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First published online 14 March 2007
doi: 10.1242/dev.000521

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Tombola, a tesmin/TSO1-family protein, regulates transcriptional activation in the Drosophila male germline and physically interacts with Always early

Jianqiao Jiang^*, Elizabeth Benson^*, Nina Bausek, Karen Doggett and Helen White-Cooper

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Aly and Tomb proteins interact. (A) 293T cells expressed HA-tagged Tomb and FLAG-tagged Aly (lanes 1 and 3) or Kr(Zn-fingers) (control, lanes 2 and 4). Binding was assessed by immunoprecipitating with anti-FLAG and blotting with anti-HA (lanes 1 and 2, top panel), or vice versa (lanes 3 and 4, top panel). Tomb and Aly co-immunoprecipitated; control assays showed no co-immunoprecipitation. Protein expression was assessed by western blotting of cell lysate (lower three panels). (B) EGFP-Tomb was immunoprecipited from Bam-GAL4-VP16, UAS-EGFP-Tomb transgenic testes and blotted with anti-Aly. Wild type was used as the negative control; the lower two panels show expression controls.

View larger version (80K): [in this window] [in a new window]   Fig. 2. The tombola region and analysis of predicted protein sequence. (A) tombola genomic region adapted from FlyBase. The tomb 5' UTR is 130-191 bp long; the 3' UTR is 71 bp long. Black boxes, coding sequence; grey boxes, UTRs; inverted triangles, insertion sites of P[EY]00456 (EY) and P[GS]12862 (GS). (B) Predicted sequence of Tomb protein. Thick underline, predicted nuclear localisation signal; bold, CXC region; light grey box, P[GS]12862 insertion site; thin underline, C-terminal conserved region. (C) Alignments of CXC domains and spacer from tomb; Drosophila mip120; human (hs) tesmin (tes) and tesmin-like (tesl); Ciona intestinalis (Ciona); Strongylocentrotus purpuratus (S. purp); C. elegans LIN-54; Arabidopsis (At) TSO1 and SOL2. Con(1) and Con(2) indicate amino acids in the first and second CXC domains: *, conserved Cys; =, residues conserved in both CXC domains; , residues conserved within CXC(1) or CXC(2), but which differ between the domains. The E(z) Cys-rich region is shown as an outgroup. (D) Alignment and predicted secondary structure (beneath) of the animal tesmin-family protein C-termini. Predictions of secondary structure are shown in the same order as the sequences (i.e. the first line is the Tomb secondary structure prediction). Black lines indicate high confidence helix predictions; the intervening region (grey line) has either no strong structural prediction (Tomb), or a strong coil prediction (Mip120, Hs-tes, Hs-tesl).

View larger version (34K): [in this window] [in a new window]   Fig. 3. tomb expression is primary spermatocyte-specific. (A) RT-PCR of tomb ORF from female bodies lacking ovaries (fb), ovaries (ov), male bodies lacking testes (mb), testes (te) and 0-16 hour embryos (em). Negative control (-ve) was without reverse transcriptase. Positive control (+ve) was gDNA. (B) Semi-quantitative RT-PCR on testis RNA showed just detectable levels of transcript in wild type (WT), and slightly elevated levels in the meiotic-arrest mutants aly and mia. Controls as in A. (C,D) RNA in situ hybridisation. (C) In wild type, tomb expression was exclusively detected in primary spermatocytes. Early primary spermatocytes showed robust staining (arrow); mRNA levels gradually declined as spermatocytes matured (arrowhead). (D) tomb mRNA expression levels in aly mutant early primary spermatocytes was similar to wild type (arrow); however, levels did not decline as spermatocytes matured (arrowhead).

View larger version (117K): [in this window] [in a new window]   Fig. 4. EGFP-Tomb rescues the tomb meiotic-arrest mutant, and localises to chromatin in wild-type primary spermatocytes. (A,B) Phase contrast of wild-type (A) and tombGS12862 (B) testes. Primary spermatocytes occupy most of the apical end. Elongating spermatid bundles are seen inside, and spilling out from, the wild-type testis, whereas tombGS12862 testes contain only stages up to mature primary spermatocytes. (C,D) EGFP-Tomb expression rescues the tomb meiotic-arrest defect; extensive spermatid elongation is apparent (D, arrows). (E-H) EGFP and phase contrast of Bam-Gal4-VP16, UAS-EGFP-Tomb testes. The driver promotes strong expression in early primary spermatocytes (E,F); expression declines as spematocytes mature (G,H). In primary spermatocytes, EGFP-Tomb was predominantly chromatin associated: each nucleus had three prominent labelled regions corresponding to the major chromosome bivalents (G, arrows).

View larger version (79K): [in this window] [in a new window]   Fig. 5. tomb is an aly-class meiotic-arrest gene. Diagnostic RNA in situ hybridisations using probes for polo (A-D), Mst87F (E-H) and Cyclin B (I-L). tombGS12862 testes (D,H,L) were more like the aly-class mutant comr (B,F,J) than the can-class mutant nht (C,G,K). The testes shown in C and K broke near the seminal vesicles during processing. (A,E,I) Wild-type control.

View larger version (80K): [in this window] [in a new window]   Fig. 6. tomb is more like aly than topi. (A-D) Hoechst 33342 labelling of primary spermatocyte DNA in live squashes and (A'-D') corresponding phase contrast images. In wild type (A,A'), the three major bivalents are decondensed, adjacent to the nuclear envelope. Chromosomes in aly (B,B') mutant primary spermatocytes are apposed to the nuclear envelope, but fuzzier and less well defined than in the wild type. Chromosomes in topi (C,C') mutant primary spermatocytes are partially condensed, and not close to the nuclear envelope. (D,D') tomb mutant primary spermatocyte chromosomes resemble those in aly mutants rather than those in wild type or topi mutants. (E-P) RNA in situ hybridisations. CG3330 (E,H,K,N), CG12907 (F,I,L,O) and CG3927 (G,J,M,P) in wild type (E-G), aly (H-J), topi (K-M) and tombGS12862 (N-P). In wild type, CG3330 message (E) persisted from primary spermatocytes until mid-elongation spermatids. CG3330 transcript was undetectable in topi testes (K), whereas aly and tomb testes had low levels of transcript (H,N). CG12907 was expressed in wild-type primary spermatocytes (F) and persisted to late elongation. This transcript was not detected in topi mutant testes (L); levels in aly and tomb spermatocytes (I,N) were similar to wild type. CG3927 in wild type was detected only in primary spermatocytes (G). aly and tomb testes showed robust expression of this gene (J,P), whereas topi testes showed low levels of CG3927 transcript (M).

View larger version (50K): [in this window] [in a new window]   Fig. 7. Aly and Topi mislocalise in tomb testes. (A-L) Anti-Topi immunostaining (A,D,G,J, green) and DNA staining (B,E,H,K, red) in mature primary spermatocytes. In wild-type primary spermatocytes (A-C), Topi protein was predominantly chromatin associated. In achi/vis cells (G-I), Topi staining was distributed throughout the nucleus, but was brighter on chromatin, whereas the nuclear Topi staining in aly (D-F) and tomb (J-L) cells was less concentrated on chromatin. (M-R) Anti-Aly immunostaining (M,P, green) and DNA staining (N,Q, red) in mature primary spermatocytes. In wild type (M-O), Aly protein was nuclear and concentrated on chromatin. Aly protein was nuclear, but excluded from chromatin in tomb cells (P-R).

View larger version (141K): [in this window] [in a new window]   Fig. 8. Tomb nuclear localisation in late primary spermatocytes depends on comr, but not achi/vis. EGFP (A,B) and phase contrast (C,D) of achi/vis; Bam-Gal4-VP16, UAS-EGFP-Tomb (A,C) or comr; Bam-Gal4-VP16, UAS-EGFP-Tomb (B,D) whole testes and (insets) mature primary spermatocytes. Initially, the EGFP-Tomb localisations in achi/vis and comr are indistinguishable (A,B, arrows). Nuclear EGFP-Tomb was retained in achi/vis, but lost from comr mature spermatocytes (A,B, arrowheads). Arrested achi/vis spermatocytes had nuclear EGFP-Tomb (A, inset), whereas arrested comr spermatocytes had low levels of exclusively cytoplasmic fusion protein (B, inset).

View larger version (26K): [in this window] [in a new window]   Fig. 9. A model for assembly of the Aly-class meiotic-arrest proteins at target promoters. (A) Normal assembly of an aly-class gene product complex is regulated at several steps. (1) Aly-Comr interaction facilitates their nuclear translocation (or possibly prevents nuclear export). Tomb, Topi and Achi/Vis proteins localise constitutively to the nucleus, and can bind with low affinity to target promoters. (2) Nuclear Aly and Comr bind to and stabilise Tomb, then interact with Topi and Achi/Vis to facilitate cooperative DNA binding. (3) Transcriptional activation requires tight association of all five components with DNA. (B) In comr (or aly) spermatocytes, Aly (or Comr) remains cytoplasmic, Tomb protein is destablised and Topi and Achi/Vis only weakly interact with DNA; transcription is not activated. (C) In tomb mutants, Aly and Comr are stable in the nucleus, but cannot promote Topi and Achi/Vis association with DNA; transcription is not activated. (D) In achi/vis (or topi) mutants, the complex is not efficiently associated with DNA; transcription is not activated.

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