tra^Hsp83.PS. (D and H) Females transformed into males due to absence of tra-2 [tra-2^B/Df(2R)Trix].

Figure 2.—

Genomic alignments and reporters for ovo expression. (A) VISTA plots showing the alignments between the 1-kb ovo control region of D. melanogaster aligned against D. simulans (Dsim), D. yakuba (Dyak), D. pseudoobscura (Dpse), and B. oleae (Bole). For each plot the lowest mapped score is 50% and the maximum is 100%. The line bisecting the plots is 75% and regions showing >80% homology are shaded. (B) D. melanogaster reporter genes are also shown. The names of the reporters are shown (left) and those showing detectable expression are indicated (boldface type). DNA present in the reporter is indicated by lines. Positions of the transcription start sites are indicated (bent arrows). On the base construct lacZ^1.1, the positions of OVO footprints (the entire protected region, not simply the consensus sites) are indicated (circles). Strong footprints (solid circles) and weak footprints (shaded circles) are also indicated, along with the coordinates.

Figure 3.—

Examples of ovo-B reporter expression in wild-type and sex-transformed flies. The sex chromosome karyotype and differentiation phenotype are shown at the top and the reporter genotype is shown at the left. (A–D) Heterozygous for ovo^ΔapΔ4. (E–H) Heterozygous for ovo^ΔapΔ5. (I–L) Heterozygous for ovo^ΔapΔ6. (M–P) Heterozygous for ovo^ΔapΔ8. (A, E, I, and M) Wild-type females. (B, F, J, and N) Wild-type males. (C, G, K, and O) Males transformed into females with tra^Hsp83.PS. (D, H, L, and P) Females transformed into males due to absence of tra-2 [tra-2^B/Df(2R)Trix].

Figure 4.—

Gel mobility shifts using the ovo-B core promoter and extracts from gonadal and somatic tissues. (A) Shifting activities from proteins extracted from female or male gonads or nongonadal soma were used. The source of protein for the gel shifts is indicated above the lane. Bands highly enriched in shifts using ovarian extracts (open arrow) or testis (solid arrow) are indicated. (B) Similar gel shifts performed with increasing amounts of unlabeled wild-type OVO binding site oligo or mutated oligo. The mobility of the shifted band in the absence of competitor is indicated (arrow).

Figure 5.—

OVO binding sites. (A) Examples of gel shifts used to test for binding activity of mutated strong OVO binding sites. The sequences of the binding sites are shown. Altered residues are in lowercase type. Two concentrations of OVO DNA-binding domain were used as well as extracts from bacteria expressing a control transcript (zo). (B) Sequence Logo showing the refined OVO binding site model. (C) The position of potential OVO binding sites in D. melanogaster (Dmel), D. simulans (Dsim), D. yakuba (Dyak), D. pseudoobscura (Dpse), and B. oleae (Bole) relative to the known (Dmel and Bole) or predicted ovo-B transcription start site (scale in base pairs shown). Nonamer scores are indicated by shading (see key). The extent of OVO footprints in D. melanogaster is shown (solid rectangles).

Figure 6.—

Examples of ovo reporter expression in an ovo^D1/+ background. The genotype with respect to ovo encoding alleles is shown at the top and the reporter genotype is shown at the left. An ovariole is shown for the +/+ micrographs, and an entire atrophic ovary is shown from the ovo^D1/+ flies. (A, C, and E) Ovarioles from females wild type for ovo. (B, D, and F) Ovaries from females heterozygous for ovo^D1. (A and B) Heterozygous for ovo^ΔapΔ4. (C and D) Heterozygous for ovo^ΔapΔ5. (E and F) Heterozygous for ovo^ΔapΔ6.

Figure 7.—

Examples of ovo-B and ovo-A reporter expression in flies with different OVO-B encoding copy numbers. The genotype with respect to ovo encoding alleles is shown at the top and the reporter genotype is shown at the left. Staining of gonads bearing the ovo-B-specific reporter (A–D) lacZ^ΔapΔ8, and the ovo-A-specific reporter (E–H) lacZ^Δb, in flies with (A and E) one, (B and F) two, (C and G) three, or (D and H) four copies of ovo alleles encoding ovo-B isoforms is shown. The ovo^Δap allele encodes only OVO-B, while the wild-type alleles also express OVO-A, albeit at a lower level.

Figure 8.—

The regulatory circuit centered on the ovo locus. Positive effects (arrows) and negative effects (blocked end) are shown. The lines represent strong (thick), moderate (thin), and weak (dashed) effects. See text for details.