A Robust Transposon-Endogenizing Response from Germline Stem Cells

Abstract

The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion remain unclear. We show that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P element transposons by activating the DNA damage checkpoint and piRNA production. We find that temperature modulates the P element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA damage checkpoint. Within 4 days, the hosts amplify P element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. We propose that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution.

Figure 1. P-element transposon invasion.

(A) Schematic diagram of experimental design. Hereinafter, the progeny from dysgenic/invasive cross are referred to as “Invaded”; the P-element-silenced offspring from noninvasive (reciprocal) cross are shortened as “Protected”. (B) Ovarian morphology and fertility of invaded and protected progeny. Invaded flies have normal size ovaries at 18°C, but rudimentary ovaries at 25°C. (C) Genome sequencing to globally probe new transposition events in invaded and protected progeny. Each dot represents one transposon family. (D) Small RNA sequencing to detect the production of piRNAs mapped to transposon. Each dot represents one transposon family. Only piRNAs targeting P-elements (green dots) decreased in invaded progeny, compared with protected controls. See also Figure S1.

Figure 2. Vigorous invasion leads to rebirth of fly oogenesis.

(A) Schematic diagram of the temperature-shift design. (B) Fertility of invaded and protected female flies after the temperature-shift/vigorous P-element invasion. (C) Ovarian morphology per day after vigorous P-element invasion.

Figure 3. P-elements are silenced in the recovered ovaries.

(A) RNA-Seq profiles for P-element from invaded progeny. (B) H3K9me3 occupancy on P-element, as measured by H3K9me3 ChIP-Seq. (C) Small RNA-Seq assay to quantify the production of P-element piRNAs. Blue, sense piRNAs. Red, antisense. Ovary morphology pictures are re-used from Figure 2C to serve as time scale. See also Figure S2.

Figure 4. Germline stem cells silence the invading P-elements via the activation of Chk2/p53.

(A) Germarial structure from invaded progeny after vigorous P-element invasion. The germ cells are labeled by Vasa protein (green). The germaria are co-stained with 1B1 (red), an antibody that targets Hu-li tai shao protein that specifies germ cell stages in germarium and outlines cell membrane. Upon intensive invasion, the early stage germ cells are arrested, as evidenced by a “gap” region (circled by dash line) that contains no Vasa positive cells. Note that germ cells in the germarium display “dot” shaped structure from 1B1 staining, indicating that they are undifferentiated germ cells. Oogenesis reinitiates at day 4 or 5, as suggested by forming the “branch” structure from 1B1 staining (pointed by arrow). (B) Germarial structure from invaded progeny that are either wild type (left), heterozygous (middle), or homozygous (right) for the chk2 gene. Mutating chk2 gene completely rescues the arrest phenotype, as evidenced by the germarium containing continuous germ cells and normal formation of cysts at different stages. (C) p53 staining to detect the activation of DNA damage response. p53 signals (arrows) are readily detectable in the germarium from 25°C incubation. See also Figure S3, S4, S5, and S6.

Figure 5. The P-element mobilization assay to quantify the effect of temperature on P-element activity.

(A) The GFP::Vasa reporter fly serves as a P-element-devoid strain to set up the invading cross. (B) Strategy for the P-element mobilization assay, used in panel (C) and (D). P-element mobilization, most likely excision, leads to disappearance of GFP signal. (C) Representative images for the mobilization assay. Germarium is co-stained by Vasa (red) and GFP (green) antibodies. P-element transposition produces GFP negative, but Vasa positive germ cells. Germ cells that have P-element mobilization events are circled by dash line. w¹ flies, which do not contain P-element transposases, serve as negative controls. (D) Quantification of P-element mobilization assay.

Figure 6. Chk2-mediated arrest response tames invading transposons at 25°C.

(A) Fertility of invaded progeny that are in either chk2 heterozygous or homozygous background. Fertility of wild-type progeny (from Figure 2B) is reused for comparison. (B) RNA-Seq profiles for P-element from invaded progeny with chk2 null mutation. Unlike wild-type flies (Figure 3A), chk2 mutants are unable to silence invading transposons. Note that Chk2 is not required for P-element silencing in protected progeny. (C) Small RNA-Seq assay to quantify the production of P-element piRNAs. Blue, sense piRNAs. Red, antisense. Without Chk2-mediated arrest response, piRNA production stays at a low level from 10 days old flies at 25°C, comparing with abundant piRNA production from age-matched wild-type flies (Figure 3C). (D) Left panel: γ-H2Av staining to probe the amount of DNA damage. Right panel: TUNEL staining displays dying cells. See also Figure S7.

Figure 7. Role of Chk2 in mediating piRNA amplification and transposon endogenization in germline stem cells.

(A) A model explains the role of Chk2 in mediating piRNA amplification. Chk2-mediated arrest restricts the newly produced piRNAs, which are most likely generated from paternally inherited piRNA clusters, within a small volume. This can quickly increase the piRNA concentration to reach a level that initiates Ping-Pong amplification, which cleaves P-element transcripts and initiates transcriptional silencing via Piwi. (B) Fertility of invaded females, which are the heterozygotes of piRNA pathway factors, after experiencing the P-element invasion. All mutant alleles were generated from the same genetic background as the w¹¹¹⁸ controls. (C) Germarial structure of the heterozygotes of piRNA pathway factors after experiencing the P-element invasion. Germaria from the heterozygotes of piRNA pathway factors contain nearly no germ cells (occasionally a few) at 10 days, suggesting that germ cells were unable to recover from P-element invasion. Note that yellow dotted lines are used to outline germaria.