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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01032

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Regulation of transcription of meiotic cell cycle and terminal differentiation genes by the testis-specific Zn-finger protein matotopetli

Lucia Perezgasga^1,*,, JianQiao Jiang^2,, Benjamin Bolival, Jr^1,, Mark Hiller^1,, Elizabeth Benson², Margaret T. Fuller¹ and Helen White-Cooper^2,

¹ Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Beckman Center B300, 279 Campus Drive, Stanford, CA 94305-5329, USA
² Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

View larger version (198K): [in a new window]   Fig. 1. The topi meiotic arrest phenotype. (A) Phase-contrast image of wild-type whole testis, apical tip in top right-hand corner. Primary spermatocytes occupy most of the apical end (arrows) Elongating spermatid bundles (arrowheads) are seen inside the testis, and spilling out from the distal end. (B) Phase-contrast image of topiZ3-2139 whole testis. Only stages up to mature primary spermatocytes (arrows) are seen. Degenerating primary spermatocytes are present in the distal region of the testis (arrowheads). (C-H) High-magnification views of mature spermatocytes from squashed preparations of wild-type (C-E) and topiZ3-2139 (F-H) testes viewed by phase-contrast (C,F) and Hoechst DNA staining (D,G) or both visualization methods simultaneously (E,H).

View larger version (70K): [in a new window]   Fig. 2. In situ hybridization to whole testis. In situ hybridization to wild-type (A,C,E,G,I), topiZ3-2139 (B,D,J) or topiZ3-0707/Df(3R)by10 (F,H) mutant testes revealed that topi is an aly-class meiotic arrest gene. The distal ends of some mutant testes were broken off during the staining. Probes were anti-sense transcripts from cyclinA (A,B), cyclinB (C,D), twine (E,F), fzo (G,H) or Mst87F (I,J).

View larger version (101K): [in a new window]   Fig. 3. Antibody stain of Aly and Comr in wild type versus topi. Immunofluorescence staining of Aly (A,D, green in merge) and DNA (B,E, red in merge) on wild-type (D-F) and topiZ3-2139 (A-C) testes revealed that Aly protein is nuclear in both wild type and mutant, but that Aly fails to accumulate on the chromatin in topiZ3-2139 mutant spermatocytes. Immunofluorescence staining of Comr (G,L, green in merge) and DNA (H,M, red in merge) on wild-type (L-N) and topiZ3-2139 (G-I) testes similarly revealed that Comr protein is nuclear in both wild type and mutant, but that Comr fails to accumulate on the chromatin in topiZ3-2139 mutant spermatocytes.

View larger version (60K): [in a new window]   Fig. 4. Molecular cloning of topi. (A) topi genetic region was defined by the right breakpoints of Df(3R)Gb104 and Df(3R)by10, an interval of about 60 kb, which contains 23 predicted genes of which five are shown. topi gene structure and the location of lesions in mutant alleles are shown. (B) topi predicted amino acid sequence (mel) aligned with the putative topi orthologues from D. pseudoobsura (pseud) and A. gambiae (gamb). Sequence identity between all three proteins is indicated by black boxes, sequence similarity is indicated by grey boxes. Predicted Zn-finger motifs are indicated with lines above the sequences. The amino acids affected in the mutant alleles are indicated with an asterisk above the sequence.

View larger version (65K): [in a new window]   Fig. 5. topi is expressed specifically in primary spermatocytes. (A) Northern blot with topi (top) and rp49 (bottom) probes. A 2.7 kb topi transcript was detected in RNA from whole males (m), but not from females (f), agametic males (ag m) or embryos (em). rp49 was detected at similar levels in all the samples. (B) RNA in situ hybridization with topi probe to wild-type testis reveals expression in primary spermatocytes (arrows) and absent from spermatogonia (small arrow) and spermatids (arrowheads). (C) RNA in situ hybridization with topi probe on topiZ3-0707/Df(3R)by10 mutant testis indicates lack of topi transcript.

View larger version (17K): [in a new window]   Fig. 6. Structural interaction between Topi and Comr proteins. (A) Mapping domains of Comr responsible for the interaction with full-length Topi. Solid bars represent protein fragments tested in the yeast two-hybrid system against Topi. The region responsible for the interaction lies in the 200-300 amno acid fragment (bracket). (B) Mapping domains of Topi responsible for the interaction with full-length Comr. Zn-finger motifs within Topi are indicated with boxes. Fragments containing either the first two, or the final five, Zn fingers can bind Comr (brackets). (C) Co-immunoprecipitation of Comr and Topi from tissue culture. Cells expressed HA-tagged Topi and FLAG-tagged Comr or Smad1. Binding was assessed by IP-ing with anti-FLAG, and blotting with anti-HA (first pair of lanes) or vice versa (second pair of lanes). Topi and Comr co-immunoprecipitate in both cases (top). Total protein expression was assessed by western blotting of the cell lysate (bottom).

View larger version (68K): [in a new window]   Fig. 7. Differential requirement for topi and achi/vis in expression of some target genes. (A) RT-PCR of CG8349 from wild-type and meiotic arrest mutant testes. No-RT is a negative control, gDNA is PCR on genomic DNA from wild-type flies. Two exposures of the same gel are shown to emphasize the differences between the mutants and wild type (top), and the lack of product in achi/vis (bottom). (B-E) In situ hybridization of CG8349 probe to testes revealed strong expression in wild-type (B) primary spermatocytes, persisting until mid-late elongation stages, weaker expression in comr (C) and topi (E), and no detectable signal in achi/vis (D) testes. (F) RT-PCR of TrxT from wild-type and meiotic arrest mutant testes. No-RT is a negative control, gDNA is PCR on genomic DNA from wild-type flies. (G-J) In situ hybridization of TrxT probe testes revealed strong expression in wild-type (G) primary spermatocytes and early-mid elongation stages. The signal in achi/vis testes was of a similar intensity to wild type (I). Weaker expression was seen in comr (H) and there was no detectable signal in topi (J) testes.

View larger version (29K): [in a new window]   Fig. 8. Model for assembly and function of an aly-class protein complex at target promoters. Aly and Comr may be imported into the nucleus together or may only form a stable complex in the nucleus. Either protein alone remains in the cytoplasm. Topi and Achi/Vis proteins would enter the nucleus independently. These putative DNA-binding proteins would recognize specific sequences at target promoters, and once bound they would together recruit the Aly/Comr complex to these promoters. Alternatively, Topi and Achi/Vis could assemble with Aly/Comr in the nucleus, and this entire complex would then bind target promoters. This complex would then recruit the NURD histone deacetylase chromatin remodelling complex and promote transcription.