The Drosophila zinc finger protein trade embargo is required for double strand break formation in meiosis

Abstract

Homologous recombination in meiosis is initiated by the programmed induction of double strand breaks (DSBs). Although the Drosophila Spo11 ortholog Mei-W68 is required for the induction of DSBs during meiotic prophase, only one other protein (Mei-P22) has been shown to be required for Mei-W68 to exert this function. We show here that the chromatin-associated protein Trade Embargo (Trem), a C2H2 zinc finger protein, is required to localize Mei-P22 to discrete foci on meiotic chromosomes, and thus to promote the formation of DSBs, making Trem the earliest known function in the process of DSB formation in Drosophila oocytes. We speculate that Trem may act by either directing the binding of Mei-P22 to preferred sites of DSB formation or by altering chromatin structure in a manner that allows Mei-P22 to form foci.

Figure 1. trem localization and expression.

(A). Schematic diagram of the trem gene and its location on 3R. trem is found in a cluster of 4 genes all of which are predicted to encode C2H2-type zinc finger proteins. (B) Black boxes depict the 5 exons in the trem gene. A piggyBac-element insertion used in this study, PBac[WH]trem^f05981, is located in the 5′ UTR 55 bp upstream of the start codon of trem. An Upstream Activation Sequence (UAS) within the PBac{WH}-element is oriented in the same direction as the trem gene. The UAS can be used to drive expression of trem. (C) Expression of trem in the germline of wild type, trem^F9 and trem^f05981 females. Maximum intensity projection of deconvolved Z-series through whole mount germarium stained with DAPI and antibodies to Trem and C(3)G, and the merge. Location of the developmental stages within the germarium is shown in the wild-type image. Each ovary is made up of approximately 16 ovarioles that are arranged according to developmental age . In region 1, the cystoblast initiates 4 rounds of pre-meiotic divisions with incomplete cytokinesis to form a 16-cell interconnected cyst. In region 2A, meiotic prophase begins, full length SC is formed (here identified by an antibody to the major transverse filament protein C(3)G) and recombination is initiated. As the cysts move posterior, pachytene continues in region 2B (recognized by pancake shaped cysts) until it reaches region 3 where the disappearance of γ-His2Av foci indicate the repair of a DSB event. The cyst then leaves the germarium and enters the vitellarium. No observable difference could be detected in either trem expression or localization between wild-type and trem^F9 germarium. Trem was not detected in trem^f05981 ovaries, and therefore we consider it to be a null allele. In addition, no defect in SC formation (using an antibody to C(3)G) was detected in either trem mutant compared to wild type. Scale bar, 15 µm.

Figure 2. Trem's localization pattern suggests it is a chromatin-associated protein.

(A) Maximum intensity projection of deconvolved Z-series through whole mount germarium stained with DAPI and antibodies to Trem and C(3)G. Trem appears to be expressed in most cells within the ovariole (shown is through stage 2 only), but is enriched in region 1 of the germarium. (B–E) Higher magnification of cells within the dashed box in image (A). Cells correspond to a cyst in late region 1 (zygotene), and show that Trem is chromatin associated and highly expressed in cells prior to the time full length SC is visible within the cyst in region 2A. In D, the nucleus containing full length SC corresponds to a pro-oocyte from a region 2A cyst. Little or no Trem protein was detected in the oocyte nucleus after Stage 2 (data not shown). Scale bars, 15 µm (A) and 5 µm (B–E).

Figure 3. Genomic alleles of trem.

(A) trem is predicted to encode a 439 amino acid protein that contains an N-terminal ZAD and 5 C2H2 zinc finger domains at the C-terminus (zf1–5). Schematic depiction of the location of 5 trem alleles obtained from TILLING trem and the original trem^F9 allele. Meiosis defective alleles are shown in purple, and alleles that do not show a meiotic phenotype are shown in blue. (B) Sequence alignment of the 5 C2H2 zinc finger domains and linker regions of Trem. Numbers next to the sequence correspond to amino acid position. Above the Trem sequence are shown the residues typically found within C2H2-type zinc fingers and the intervals between them. The amino acid residues in grey correspond to the conserved residues within the 5 zinc fingers in Trem. The locations of the genomic trem alleles (trem¹²⁷², trem³¹⁰⁰ and trem^F9) are underlined, and italics denote the transgenic alleles. The amino acid residues in red correspond to fertile alleles, and those in green correspond to semi-sterile alleles. All linker domain mutations correspond to proline to leucine changes, and zinc finger mutations in the second conserved histidine correspond to a histidine to tyrosine change. The residues at position −1, 2, 3 and 6 are important for specifying the DNA target .

Figure 4. trem^f05981 females are able to respond to breaks created by X-ray.

Maximum intensity projections of deconvolved Z-series through pro-oocytes in region 2A in trem^f05981 females either unexposed or exposed to 1000 Rad of X-ray stained with DAPI and antibodies to γ-His2Av and C(3)G and the merge. Unexposed wild-type pro-oocytes in region 2A are shown for comparison of qualitative differences in staining. Two adjacent cells within the cyst show the predicted thread-like pattern of SC. Average number of DSBs in region 2A in trem^f05981 females is 0.4, N = 57. Average number of DSBs in trem^f05981 females 5 hours after exposure to X-ray is 11.3, N = 13. Average number of DSBs in wild-type females is 12.5, N = 20 (see Table 4). Scale bar, 5 µm.

Figure 5. trem^f05981 females fail to accumulate any DSBs in region 3 in the absence of DNA repair.

Using an antibody to γ-His2Av, okra^AA/okra^RU; trem^f05981 females fail to accumulate any DSBs in region 3 (average 0.1 compared to 21.8 for okra^AA/okra^RU). Images are maximum intensity projections of deconvolved Z-series through the pro-oocytes in region 3 of the germarium. Stage of oocyte was determined by position and shape of cyst within the germarium, and the pro-oocyte(s) was identified by staining with antibodies to C(3)G and Orb. okra^AA/okra^RU; trem^f05981 females were able to rescue the two-oocyte phenotype associated with okra^AA/okra^RU indicating that the pachytene checkpoint is not activated in the absence of Trem (see Discussion). Scale bar, 15 µm.

Figure 6. Mei-P22 fails to localize to discrete foci in trem^F9 females.

Mei-P22^3XHA overexpression in the germline of trem^F9 females fails to localize to discrete foci in early pachytene. Maximum intensity projection of deconvolved Z-series through whole mount germarium stained with DAPI and antibodies to HA. (A) Females of the genotype P{nos-Gal4::VP16}/+; PUASp-mei-P22^3XHA/+; mei-P22¹⁰³ show both chromatin-associated Mei-P22^3XHA, as well as its localization to discrete foci in 50/50 images. (B) Females of the genotype P{nos-Gal4::VP16}/+; PUASp-mei-P22^3XHA/+; trem^F9 only show the chromatin associated localization pattern. No discrete foci were detected in 60 images analyzed. (C) Females of the genotype P{nos-Gal4::VP16}/+; PUASp-mei-P22^3XHA/+; trem^F9/TM3, Sb show both chromatin-associated Mei-P22^3XHA, as well as its localization to discrete foci in 8/8 images. (+^*) chromosome is the 3rd chromosome balancer chromosome, TM3 Sb, and the females used were the sisters of the P{nos-Gal4::VP16}/+; PUASp-mei-P22^3XHA/+; trem^F9 females in B. Germarium regions 1, 2A and 2B are shown in (A–C). Scale bars, 10 µm (A–B) and 15 µm (C).

Figure 7. X-rays partially rescue prometaphase and metaphase I spindle defects associated with the trem^F9 mutation.

Images are maximum intensity projections of the optical sections through the tubulin staining region. Meiotic figures are stained with DAPI and tubulin, shown in gray scale. Shown are representative images from trem^F9 oocytes. All images are from non-X-ray treated trem^F9 females with the exception of the 3-spindle image which is from X-ray treated trem^F9 females. Note that 1-spindle images in trem^F9 oocytes did not necessarily have the proper DNA configuration as is seen in wild type. See Table 7 for quantification. Scale bar, 15 µm.