Direct inhibition of Pumilo activity by Bam and Bgcn in Drosophila germ line stem cell differentiation

Abstract

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. Here, we show that Bam and Bgcn inhibit Pum function through direct binding. We identified a ternary complex involving Bam, Bgcn, and Pum in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells.

FIGURE 1.

A ternary complex involving Bam, Bgcn, and Pum. A yeast three-hybrid assay revealed a ternary complex involving Bam, Bgcn, and Pum. A, a schematic diagram depicting a yeast three-hybrid assay where Bam is fused to the LexA DNA-binding domain (DBD), Pum or its derivatives (Puf or the N terminus of Pum (Pum_N)) is fused to the B42 TAD, Nos is fused to the Gal4 TAD, and Bgcn is added in a non-fusion form. Interaction was monitored with LacZ expression. B, left panel, schematic diagrams of Pum, the C-terminal Puf domain, and the N-terminal region lacking the Puf domain used for yeast three-hybrid assay. Right panel, yeast patches yielded a blue color on X-gal plates only when Bgcn was co-expressed with Bam and Pum or the N terminus of Pum. Co-expression of Bam with either Nos or Puf did not result in a blue color with or without Bgcn.

FIGURE 2.

A weak Bam-Pum interaction and a strong ternary complex involving Bam, Bgcn, and Pum. A, Bam and Pum were fused to the mKG_N and mKG_C fragments of the monomeric Kusabira-Green, respectively (Bam-mKG_N and Pum-mKG_C), which alone do not yield fluorescence (left panel). Interaction between these proteins yielded fluorescent green signal in the cytoplasm in the absence of Bgcn, but co-expression of intact Bgcn with these Bam and Pum constructs resulted in a great increase in signal (center panels). The cells expressing the control p50-mKG_N and p65-mKG_C plasmids yielded fluorescence in the nucleus (right panel). B, cells emitting GFP signals after transfection were counted. The number of GFP-positive cells co-expressing Bam-mKG_N and Pum-mKG_C was compared with the number of GFP-positive cells co-expressing Bam-mKG_N, Bgcn, and Pum-mKG_C, which is normalized to 100%.

FIGURE 3.

A protein complex containing Bam, Bgcn, Pum, and Nos. Lysates from S2 cells expressing a combination of FLAG-tagged Bam, Myc-tagged Bgcn, and HA-tagged Pum were subjected to Western blot either directly (lanes 1–4) or after being immunoprecipitated (IP) via anti-FLAG-conjugated agarose beads (lanes 5–8) with specific antibodies as indicated.

FIGURE 4.

Bam and Bgcn interact without Pum. A, diagram showing the regions (underlined) used to generate anti-PumT803 and anti-PumT980 antibodies. The Puf domain is indicated by a gray box. Lysates from S2 cells were immunoprecipitated (IP) with the combined anti-PumT803 and anti-PumT980 antibodies and then analyzed by Western blot with anti-Pum1637 (37). As a control, unrelated IgG was used. B, lysates from S2 cells expressing FLAG-tagged Bam and Myc-tagged Bgcn were subjected to Western blot analysis either directly (lanes 1 and 2) or after being precipitated with anti-FLAG M2 antibody-conjugated agarose beads (lanes 3–5). The extracts were premixed either with control antibody (IgG) or anti-Pum antibodies (anti-PumT803 and anti-PumT980) before precipitation with anti-FLAG M2 antibody-conjugated agarose beads. C, quantification of Pum and Bgcn precipitated by Bam. The Western blot bands (lanes 4 and 5 in Fig. 4B) were quantified using the Scion Image program (25). Precipitated protein levels were compared between preincubation (+) and no preincubation (−) with anti-pum antibodies, which were normalized to 100%. The results are presented as the means ± S.E. obtained from three independent experiments.

FIGURE 5.

Bam inhibition of NRE-mediated translational repression of Pum. A, schematic diagram of the luciferase-NRE (LUC/NRE) reporter that contains the CMV promoter (black arrow), the luciferase coding sequence, and one copy of the hb NRE sequence (black square box) at its 3′-untranslated region. LUC/NREmt contains a mutation in the NRE sequence as underlined (gray square box). B–D, HEK 293 cells were transfected with expression vectors and the luciferase reporter in several combinations. Luciferase activities were obtained from at least three independent experiments performed in triplicate. B, expression of Pum, but not Bam or Bgcn, repressed Luc expression of the reporter. C, expression of Bam, but not Bgcn, abrogated Pum repression of Luc expression. D, Pum failed to repress Luc expression when the NRE sequence was mutated. Puf repressed Luc expression but was not derepressed by Bam.

FIGURE 6.

Model depicting Bam/Bgcn binding and inhibition of Pum/Nos activity. In the germ line stem cells (GSC), the absence of Bam allows for Pum/Nos repression of differentiation-promoting genes. In the cystoblast (CB), Bam in conjunction with Bgcn binds to Pum at the N-terminal region and inhibits Pum/Nos repression of differentiation. CC denotes cap cells.