Tip60 complex promotes expression of a differentiation factor to regulate germline differentiation in female Drosophila

Abstract

Germline stem cells (GSCs) self-renew and differentiate to sustain a continuous production of gametes. In the female Drosophila germ line, two differentiation factors, bag of marbles ( bam) and benign gonial cell neoplasm ( bgcn), work in concert in the stem cell daughter to promote the generation of eggs. In GSCs, bam transcription is repressed by signaling from the niche and is activated in stem cell daughters. In contrast, bgcn is transcribed in both the GSCs and stem cell daughters, but little is known about how bgcn is transcriptionally modulated. Here we find that the conserved protein Nipped-A acts through the Tat interactive protein 60-kDa (Tip60) histone acetyl transferase complex in the germ line to promote GSC daughter differentiation. We find that Nipped-A is required for efficient exit from the gap phase 2 (G2) of cell cycle of the GSC daughter and for expression of a differentiation factor, bgcn. Loss of Nipped-A results in accumulation of GSC daughters . Forced expression of bgcn in Nipped-A germline-depleted ovaries rescues this differentiation defect. Together, our results indicate that Tip60 complex coordinates cell cycle progression and expression of bgcn to help drive GSC daughters toward a differentiation program.

FIGURE 1:

Nipped-A is required in the germ line for differentiation of the germline stem cell daughter. (A) Schematic representation of a Drosophila germarium where germ cells (blue) are surrounded by somatic cells (gray). The germline stem cells (GSCs) reside in the germarium and are maintained by a somatic niche. The GSC divides to give rise to a daughter, called a precystoblast (pre-CB). The pre-CB turns on differentiation factors and is then called the cystoblast (CB). The CB undergoes incomplete mitotic divisions to give rise to 2-, 4-, 8-, and 16-cell cysts. Single cells are marked by punctate structures called spectrosomes (red), and cysts are marked by the elongated, branched spectrosomes called fusomes (red). The 16-cell cyst migrates, buds off from the germarium, and is encapsulated by the soma (gray) to generate egg chambers. Developing egg chambers will have one germ cell that becomes the oocyte (yellow), and the other germ cells will be support cells (blue). (B) Schematic of Nipped-A function. Nipped-A (teal) can associate with transcriptional activators (light green) to recruit SAGA and Tip60 complexes. These complexes can acetylate lysines on histones (dark green circle) to regulate transcription (dark green arrow). Nipped-A cartoon is based on the Cryo-EM structure of Tra1 in SAGA complex in Pichia pastoris yeast (Sharov et al., 2017). (C, C′) Control and (D, D′) germline-depleted Nipped-A (RNAi line #1) germaria stained with Vasa (blue) and 1B1 (red). Germaria depleted of Nipped-A show accumulation of single cells (yellow dashed line). 1B1 channel is shown in C′ and D′. (E) Quantitation of the number of single cells in germaria of control and germline-depleted Nipped-A using three RNAi lines (34.64 ± 15.04 in Nipped-A RNAi #1, 27.96 ± 12.17 in Nipped-A RNAi #2, and 12.56 ± 4.94 in Nipped-A RNAi #3 compared with 3.04 ± 0.68 in UAS-Dcr2;nosGAL4 control; n = 25 for all, ****P < 0.0001). (F, F′) Control and (G, G′) germline-depleted Nipped-A germaria stained with pMAD (green), Vasa (blue), and 1B1 (red). Germaria depleted of Nipped-A do not accumulate pMAD-positive germ cells (yellow dashed circle) (n = 20 for both, P < 0.0001). pMAD channel is shown in F′ and G′. (H, H′) Control and (I, I′) germline-depleted Nipped-A germaria stained with BamC (red) and Vasa (blue). Germaria depleted of Nipped-A do not accumulate BamC-positive germ cells (yellow dashed line in control) (n = 25 for both). BamC channel is shown in H′ and I′. (J, J′) Control and (K, K′) pgcGFP with Nipped-A germline-depletion germaria stained with GFP (green), Vasa (blue), and 1B1 (red). Germaria depleted of Nipped-A accumulate a higher number of Pgc-positive germ cells (n = 25 for both, P < 0.0001). Pgc expression is marked by GFP (yellow dashed circle/line in control and knockdown, respectively). GFP channel is shown in J′ and K′. Statistical analysis performed with Student’s t test for all except for Chi-square for H–I′. Scale bar for all images is 20 μm.

FIGURE 2:

Tip60 complex members and HAT activity are required for timely differentiation. (A, A′) Control and germline-depleted Tip60 complex members (B,B′) Bap55, (C, C′) dom, (D, D′) E(Pc), (E, E′) Tip60, and (F, F′) YL-1 germaria stained with Vasa (blue) and 1B1 (red). Tip60 complex member–depleted germaria show accumulation of single cells (yellow dashed line). 1B1 channel is shown in A′, B′, C′, D′, E′, and F′. (G) Quantitation of the number of single cells in control and germline-depleted Tip60 complex members Act87E, Bap55, Brd8, DMAP1, dom, E(Pc), Eaf6, Mrg15, Nipped-A, pont, rept, Tip60, and YL-1 germaria (3.20 ± 1.50 in Act87E, 16.04 ± 5.93 in Bap55 RNAi, 4.96 ± 2.15 in Brd8 RNAi, 8.52 ± 4.02 in DMAP1 RNAi, 20.76 ± 8.54 in dom RNAi, 21.76 ± 7.42 in E(Pc) RNAi, 4.80 ± 2 in Eaf6 RNAi, 5.36 ± 2.16 in Mrg15 RNAi, 34.64 ± 15.04 in Nipped-A RNAi, 6.36 ± 2.58 in pont RNAi, 0 ± 0 in rept RNAi, 32.72 ± 19.86 in Tip60 RNAi, and 8.16 ± 3.57 in YL-1 RNAi compared with 3.04 ± 0.68 in UAS-Dcr2;nosGAL4 control; n = 25 for all, P = 0.3110 for Act87E; ****P < 0.0001). (H, H′) Nipped-A heterozygote and (I, I′) Tip60;Nipped-A trans-heterozygote germaria stained with Vasa (blue) and 1B1 (red). Trans-heterozygote germaria show accumulation of single cells in (yellow dashed line). 1B1 channel is shown in H′ and I′. (J) Quantitation of the number of single cells in germaria of Tip60 heterozygote, Nipped-A heterozygote, and Tip60;Nipped-A trans-heterozygote (8.80 ± 2.33 in Tip60;Nipped-A trans-heterozygote compared with 3.36 ± 0.91 in Tip60 heterozygote control and 3.56±0.87 in Nipped-A heterozygote control; n = 25 for both; ****P < 0.0001). (K, K′) Control and (L, L′) nosGAL4-driven UAS-Tip60^DN germaria stained with Vasa (blue) and 1B1 (red). Germaria with overexpression of Tip60^DN show accumulation of single cells (yellow dashed line). 1B1 channel is shown in K′ and L′. (M) Quantitation of the number of single cells in control and nosGAL4-driven UAS-Tip60^DNgermaria (17.5 ± 7.47 in UAS-Tip60^DN compared with 2.95 ± 0.60 in nosGAL4 control; n = 20 for both; ****P = 0.0009). Statistical analysis performed with Student’s t test. Scale bar for all images is 20 μm.

FIGURE 3:

Tip60 complex members are required for acetylation of H4K16. (A, A′) UAS-Dcr2;nosGAL4, (B, B′) germline-depleted bam, (C, C′) germline-depleted Nipped-A, and (D, D′) nosGAL4-driven UAS-Tip60^DN germaria stained with H4K16ac (green), Vasa (blue), and 1B1 (red). Germaria with depletion of Nipped-A or Tip60 HAT activity show lower levels of H4K16ac levels in the germ line. Stem cell daughters are outlined with yellow dashed lines in all images. H4K16ac channel is shown in A′, B′, C′, and D′. (E) Quantitation of somatic H4K16ac intensity in UAS-Dcr2;nosGAL4, germline-depleted bam and Nipped-A, and nosGAL4-driven UAS-Tip60^DN(1.05 ± 0.60 in UAS-Dcr2;nosGAL4, 0.85 ± 0.65 in bam RNAi, 0.94 ± 0.47 in Nipped-A RNAi, and 1.18 ± 0.09 in UAS-Tip60^DN; n = 35 for all; P = 0.0924). (E′) Quantitation of normalized germline H4K16ac intensity in UAS-Dcr2;nosGAL4, germline-depleted bam and Nipped-A, and nosGAL4-driven UAS-Tip60^DNgerm cells (0.52 ± 0.20 in Nipped-A RNAi and 0.45 ± 0.27 in UAS-Tip60^DNcompared with 0.69 ± 0.41 in UAS-Dcr2;nosGAL4 and 0.96 ± 0.43 in bam RNAi controls; n = 35 for all; *P < 0.03, **P = 0.0093, and ***P = 0.004). Statistical analysis performed with Student’s t test for all except for one-way analysis of ANOVA for E. Scale bar for all images is 20 μm.

FIGURE 4:

Nipped-A regulates bgcn levels. (A) Volcano plot of log₂ fold change (FC) vs. –log₁₀ P value. Red dots represent significantly down-regulated transcripts, and blue dots represent significantly up-regulated transcripts in Nipped-A RNAi ovaries compared with bam RNAi ovaries (FDR = 0.05). Genes with threefold or higher change were considered significant. Two-hundred eighty genes were significantly down-regulated while 470 genes were significantly up-regulated. (B) Table representing the Gene Ontology analysis carried out on differentially expressed genes in Nipped-A–depleted ovaries compared with bam-depleted ovaries. GO: 0040003 was identified from significantly down-regulated genes (red), while GO: 0006508, GO: 0006022, GO: 0006517, GO: 0008236, GO: 0004175, GO: 0016787, GO: 0140096, and GO: 0004180 were identified from significantly up-regulated genes (blue). (C) UCSC genome browser view of bgcn locus and pym intervening locus. Germline-depleted bam and Nipped-A reads shown on top (purple and teal, respectively). RefSeq annotations (bottom, blue) indicate that germline-depleted Nipped-A has reads mapping to Partner of Y14 and Mago (pym); pym reads do not change but reads mapping to bgcn are lower in germline-depleted Nipped-A. (D, D′) Control and (E, E′) germline-depleted bgcn germaria stained with Vasa (blue) and 1B1 (red). Germaria depleted of bgcn show accumulation of single cells (yellow dashed line). 1B1 channel is shown in D′ and E′. (F) Quantitation of the number of single cells in control and germline-depleted bgcn germaria (67.36 ± 13.99 in bgcn RNAi compared with 2.28 ± 0.84 in UAS-Dcr2;nosGAL4 control; n = 25 for both, ****P < 0.0001). (G, G′) Control and (H, H′) germline-depleted bgcn germaria stained with pMAD (green), Vasa (blue), and 1B1 (red). Germaria depleted of bgcn do not accumulate pMAD-positive germ cells (yellow dashed circle) (n = 25, P = 0.1189). pMAD channel is shown in G′ and H′. (I, I′) Control and (J, J′) germline-depleted bgcn germaria stained with BamC (red) and Vasa (blue). Germaria depleted of bgcn do not accumulate BamC-positive germ cells (yellow dashed line in control) (n = 25 for both). BamC channel is shown in I′ and J′. (K, K′) bgcn heterozygote and (L, L′) bgcn/Nipped-A trans-heterozygote germaria stained with Vasa (blue) and 1B1 (red). Trans-heterozygote shows accumulation of single cells (yellow dashed line) (n = 25). 1B1 channel is shown in K′ and L′. Statistical analysis performed with Student’s t test for all except for Chi-square for I–J′. Scale bar for all images is 20 μm.

FIGURE 5:

Ectopic expression of bgcn rescues Nipped-A RNAi early defect. (A, A′) Germline-driven bgcn^EP, (B, B′) depletion of Nipped-A, and (C, C′) rescue germaria stained with Vasa (blue) and 1B1 (red). Germline-driven bgcn^EPand rescue germaria show fusome formation (yellow dashed line). Germaria depleted of Nipped-A accumulate only spectrosome-containing cells (white arrow). 1B1 channel is shown in A′, B′, and C′. (D) Quantitation of the number of single cells in germline-driven bgcn^EP, depletion of Nipped-A, and rescue germaria (2.78 ± 1.38 in bgcn^EP; 5.72 ± 2.37 in rescue compared with 16.52 ± 5.02 in Nipped-A RNAi; n = 50 for all ****P < 0.0001). (E) Schematic showing Tip60 complex regulates differentiation in the precystoblast (GSC daughter). In G2 phase, Pgc is expressed prior to differentiation, we postulate that Tip60 complex modulates bgcn mRNA levels to promote differentiation. Bgcn and Bam proteins then complex to stimulate differentiation in the GSC daughter. Statistical analysis performed with Student’s t test. Scale bar for all images is 20 μm.