Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line

Abstract

Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, we purified the protein that specifically binds to the promoter of spermatid-differentiation gene Sdic and identified it as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G(2)/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line.

Fig. 1.

Identification of the TSE-binding activity as Modulo. (A Left) TSE-binding activity in testes (tes) and in the whole gonadectomized males (as shown at the top) analyzed by EMSA using the TSE oligonucleotide probe. The prominent DNA–protein complex identified in testes is indicated by the arrowhead. (A Right) Reactions with protein extracts from testes performed in the presence of the unlabeled TSE oligonucleotide (SC), or the nonspecific oligonucleotide competitor 2 (nsc). (B) Outline of purification of the TSE-binding activity. The media used for purification are indicated at the left. (C) Final stage of purification of the TSE-binding protein. (Upper) Fractions eluted from the DNA-covered beads with the concentrations of KCl indicated at the top, in millimoles, were analyzed by EMSA using the TSE oligonucleotide probe. (Lower) The fractions as above analyzed by SDS/PAGE in 4–15% gradient gel, followed with silver staining. SW, Southwestern blot analysis of the fraction eluted with 300 mM KCl, using the ³²P-labeled TSE probe. (D) Immuno-EMSA of the purified TSE-binding protein confirms its identity as Modulo. (Left) Reactivity of the anti-Modulo IgY with the total adult fly protein (Upper) and the purified recombinant full-size 62-kDa Modulo protein (Lower). (Right) EMSA analysis of the purified TSE-binding protein preincubated with increasing amounts (0.1, 0.3, and 1.0 μg) of the anti-Modulo IgY (anti-mod AB) or Modulo-depleted IgY used as negative control (mod-depl.AB). The control reactions (C) did not contain any antibody.

Fig. 2.

Modulo variant expressed in testes carries an acidic activator domain. (A Upper) Western blot analysis shows the difference in size between the Modulo variants expressed in testes (wt tes) and in somatic tissues (gonadectomized males). Absence of the signal in testes of the male sterile mod⁰⁷⁵⁷⁰ mutant (mod-tes) confirms specificity of analysis. (A Lower) Silver staining of the duplicate gel shows total protein loading on the lanes. (B) Staining of the SDS/PAGE gel with Coomassie blue (P) and Western blot analysis using anti-Modulo IgY (WB) show purity of the recombinant full-size His₆-tagged Modulo protein used for EMSA. (C) Binding of the recombinant full-size Modulo to testes-specific promoters is sequence-specific. PCR-amplified core promoter fragments of Sdic and β(2)Tubulin were used as the probes for EMSA. The complexes formed by Modulo are indicated by the arrowhead. The specific competitor (the TSE oligonucleotide, sc) and the nonspecific competitor 2 (nsc) were added to reactions as indicated at the top. (D) Modulo coimmunoprecipitates with the testes-specific TFIID subunit Sa. Samples of the total testes lysates, immunoprecipitated proteins (IP), and mock-precipitated samples (mock) analyzed by Western blotting. The antibodies used for immunoprecipitation (IP) and Western blot analysis (WB) are indicated at the left. (E) Characterization of the 50-kDa somatic Modulo variant using the nano-LC/MS/MS data. The full-size Modulo sequence is shown. The peptides identified by MS are underlined. The unusual peptide flanked with the trypsin cleavage site only at its C terminus is highlighted in black; this peptide probably marks the N terminus of the 50-kDa Modulo variant. The acidic domain present only in the full-size Modulo but not in the 50-kDa variant is shown in bold; the acidic residues (D, E) are highlighted.

Fig. 3.

Modulo is expressed in spermatogenesis before up-regulation of the spermatid-differentiation genes, similar to the general regulators of transcription. (A–C) Localization of Modulo in adult testes by immunofluorescense (red channel, A) shows that Modulo's up-regulation precedes activation of the Sdic::GFP transgene (green channel, C). The tip of the testis containing stem cells, spermatogonia, and spermatocytes is shown. (B) The red and the green channels merged with the signal from the chromatin DAPI stain (blue). Note condensation of small compact nuclei of spermatogonia and stem cells at the tip of the testis. (Scale bars, 30 μm.) (D) Up-regulation of the meiotic-arrest genes precedes transcriptional activation of the spermatid-differentiation genes in testis development. The bar graph shows the relative amounts of the transcripts of the meiotic-arrest genes (gray, average of the data for aly, can, and mia) and of the spermatid-differentiation genes [black, average of the data for Sdic, dhod, β(2)Tubulin, fzo, ocn, and dj] in testes of larvae and pupae of different ages (as indicated at the bottom). Error bars show the range of the real-time RT-PCR data for individual genes within each group.

Fig. 4.

Regulation of Modulo expression in testes by the meiotic-arrest genes and Boule links the pathways leading to meiosis and spermatid differentiation. (A) Modulo expression in testes of the wild type (wt) and of the bam^Δ[supi]86 (bam), achi¹ (ach), sa¹ (sa), Taf12L^KG00946 (rye), and bol¹ (bol) analyzed by Western blotting. (B) The duplicate gels stained with silver (Left) or Coomassie blue (Right) show total-protein loading on the lanes. (C) Model for the role of Modulo in cross-communication between the pathways leading to the G₂/M transition and spermatid differentiation. Arrows indicate positive regulation.