Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila
- Stowers Institute for Medical Research, United States
- Kansas University Medical Center, United States
Figures
Figure 1 with 4 supplements
vilya encodes a RING domain-containing protein required for DSB formation.
(A) Schematic diagram of a germarium showing the timing of SC and DSB formation. (B) vilya826 homozygotes and Df/vilya826 transheterozygotes cause high levels of X chromosome nondisjunction. The …
Figure 1—figure supplement 1
vilya, CG2709, encodes a RING domain-containing protein.
(A) mei-826 (Collins et al., 2014) was mapped to CG2709 (Materials and Methods) and renamed vilya826. (B) vilya is predicted to encode a 237 amino acid protein with a RING domain and a potential …
Figure 1—figure supplement 2
Centromere clustering and homolog pairing is not affected in vilya826.
(A) Using an antibody to the CENP-A homolog, CID, clustering of centromeres is unaffected in vilya826 compared to wild type in region 2A. 100% of region 2A oocytes analyzed (n) for both wild type …
Figure 1—figure supplement 3
C(3)G and Orb staining appears normal in vilya826.
Immunofluorescence analysis of wild-type (A) and vilya826mutant (B) germaria showing the timing of SC formation, SC structure and oocyte determination. The SC is labeled with an antibody to C(3)G …
Figure 1—figure supplement 4
Vilya plays a direct role in DSB formation in early pachytene.
Immunofluorescence analysis comparing the induction and location of DSBs as marked by an antibody to γH2AV (green) in both surrounding nurse cells and oocyte nuclei (identified with an antibody to …
Figure 2 with 2 supplements
vilya826 rescues the fertility and karyosome defects of the DSB repair-deficient mutant okra.
(A) vilya826 rescues the fertility defect of the DSB repair-deficient mutant okra and displays an increase in chromosome nondisjunction in the double mutant, similar to that of the single vilya826 …
Figure 2—figure supplement 1
vilya826 rescues the karyosome defect of the DSB repair-deficient mutant okra.
The karyosome structure of a wild-type stage 8 egg chamber is shown for comparison purposes. A schematic of the egg chamber is shown below each image to identify the region highlighted in the image. …
Figure 2—figure supplement 2
vilya826 is defective in meiotic recombination.
(A) vilya826 is defective in meiotic recombination as assayed for intervals cv-v and v-f on the X chromosome. (B) Recombination frequency across the entire 3rd chromosome in vilya826 is reduced over …
Figure 3 with 2 supplements
Vilya localizes to the central region of the SC in both linear elements and discrete foci.
(A) Localization of Vilya3XHA throughout early pachytene as assayed by germline expression of vilya3XHA using antibodies to the transverse filament protein C(3)G (green) and an antibody to HA (red). …
Figure 3—figure supplement 1
Localization of Vilya3XHA within pachytene nuclei.
(A) Immunolocalization of Vilya3XHA in a mid-pachytene (region 3) nucleus that did not have any distinct Vilya3XHA foci and a late pachytene (Stage 6) nucleus showing that once the discrete foci …
Figure 3—figure supplement 2
Vilya3XHA foci are not found at centromeres in early/mid-pachytene.
Immunolocalization of Vilya3XHA and CID in vilya3XHA expressing early/mid-pachytene oocytes showing the absence of foci at centromeres. Pachytene nuclei in the specified regions were labeled with …
Figure 4
Immuno-EM of Vilya3XHA shows localization to both the RNs and to the central region of the SC.
Immuno-gold labeling of Vilya3XHA from germline-expressed PUASp-vilya3XHA ovaries. (A) A low magnification image of a section from a single nucleus with two RNs (outlined with red dashed box). A …
Figure 5
Localization of Vilya3XHA to discrete foci is dependent on the process of DSB formation.
(A-D) Immuno-localization of Vilya3XHA foci in the presence and absence of DSB formation. Pachytene nuclei in the specified regions were labeled with antibodies to HA (red), C(3)G (green) and …
Figure 6
A subset of Vilya3XHA foci localize near γH2AV staining at DSB sites.
(A,B) Immunofluorescence analysis of Vilya3XHA (red) localization at the sites of programmed DSBs that are recognized with the γH2AV modification (green) and Corolla (blue) in region 2A (A) or …
Figure 7
Localization of Vilya3XHA to discrete foci in early pachytene is not dependent on the SC.
Immunofluorescence analysis of vilya3XHA-expressing region 2A oocytes in the presence and absence of c(3)G. Early pachytene nuclei in region 2A were labeled with antibodies to HA (red), γH2AV …
Figure 8
Some DSBs created by X-ray can recruit Vilya3XHA to discrete foci.
Immunofluorescence analysis of germline expression of PUASp- vilya3XHA in the absence of functional mei-W68 with and without X-ray treatment. Ovaries were stained with antibodies to HA (red), γH2AV …
Figure 9 with 1 supplement
Vilya and Mei-P22 interact by yeast two-hybrid.
Yeast two-hybrid was used to test for an interaction between Vilya and Mei-P22. All diploid strains, where the OD600 was equalized before plating, grow equally well under selection for both the bait …
Figure 9—figure supplement 1
A functional RING domain is required for Vilya to interact with Mei-P22 in yeast-two hybrid assay.
(A) Mutations in any of the key residues in the RING domain ablate the ability of Vilya to interact with Mei-P22. (B) Western blot analysis showing that the RING domain mutants are expressed in the …
Figure 10 with 1 supplement
Model of DSB formation in Drosophila female oocytes.
(Left) In wild-type oocytes, Mei-P22, which localizes to discrete foci prior to the time γH2AV foci are present, is located at chromatin adjacent to the SC (Liu et al., 2002). Vilya localizes to the …
Figure 10—figure supplement 1
Protein alignment of Vilya to Zip3 and HEI10 homologous proteins.
(A) Protein alignment of Drosophila melanogaster (Dm) Vilya (AAF45818), Caenorhabditis elegans (Ce) ZHP-3 (NP_001250801), Saccharomyces cerevisiae (Sc) Zip3 (NP_013498), Mus musculus (Mm) RNF212 …
Videos
Video 1
Rotation of an early/mid-pachytene nuclei.
Movie showing X and Y rotation of the nucleus in Figure 3C-b. Each chromosome arm is marked with a separate color. The SC is labeled using an antibody to C(3)G (green), and Vilya3XHA foci are …
