Environmentally-induced epigenetic conversion of a piRNA cluster
Abstract
Transposable element (TE) activity is repressed in animal gonads by PIWI-interacting RNAs (piRNAs) produced by piRNA clusters. Current models in flies propose that germinal piRNA clusters are functionally defined by the maternal inheritance of piRNAs produced during the previous generation. Taking advantage of an inactive, but ready to go, cluster of P-element derived transgene insertions in Drosophila melanogaster, we show here that raising flies at high temperature (29°C) instead of 25°C triggers the stable conversion of this locus from inactive into actively producing functional piRNAs. The increase of antisense transcripts from the cluster at 29°C combined with the requirement of transcription of euchromatic homologous sequences, suggests a role of double stranded RNA in the production of de novo piRNAs. This report describes the first case of the establishment of an active piRNA cluster by environmental changes in the absence of maternal inheritance of homologous piRNAs.
Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
https://doi.org/10.7554/eLife.39842.001Introduction
Transposable element (TE) activity needs to be repressed to avoid severe genome instability and gametogenesis defects. In humans, growing evidence has implicated TE in several disorders such as cancers defining a new field of diseases called transposopathies (Wylie et al., 2016a; Wylie et al., 2016b). In the animal germline, TE activity is controlled at both transcriptional and post-transcriptional levels by small RNAs called piRNAs associated with the PIWI clade of germline Argonaute proteins (Piwi, Aub and Ago3 in Drosophila) (Brennecke et al., 2007; Gunawardane et al., 2007; Le Thomas et al., 2013; Sienski et al., 2012). piRNAs are processed from transcripts produced from specific heterochromatic loci enriched in TE fragments, called piRNA clusters (Brennecke et al., 2007; Gunawardane et al., 2007). These loci undergo non-canonical transcription, ignoring splicing and transcription termination signals, licensed by specific protein complexes such as Rhino-Deadlock-Cutoff (Mohn et al., 2014; Zhang et al., 2014) and Moonshiner-TRF2 (Andersen et al., 2017). Thus, when a new TE inserts into a naive genome, it will freely transpose until one copy gets inserted into a piRNA cluster leading to the production of homologous new TE piRNAs that will then repress transposition (Brennecke et al., 2008). In support of this idea, exogenous sequences inserted into preexisting piRNA clusters lead to the production of matching piRNAs (de Vanssay et al., 2012; Hermant et al., 2015; Marie et al., 2017; Muerdter et al., 2012; Pöyhönen et al., 2012). The specificity of the efficient repression mediated by piRNAs appears to be determined solely by the piRNA cluster sequences. Thus, it raises the question of how piRNA cluster loci are themselves specified. Histone H3 lysine nine tri-methylation (H3K9me3) that is recognized by Rhino, a paralog of heterochromatin protein HP1 (Klattenhoff et al., 2009), is a shared feature of piRNA clusters. Enrichment of H3K9me3, however, is not specific to piRNA clusters and tethering Rhino onto a transgene leads to the production of piRNAs only when both sense and antisense transcripts are produced (Zhang et al., 2014). This suggests that neither H3K9me3 marks nor having Rhino-bound is sufficient to induce piRNA production. One current model proposes that piRNAs clusters are defined and activated at each generation by the deposition in the egg of their corresponding piRNAs from the mother (Huang et al., 2017). In support of this model, we previously described the first case of a stable transgenerational epigenetic conversion known as paramutation in animals (de Vanssay et al., 2012). This phenomenon was first described in plants and defined as "an epigenetic interaction between two alleles of a locus, through which one allele induces a heritable modification of the other allele without modifying the DNA sequence" (Brink, 1956; Chandler, 2007). In our previous study, we showed that an inactive non-producing piRNA cluster of P transgene insertions inherited from the father can be converted into a piRNA-producing cluster by piRNAs inherited from the mother (de Vanssay et al., 2012). This attractive model, however, does not answer the question of how the first piRNAs were produced.
To address this paradox, we used the same BX2 cluster of seven P(lacW) transgenes, which resulted from multiple and successive P(lacW) transposition events, thus resembling the structure of natural piRNA clusters (Dorer and Henikoff, 1994). The key advantage of the BX2 locus is that it can exist in two epigenetic states for the production of germline piRNAs: 1) the inactive state (BX2OFF) does not produce any piRNAs and thus is unable to repress the expression of homologous sequences, and 2) the active state (BX2ON) produces abundant piRNAs that functionally repress a homologous reporter transgene in the female germline (de Vanssay et al., 2012; Hermant et al., 2015). We therefore used BX2 in an inactive state to search for conditions that would convert it into an active piRNA-producing locus, without pre-existing maternal piRNAs. In this report, we describe how culturing flies at high temperature, 29°C instead of 25°C, induces the conversion of an inactive BX2 locus (BX2OFF) into a stable piRNA cluster exhibiting repression properties (BX2ON). It should be noted that flies in their natural habitat exhibit this range of temperature, especially in the context of global warming. These data provide the first report of a de novo piRNA cluster establishment independent of maternal inheritance of homologous piRNAs and highlight how environmental changes can stably induce transgenerational modification of the epigenome.
Results
Germline silencing induced at high temperature
Earlier studies of hybrid dysgenesis reported that high temperature enhances P-element repression (Ronsseray et al., 1984) and that thermic modification of P repression can persist over several generations (Ronsseray, 1986). Moreover, P-element repression in a strain carrying two P-elements inserted into a subtelomeric piRNA cluster can be stimulated by heat treatment (Ronsseray et al., 1991). Very recently, the tracking of natural invasion of P elements in Drosophila simulans confirmed the key role of high temperature in the establishment of repression through generations (Kofler et al., 2018). These results suggested that temperature may influence the activity of some piRNA clusters. To investigate whether high temperature (29°C) could affect the stability of BX2 epialleles (BX2OFF and BX2ON) across generations, we generated flies carrying, on the same chromosome, each of the BX2 epialleles and an euchromatic reporter transgene sharing P and lacZ sequences with BX2 (made of seven P(lacW), Figure 1—figure supplement 1A). This transgene promotes the expression of ß-Galactosidase both in the germline and in the somatic cells of the ovary and thus will hereinafter be referred to as 'P(TARGET)GS' (Figure 1—figure supplement 1B). As was previously described (de Vanssay et al., 2012), at 25°C BX2OFF does not synthesize functional piRNAs complementary to P(TARGET)GS resulting in ß-Galactosidase expression in whole ovaries of BX2OFF, P(TARGET)GS lines (Figure 1A). Whereas in BX2ON, P(TARGET)GS lines, functional lacZ piRNAs are synthesized in the germline where they specifically repress the P(TARGET)GS ß-Galactosidase expression (Figure 1B). Both BX2OFF, P(TARGET)GS and BX2ON, P(TARGET)GS lines incubated at 25°C for 23 generations maintained their epigenetic state, showing that both epialleles are stable (Figure 1D, Supplementary file 1). At 29°C, the repression capacity of BX2ON, P(TARGET)GS lines remained stable through 25 generations. Among the BX2OFF, P(TARGET)GS lines, 24.7% of females analyzed during 25 generations showed a complete and specific germline ß-Galactosidase repression (n = 3812, Figure 1E and Supplementary file 1), suggesting a conversion of the BX2OFF epiallele into BX2ON. Interestingly, the appearance of females showing ß-Galactosidase repression was gradual and stochastic, resulting in a global frequency that increased with the number of generations (Supplementary file 1). To test whether the temperature-induced conversion was stable, a set of five lines showing full repression capacity after 23 generations at 29°C, obtained from an independent experiment, were transferred to 25°C and tested for their silencing capacities for several generations. In all cases, the silencing capacities of the BX2ON epiallele induced at 29°C remained stable during 50 additional generations at 25°C (Supplementary file 2). These stable BX2ON lines converted by high temperature were named hereafter BX2Θ (Greek theta for temperature) to distinguish them from the BX2* lines converted by maternally inherited piRNAs (de Vanssay et al., 2012). Taken together, our data show that BX2OFF can be functionally converted by high temperature (29°C), strongly suggesting that de novo piRNA production can occur in the absence of maternal inheritance of homologous piRNAs.

Functional assay of the BX2 epigenetic state.
Females carrying either one of the BX2 epialleles and P(TARGET)GS were analyzed. (A) When BX2 is OFF for production of piRNAs (BX2OFF), no repression of P(TARGET)GS occurs, allowing expression of ß-Galactosidase in both germline and in somatic lineages in ovaries (ß-Galactosidase staining). BX2OFF is illustrated by a blue fly. (B) When BX2 is ON for production of piRNAs (BX2ON), repression of P(TARGET)GS occurs only in the germline lineage. BX2ON is illustrated by a light brown fly. (C) Drawing of an intermediate egg chamber showing germ cells (nurse cells and oocyte in blue) surrounded by somatic follicular cells (in pink), adapted from Figure 1A from Frydman and Spradling (2001). (D) At 25°C, BX2OFF and BX2ON are stable over generations. (E) At 29°C, BX2OFF can be converted into BX2ON, while BX2ON is stable over generations.
BX2 lines converted by high temperature or by maternal homologous piRNA inheritance present identical functional and molecular properties
We further characterized the functional and molecular properties of BX2Θ activated by temperature and compared them to BX2* activated by maternal inheritance of homologous piRNAs. Firstly, we compared the maternal and paternal BX2 locus inheritance effect of three BX2Θ lines and three BX2* at 25°C (Figure 1—figure supplement 2A). Maternal inheritance of either BX2Θ or BX2* loci leads to complete and stable repression of ß-Galactosidase expression (n flies = 152 and 159, respectively, Supplementary file 3), whereas paternal inheritance of either BX2Θ or BX2* loci, that is in absence of maternal piRNA deposition, results in ß-Galactosidase expression, and thus a definitive loss of BX2 silencing capacities (n flies = 156 and 155, respectively, Supplementary file 3). Secondly, we previously showed that progeny with a paternally inherited BX2OFF locus and maternally inherited BX2* piRNAs, but lacking the maternally BX2* genomic locus, have 100% conversion (de Vanssay et al., 2012). This process of recurrent conversions of an allele that is heritable without DNA modification is known as paramutation, thus BX2* females are paramutagenic, that is able to trigger paramutation. To test this property on BX2 lines converted by temperature, BX2Θ females were crossed with BX2OFF males. The progeny that inherited the paternal BX2OFF locus but not the maternal BX2Θ locus were selected and three independent lines were established (Figure 1—figure supplement 2B). Silencing measured over 20 generations revealed 100% of repression capacity showing that BX2Θ is also paramutagenic (n flies = 159, Supplementary file 4).
To determine whether the silencing capacities of BX2Θ involved piRNAs, small RNAs from BX2OFF, BX2Θ and BX2* ovaries were extracted and sequenced (Supplementary file 5). Unique reads matching the P(lacW) sequences were identified only in the BX2Θ and BX2* libraries (Figure 2A). Most of these small RNAs display all of the characteristics of bona fide germline piRNAs, that is a high proportion of 23–29 nt with a strong U bias on the first 5' nucleotide, an enrichment of a 10 nucleotide overlap between sense and antisense piRNAs, also known as the ping-pong signature, and a high proportion of reads with A at the tenth position among the 10 nt overlapped reads (Figure 2B–C) (Brennecke et al., 2007; Gunawardane et al., 2007). As a control, the 42AB piRNA cluster, a canonical germline dual-strand piRNA cluster, presented no significant difference between the three genotypes (Figure 2—figure supplement 1). Therefore, these results show that high temperature can initiate piRNA production from BX2 naive sequences (BX2OFF) and strongly suggest that once a piRNA cluster is activated for piRNA production, the 'ON' state is maintained at each generation by maternal inheritance of piRNAs.

BX2Θ and BX2* produce piRNAs and are enriched in H3K9me3.
(A) Size distribution of ovarian small RNAs matching BX2 transgene sequences reveals that both BX2Θ and BX2* but not BX2OFF produce 21 nt siRNAs and 23–29-nt piRNAs (upper panels, pBR = backbone plasmid pBR322). Positive and negative values correspond to sense (red) and antisense (blue) reads, respectively. Unique 23–29 nt mappers are shown on the BX2 transgene sequences (lower panels). (B) Percentage of 23–29 nt small RNAs from BX2Θ and BX2* matching transgene sequence with a U at the first position are shown. n.d.: not determined due to low number of reads. (C) Relative frequency (z-score) of overlapping sense-antisense 23–29 nt RNA pairs reveals an enrichment of 10 nt overlapping corresponding to the ping-pong signature. (D) H3K9me3 and (E) Rhino binding on the BX2 transgene in ovaries of BX2OFF, BX2Θ and BX2* strains revealed by chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) on specific white sequences. In both ‘ON’ strains, BX2Θ and BX2*, H3K9me3 and Rhino levels over the transgene are very similar and higher than in the BX2OFF strain (unpaired t-test was used to calculate significance of the differences (p<0.05, n = 5).
BX2 is inserted into the first intron of the AGO1 gene (de Vanssay et al., 2012) and we looked at the piRNA production from this region in the different BX2 epigenetic contexts. No significant amount of piRNAs coming from the AGO1 gene region can be detected whatever the BX2 state (Figure 2—figure supplement 2). These findings indicate that the AGO1 gene region is not a natural piRNA cluster. To test whether other non-piRNA producing genomic loci have started to produce piRNAs following high temperature treatment, we looked for specific piRNAs (23–29 nt) matching at unique positions on Drosophila chromosomes and compared them between BX2Θ and BX2OFF. The reads were then resampled per 50 kilobases windows. To eliminate background noise, only regions that produced more than five piRNAs per kilobase on average in both libraries were considered. Only exons of the white gene present in the P(lacW) transgenes of BX2 showed differential piRNA expression (log2 ratio >8.5, Figure 2—figure supplement 3). This analysis revealed that the activation of piRNA production after thermic treatment is restricted to the BX2 locus, suggesting that all other loci able to produce piRNAs are already active.
Previous studies had suggested that the chromatin state plays a role in the differential activity of BX2 (Le Thomas et al., 2014). We therefore profiled H3K9me3 marks and Rhino binding on the P(lacW) transgene in ovaries from BX2OFF, BX2Θ and BX2* strains by chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR). In both strains BX2Θ and BX2*, H3K9me3 and Rhino were similarly enriched over the P(lacW) transgene compared to the BX2OFF strain, significantly for H3K9me3 (Figure 2D–E). Taken together, these results show that de novo activation of BX2OFF by 29°C treatment (BX2Θ) or paramutation by maternal inheritance of homologous piRNAs (BX2*) lines leads to similar functional and molecular properties.
Epigenetic conversion at 29°C occurs at a low rate from the first generation
To explain the low occurrence and the generational delay of BX2 conversion at 29°C (Supplementary file 1), we propose that conversion is a complete but rare event occurring in a small number of egg chambers at each generation. Under this hypothesis, the sampling size of tested females should be crucial to observe such stochastic events. We therefore increased the number of analyzed females raised at 29°C during one generation. For this, eggs laid by females maintained at 25°C carrying the P(TARGET)GS reporter transgene and the BX2OFF locus were collected during three days. These eggs were then transferred at 29°C until adults emerged (Figure 3A). To follow their offspring, we individually crossed 181 G1 females with two siblings and let them lay eggs for three days at 25°C. These 181 G1 females were then stained for ß-Galactosidase expression. Strikingly, repression occurred only after one generation at 29°C in a few of the egg chambers of 130 G1 females (≈2.7% of the estimated total number of G1 egg chambers n≈21700, Figure 3B, right panel). These results support our hypothesis whereby epigenetic conversion of BX2OFF into BX2Θ is an instantaneous and complete event occurring at a low frequency per egg chamber and at each generation that is kept at 29°C.

BX2 conversion at 29°C occurs in one generation at a low rate.
(A) G0 females carrying the P(TARGET)GS reporter and BX2OFF laid eggs at 25°C during three days. The BX2OFF state of these females was confirmed after the three days at 25°C by ß-Galactosidase staining (number of egg chambers ≥ 1200). (B) Their eggs were allowed to develop at 29°C until emergence of the next generation. G1 females (n = 181) were individually mated with two siblings and left to lay for three days at 25°C. G1 females were then individually stained for ß-Galactosidase expression. Strikingly, 130 females (71.8%) show ß-Galactosidase repression in some egg chambers (586 among ≈21700 - estimation of the total egg chamber number among 181 females). The BX2OFF into BX2ON conversion frequency is ≈2.7%. (C) Analysis of each G1 female progeny developed at 25°C by ß-Galactosidase staining. The progeny of the 51 G1 females that did not present repression maintained BX2OFF state (n flies = 342). Most of the progeny of the 130 G1 females presenting conversion show no repression (97.2%, n flies = 1488) while 41 females present partial (n = 24) or complete (n = 17) repression of the germline expression of ß-Galactosidase.
To test the stability of the epigenetic BX2Θ states observed in G1 females, offspring daughters (G2) were raised at 25°C and their ovaries examined for ß-Galactosidase expression. Among G2 females, partial (n = 24) or complete (n = 17) repression of ß-Galactosidase expression in the germline was observed only in the progeny of those 130 G1 females in which partial repression was previously detected (Figure 3C). The proportion of 2.2% of converted G2 females (41/1830) is reminiscent with the proportion of repressed egg chambers observed in G1. The progeny of the 51 G1 females that did not present repression (Figure 3B left panel) did not show spontaneous conversion (Figure 3C). Taken together, these observations strongly suggest that newly converted BX2ON egg chambers give rise to adult females with complete or partial silencing capacities. The low conversion rate observed in thousands of flies after one generation raised at high temperature and its stability through the next generation might explain the apparent delay of BX2ON conversion of dozens of flies continuously raised at 29°C observed in the first set of experiments (see Figure 1E and Supplementary file 1). We more finely analyzed the silencing capacities of eight independent BX2OFF lines throughout generations at 29°C (Supplementary file 1) by monitoring P(TARGET)GS repression in each egg chamber instead of whole ovaries (Supplementary file 6). BX2 conversion occurred in each tested line with various dynamics (Figure 1—figure supplement 3A), likely reflecting a genetic drift due to the low conversion occurrence coupled to an important sampling effect at each generation. Globally, the mean of repression frequencies seems to indicate a progressive increase of BX2 conversion over generations (Figure 1—figure supplement 3B). Altogether, our results illustrate how environmental modifications like high temperature experienced during one generation might stably modify the epigenome of the future ones. Such a newly acquired epigenetic state may spread in a given population within a few generations.
High temperature increases BX2 antisense RNA but not piRNAs
Previously, we showed that BX2OFF and BX2ON produce similar amounts of sense and antisense transcripts (de Vanssay et al., 2012). However, these transcripts do not lead to ß-Galactosidase expression in the germline nor piRNA production in the BX2OFF line. We wondered if high temperature might change the RNA steady-state level of BX2. To ensure that we were detecting RNA specifically from BX2, qRT-PCR experiments targeting the lacZ gene were carried out on ovarian RNA extracted from the BX2OFF line that did not contain P(TARGET)GS. We observed a significant increase in the steady-state BX2 RNA levels at 29°C compared to 25°C (Figure 4A). Remarkably, strand-specific qRT-PCR experiments revealed that only BX2 antisense transcripts, corresponding to antisense lacZ transcripts, increase at 29°C (Figure 4B). As BX2 is inserted into the AGO1 gene in a convergent transcription manner (de Vanssay et al., 2012), Figure 4C), we compared AGO1 steady-state RNA level at 29°C and 25°C. AGO1 transcript isoforms that are initiated upstream the BX2 insertion point are significantly increased at 29°C (Figure 4D and Figure 4—figure supplement 1A–B). Thus, it is possible that, at 29°C, an increase of transcription from the AGO1 promoters located upstream the BX2 insertion point could lead to an increase of BX2 antisense RNA transcription.

BX2OFF antisense RNA increase at 29°C.
(A) RT-qPCR experiments revealed that the steady-state level of ovarian lacZ RNAs from BX2 is more abundant at 29°C (n = 5) compared to 25°C (n = 6). (B) Sense-specific RT-qPCR experiments revealed that only antisense transcripts from BX2, corresponding to antisense lacZ transcripts, are increased (25°C n = 6, 29°C n = 4). Significant p-values are given (bilateral Student's t-test). ns: not significant. (C) Map of the BX2 locus containing seven P(lacW) transgenes inserted into the AGO1 gene. P(lacW) and AGO1 are drawn to scale. The lacZ gene contained in P(lacW) and AGO1 are transcriptionally in opposite directions. Black arrows show lacZ primers used for (A) and (B) experiments. White arrows show primers used for sense-specific reverse transcription in (B) experiment. Grey arrows show AGO1 primers used for (D) experiment: these primers are specific for AGO1 transcripts (RA, RC and RD) that originate from promoters located upstream the BX2 insertion point and, thus, are potentially convergent to BX2. Red arrows show primers used to measure steady-state of all AGO1 isoforms (see Figure 4—figure supplement 1D). (D) RT-qPCR experiments performed on flies devoid of P(lacW) transgenes (w1118 context) revealed that the steady-state level of ovarian AGO1-RA, -RC and -RD RNA isoforms is more abundant at 29°C (n = 5) compared to 25°C (n = 6). (E–I) To compare small RNAs at 25 versus 29°C, total RNAs were extracted from BX2OFF ovaries dissected from adults incubated at 25°C or 29°C. Three samples were tested for each temperature. Small RNAs from 18 to 30 nt were deep sequenced. For each library, normalization has been performed for 1 million reads matching the Drosophila genome (rpm, Supplementary file 7). Size distributions of unique reads that match reference sequences are given. (E) Small RNAs matching the Drosophila genome present similar profiles in both temperatures except for 22 nt RNAs that are more represented at 25°C. (F) The 21 to 25 nt reads matching the 42AB piRNA cluster that range from 21 to 25 nt are slightly more abundant at 25°C. (G) Strikingly, almost only 21 nt RNAs match BX2 sequence. They are equally distributed among sense (H) and antisense (I) sequences at both temperatures. (J) No small RNAs corresponding to the AGO1 gene can be detected whatever the temperature. *=p < 0.05, bilateral Student's t-test.
We then examined whether the increase of BX2 antisense RNAs leads to an increase of antisense small RNAs. Ovarian small RNAs (18 to 30 nucleotides) of BX2OFF flies (without P(TARGET)GS) raised at 25°C and at 29°C for one generation were sequenced and the read numbers normalized (Supplementary file 7). A slight, yet statistically significant, decrease is observed at 29°C for small RNAs matching the whole genome (Figure 4E) and for the 42AB piRNA cluster (Figure 4F). Strikingly, no piRNAs were produced from the BX2 locus at 25°C nor after one generation at 29°C. Thus, the increase of BX2 antisense transcripts observed at 29°C (Figure 4B) did not correlate with an increase of corresponding antisense piRNAs. At 25°C and 29°C, BX2OFF produced the same low amount of 21 nt small RNAs, equally distributed between sense and antisense (Figure 4G,H,I), suggesting that BX2 transcripts are processed into siRNAs. These results confirm that AGO1 is not a piRNA producing locus (as shown in Figure 2—figure supplement 2) and showed that, at 29°C, AGO1 is still not producing small RNAs (Figure 4J). These data indicate that 21 nt small RNA production was restricted to BX2 sequences.
Heat conversion requires a transcribed homologous sequence in trans
Quantitative RT-PCR experiments described above were carried out on flies bearing only the BX2 locus while all conversion experiments at 29°C were done with flies bearing the BX2 and the P(TARGET)GS locus. Interestingly, the amount of P(TARGET)GS transcripts is affected by temperature but in the opposite way to BX2, as less transcripts were measured at 29°C compared to 25°C (Figure 4—figure supplement 1C). We next asked whether the P(TARGET)GS transgene could participate in the conversion process of BX2. For this, 'heat-activated-conversion' experiments of BX2 were done in flies not carrying the P(TARGET)GS (Figure 3—figure supplement 1). To assess the BX2 epigenetic state of the G1 raised at 29°C, 157 G1 females were individually crossed at 25°C with males harboring the P(TARGET)GS transgene. Among the 1137 G2 females analyzed, only one female presented partial repression of the ß-Galactosidase expression and none presented complete repression (Figure 3—figure supplement 1). If we compare these results with those obtained with the BX2, P(TARGET)GS lines (Figure 3), the difference was highly significant (p=8.5×10−6, homogeneity χ2 = 23.35 with 2 degrees of freedom, Supplementary file 8). To further validate the requirement of the euchromatic homologous transgene P(TARGET)GS in establishing the temperature-dependent BX2 conversion, we generated eight independent lines in which BX2 was recombined into the same P(TARGET)GS genetic background but without the P(TARGET)GS transgene. After 30 generations at 29°C, no female showing ß-Galactosidase repression was observed (Supplementary file 9). A homogeneity χ2 test comparing, at G13, the repression occurrence in BX2, P(TARGET)GS lines (31/161, Supplementary file 1) and in recombined BX2 lines (0/975, Supplementary file 9) is highly significant (p=7.04×10−44, homogeneity χ2 = 192.9 with 1 degree of freedom), arguing against a background effect of the P(TARGET)GS line in the conversion phenomenon. Additionally, we reproduced the experiment with an euchromatic P(TARGET) expressed only in the germline and referred to hereinafter as 'P(TARGET)G' (Figure 1—figure supplement 1A–B). In the presence of P(TARGET)G, BX2 was converted at 29°C at a rate comparable with that observed with P(TARGET)GS (Supplementary file 10). These data show that BX2 conversion by temperature cannot be attributed to any specificity linked to the P(TARGET)GS insertion. To know if the transcription of the P(TARGET)GS (or of the P(TARGET)G) is required, we reproduced the same experiment with another euchromatic transgene that is not expressed in the germline but in the somatic cells surrounding the germ cells and therefore referred to hereinafter as 'P(TARGET)S' (Figure 1—figure supplement 1A–B). In the presence of P(TARGET)S, BX2 was not converted at 29°C (0/784, Figure 3—figure supplement 2). A homogeneity χ2 test comparing these results with those obtained with P(TARGET)GS considering only the complete BX2ON G2 females (17/1464 females, Figure 3) is significant (p=0.0055, homogeneity χ2 = 7.7 with 1 degree of freedom). We conclude that the transcription of a reporter transgene sharing homologous sequences (i.e. lacZ) with BX2 is required in the germline for BX2 conversion at 29°C.
To summarize, in the absence of P(TARGET) sequences, at 29°C BX2OFF produces an elevated number of antisense transcripts, no piRNAs and is unable to be converted to BX2ON. In contrast, when a P(TARGET) is present and transcribed in the germline, BX2 conversion and piRNA production are observed at 29°C. Although much of the mechanistic aspects of BX2 conversion remain unknown, these findings lead us to propose that, at 29°C, double strand RNA (dsRNA) made of the excess of BX2 antisense transcripts and the sense P(TARGET) transcripts could be a prerequisite for the production of de novo piRNAs and the conversion of BX2 into an active piRNA cluster (see recapitulative model Figure 5).

Model of BX2 activation at 29°C.
(A) At 25°C, a low bidirectional transcription of the BX2 cluster leads to a small production of 21 nt RNAs. BX2OFF is stable at 25°C, no conversion event is observed and the lacZ reporter gene from P(TARGET)GS remains active throughout generations. (B) At 29°C, a specific increase in antisense transcription occurs, presumably due to a higher activity of the promoter of the AGO1 gene (orange box). This excess of BX2 antisense RNA could interact with sense P(TARGET)GS transcripts to produce double stranded RNA. Through a yet unknown mechanism, such dsRNA could lead to the formation of de novo BX2 piRNAs. These piRNAs could in turn trigger the conversion of BX2OFF into an active piRNA cluster, a phenomenon observed based on the repression of the lacZ reporter gene of P(TARGET)GS. The BX2 conversion is a rare event (≈2% per generation) but once achieved, BX2ON remains active throughout generations due to the maternal inheritance of homologous piRNAs and the paramutation of the paternal BX2OFF allele.
Discussion
Here, we report on the heritable establishment of a new piRNA cluster associated with silencing properties induced by high temperature during development. The epigenetic response to heat exposure has been studied in several model species: in Arabidopsis for instance, increasing temperature induces transcriptional activation of repetitive elements (Ito et al., 2011; Pecinka et al., 2010; Tittel-Elmer et al., 2010). Whether these changes involve chromatin modifications is not clear but none of these modifications have been found to be heritable through generations except in mutants for siRNA biogenesis where high frequency of new TE insertions was observed in the progeny of stressed plants (Ito et al., 2011). In animals, response to heat can result in modification of DNA methylation at specific loci in reef building coral (Dimond and Roberts, 2016), chicken (Yossifoff et al., 2008) and wild guinea pigs (Weyrich et al., 2016). In the latter, modifications affecting ≈50 genes are inherited in G1 progeny (Weyrich et al., 2016). The mechanisms of this heritability, however, are not yet understood. In Drosophila, heat-shock treatment of 0–2 hr embryos for one hour at 37°C or subjecting flies to osmotic stress induce phosphorylation of dATF-2 and its release from heterochromatin (Seong et al., 2011). This defective chromatin state is maintained for several generations before returning to the original state. We have tested if such stresses were able to convert BX2OFF into BX2ON in one generation but neither heat-shock nor osmotic stress induces BX2 conversion (Supplementary file 11), suggesting that BX2 activation does not depend on dATF-2. In a more recent paper, Fast et al. (Fast et al., 2017) found less piRNAs at 29°C than at 18°C. However, RNAseq analyses of differentially expressed genes involved in the piRNA pathway were not conclusive, as some piRNA genes (ago3, aub, zuc, armi) were more expressed at 29°C while others (shu, hsp83, Yb) were less expressed as compared to the levels at 18°C (Fast et al., 2017). Overall, the enhancement of the piRNA ping-pong amplification loop observed at 29°C was attributed to RNA secondary structures, because of a lack of specificity for any particular class of TE (Fast et al., 2017). Furthermore, in contrast to our RT-qPCR results obtained at 25°C and 29°C (Figure 4D and Figure 4—figure supplement 1A–B), AGO1 was not differentially expressed between 18°C and 29°C (Fast et al., 2017). This difference can be explained, because, in our experiments, we checked for specific spliced transcripts of AGO1 (RA, RC and RD) that originate upstream the BX2 insertion point (Figure 4C). In agreement with Fast et al., no statistical difference in the global amount of AGO1 transcripts between 25°C and 29°C is observed using primers located downstream the BX2 insertion point (Figure 4—figure supplement 1D). This suggests that the AGO1 promoter located downstream the BX2 insertion point, and responsible for the production of the RB isoform (see Figure 4), is not sensitive to temperature. Taken together, these observations show that temperature modification might induce epigenetic changes in several species but the underlying mechanisms remain largely unsolved.
Whole genome comparison of small RNA sequencing between BX2OFF and newly BX2ON heat-converted flies (BX2Θ) did not reveal additional regions stably converted for piRNA production (Figure 2—figure supplement 3). This suggests that no other loci are metastable for piRNA production, that is all potential piRNA clusters are already active at 25°C. This observation raises the question of what makes BX2 locus competent for piRNA activation at high temperature. The BX2 locus is the result of successive induced transpositions of P(lacW) used to screen for white-variegating phenotype (Dorer and Henikoff, 1994). Thus, BX2 resembles natural TE clusters where TEs have the capacity to transpose into each other assembling structures named nested TE, as described in numerous genomes (Gao et al., 2012; Liu et al., 2007; Zanni et al., 2013). Some nested TE loci might have the capacity to be activated and respond to a new TE invasion. In Drosophila, BX2-like tandemly inserted transgenes were shown to be new sites of HP1 enrichment in larval salivary glands, emphasizing a heterochromatic structure at the BX2 locus in somatic cells (Fanti et al., 1998), and to cause pairing-dependent silencing (Dorer and Henikoff, 1997). When tested for repressing capacities, however, this strain is inactive for BX2 piRNA production (de Vanssay et al., 2012; Josse et al., 2008). We have shown that maternally inherited P(lacW) piRNAs are able to paramutate with complete and stable penetrance from an inactive BX2 locus into an active locus for piRNA production (de Vanssay et al., 2012). The paramutated BX2 locus appeared to be a genuine piRNA cluster since it is sensitive to a number of factors known to be involved in piRNA biogenesis such as aub, rhi, cuff, zuc (Hermant et al., 2015) and moonshiner (Figure 3—figure supplement 3). The number of transgene copies appears to be crucial in the process since smaller number of transgenes results in less somatic heterochromatinization (Dorer and Henikoff, 1994; Fanti et al., 1998), less pairing-dependent silencing (Dorer and Henikoff, 1997) and unstable paramutation (de Vanssay et al., 2012). Taken together, these data suggest that the heterochromatic structure of a cluster precedes piRNA production. This is supported by our ChIP experiments showing that H3K9me3 levels on BX2OFF are slightly below the H3K9me3 level of piRNA producing states (BX2ON and BX2Θ, Figure 2D). The same observation can be made for Rhino (Figure 2E), suggesting that Rhino may be already present on the BX2OFF locus but below the threshold required for piRNA production as suggested by Akulenko et al. (2018). Thus, a locus made of repeated sequences and being likely heterochromatic (H3K9me3, Rhino) is a necessary but not sufficient condition to specify an active piRNA cluster.
In the germline, piRNA clusters produce piRNAs from both strands and it was recently shown that, in most cases, transcription initiates within clusters on both strands through the interaction of Rhino and Moonshiner (Andersen et al., 2017). In few cases, however, piRNA cluster transcription may take advantage of the read-through from a flanking promoter (Andersen et al., 2017). Zhang et al. (Zhang et al., 2014) have shown that tethering Rhino onto a transgene leads to its repression but the production of piRNA depends on the presence of another transgene producing antisense RNA. Moreover, in the context of the Pld promoter deletion, a gene flanking the 42AB piRNA cluster, flies can produce Pld piRNAs only if a Pld cDNA is expressed in trans (Andersen et al., 2017). From all of these observations, a model emerges predicting that simultaneous production of sense and antisense RNA is a shared requirement for piRNA production. However, even if BX2OFF is transcribed on both strands, without additional signals, it still remains inactive for piRNA production.
In addition to having a number of heterochromatic repeats and a double stranded transcription, the production of de novo piRNAs from BX2 requires a triggering signal. From our experiments, BX2 conversion relies on the simultaneous increase of both sense and antisense RNAs. An active role of euchromatic copies in the establishment of new piRNA clusters by high temperature appears to be consistent with what would naturally happen during the invasion of a naive genome by new TEs or when chromosomal breakages occur leading to the loss of piRNA cluster loci (Asif-Laidin et al., 2017). At first, uncontrolled euchromatic TE transposition takes place before the establishment of repression. Such repression would occur after a copy integrates into a preexisting piRNA cluster or by the generation of a new cluster made by successive insertion of nested copies. Consequently, clusters of elements cannot exist without transcriptionally active euchromatic copies. The increase of germline antisense transcripts upon stress or environmental factors, depending on the neighboring genomic environment, and the concomitant presence of numerous sense transcripts from euchromatic active copies, appear to be the starting signals for new piRNA production. These piRNAs can then be inherited at the next generation where they will stably paramutate the corresponding DNA locus with repetitive nature. At that time, the triggering signal is no longer necessary since BX2 remains activated once flies get back at 25°C. Future generations thus remember what was once considered a threat only through the legacy of maternal piRNAs.
Materials and methods
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Drosophila melanogaster) | AGO1 | NA | FLYB: FBgn0262739 | |
Gene (D. melanogaster) | RpL32 | NA | FLYB: FBgn0002626 | |
Gene (D. melanogaster) | eEF5 | NA | FLYB: FBgn0285952 | |
Gene (D. melanogaster) | Moonshiner | NA | FLYB: FBgn0030373 | |
Strain, strain background (D. melanogaster) | w1118 | Laboratory Stock | FLYB: FBal0018186 | |
Strain, strain background (D. melanogaster) | BX2 | Dorer and Henikoff, 1994 PMID:8020105 | FLYB: FBti0016766 | |
Strain, strain background (D. melanogaster) | P(TARGET)GS | Bloomington Drosophila Stock Center | FLYB: FBst0011039 | also called P-1039 |
Strain, strain background (D. melanogaster) | P(TARGET)G | Bloomington Drosophila Stock Center | FLYB: FBti0003435 | also called BQ16 |
Strain, strain background (D. melanogaster) | P(TARGET)S | Bloomington Drosophila Stock Center | FLYB: FBti0003418 | also called BA37 |
Strain, strain background (D. melanogaster) | nosGAL4 | Bloomington Drosophila Stock Center | FLYB: FBti0131635, RRID:BDSC_32180 | |
Genetic reagent (D. melanogaster) | P(lacW) | PMID: 2558049 | FLYB: FBtp0000204 | |
Genetic reagent (D. melanogaster) | P(PZ) | Mlodzik and Hiromi, 1992 doi: 10.1016/B978-0-12-185267-2.50030–1 | FLYB: FBtp0000210 | |
Genetic reagent (D. melanogaster) | P(A92) | PMID: 2827169 | FLYB: FBtp0000154 | |
Genetic reagent (D. melanogaster) | Moon shRNA PA61 | Andersen et al., 2017 doi:10.1038/nature23482 | Dr. Julius Brennecke (Institute of Molecular Biotechnology, Vienna) | |
Genetic reagent (D. melanogaster) | Moon shRNA PA62 | Andersen et al., 2017 doi:10.1038/nature23482 | Dr. Julius Brennecke (Institute of Molecular Biotechnology, Vienna) | |
Antibody | Mouse IgG polyclonal antibody | Merck (ex-Millipore) | Cat# 12-371B, RRID:AB_2617156 | |
Antibody | Rabbit polyclonal antibody against H3K9me3 | Merck (ex-Millipore) | Cat# 07–442 | |
Antibody | Rabbit polyclonal antibody against Rhino | PMID: 19732946 | Dr. William Theurkauf (University of Massachusetts Medical School, Worcester) | |
Sequence-based reagent | RT-qPCR primers | Sigma-Aldrich | ||
Sequence-based reagent | RT-qPCR primers | Eurogentech | ||
Commercial assay or kit | RNeasy kit | Qiagen | Cat# 74104 | |
Commercial assay or kit | Illumina TruSeq Small RNA library preparation kits | Fasteris | http://www.fasteris.com | |
Commercial assay or kit | Revertaid RT | Thermo Scientific | EP0442 | |
Commercial assay or kit | Random Hexamers | Invitrogen | N8080127 | |
Commercial assay or kit | DNaseI (Rnase free) | New Englands Biolabs | M0303S | |
Commercial assay or kit | dNTPs solution Mix | New Englands Biolabs | N0447S | |
Commercial assay or kit | Ribolock RNA inhibitor | Thermo Scientific | EO0381 | |
Commercial assay or kit | Ssofast Evagreen Supermix | Biorad | Cat# 172–5204 | |
Commercial assay or kit | qPCR kit | Roche | Cat# 04887352001 | |
Chemical compound, drug | TRIzol | Invitrogen | Cat# 15596026 | |
Chemical compound, drug | Chloroform | Carlo Erba Reagents | Cat# 438601 | |
Chemical compound, drug | Chloroform | Sigma-Aldrich | C2432 | |
Chemical compound, drug | Ethanol (EtOH) | Merck millipore | Cat# 100983 | |
Chemical compound, drug | Ethanol (EtOH) | Honeywell | Cat# 32221 | |
Chemical compound, drug | Glycerol | VWR AnalaR NORMAPUR | Cat# 24388.295 | |
Chemical compound, drug | Glutaraldehyde | Sigma Aldrich | G-5882 | |
Chemical compound, drug | Potassium hexacyanoferrate(III) | Sigma Aldrich | P3667 | |
Chemical compound, drug | Potassium hexacyanoferrate(II) trihydrate | Sigma Aldrich | P3289 | |
Chemical compound, drug | X-Gal | Dutscher | Cat# 895014 | |
Chemical compound, drug | NaCl | VWR AnalaR NORMAPUR | Cat# 27810.295 | |
Chemical compound, drug | NaCl | Sigma-Aldrich | Cat# 31432 | |
Chemical compound, drug | Formaldehyde | Sigma-Aldrich | Cat# 252549 | |
Chemical compound, drug | Schneider Medium | Gibco | Cat# 21720–024 | |
Chemical compound, drug | Insulin | Sigma-Aldrich | I4011 | |
Chemical compound, drug | PBS | Ambion | AM9625 | |
Chemical compound, drug | Triton | Sigma-Aldrich | T8787 | |
Chemical compound, drug | KCl | Ambion | AM9640G | |
Chemical compound, drug | HEPES | Fisher Scientific | BP299 | |
Chemical compound, drug | IPEGAL | Sigma-Aldrich | Cat# 18896 | |
Chemical compound, drug | DTT | Fisher Scientific | R0861 | |
Chemical compound, drug | Na Butyrate | Sigma-Aldrich | Cat# 07596 | |
Chemical compound, drug | EDTA free protease inhibitor | Roche | Cat# 04693159001 | |
Chemical compound, drug | N lauryl sarkosyl | Sigma-Aldrich | L-5125 | |
Chemical compound, drug | BSA | Fisher Scientific | BP9703 | |
Chemical compound, drug | SDS 20% | Euromedex | EU0660-B | |
Chemical compound, drug | Tris HCl | Invitrogen | Cat# 15504–020 | |
Chemical compound, drug | Dynabeads A | Invitrogen | 10002D | |
Chemical compound, drug | Glycine | Sigma-Aldrich | G8898 | |
Chemical compound, drug | Isopropanol | VWR | Cat# 20842.298 | |
Software, algorithm | Galaxy Server | ARTBIO | https://mississippi.snv.jussieu.fr/ | |
Software, algorithm | Weblogo | Crooks et al., 2004 doi:10.1101/gr.849004 |
Transgenes and strains
Request a detailed protocolAll transgenes are in the w1118 background. The BX2 line carries seven P-lacZ-white transgenes, (P(lacW), FBtp0000204) inserted in tandem and in the same orientation at cytological site 50C on the second chromosome (Dorer and Henikoff, 1994). The transgene insertion site is located in an intron of the AGO1 gene (de Vanssay et al., 2012). Homozygous individuals are rare and sterile and the stock is maintained in heterozygous state with a Cy-marked balancer chromosome. ß-Galactosidase activity from these transgenes cannot be detected in the germline. P(TARGET)GS corresponds to P(PZ) (FBtp0000210), a P-lacZ-rosy enhancer-trap transgene inserted into the eEF5 gene at 60B7 and expressing ß-Galactosidase in the germline and somatic cells of the female gonads (Bloomington stock number 11039 (FBst0011039). Homozygous flies are not viable and the stock is maintained over a Cy-marked balancer chromosome. P(A92) (FBtp0000154) is another P-lacZ-rosy enhancer-trap transgene that has been used in this study: P(TARGET)G corresponds to BQ16 (FBti0003435) expressing lacZ only in the germline and P(TARGET)S corresponds to BA37 (FBti0003418) expressing lacZ only in the somatic follicle cells that surround the nurse cells. In both lines, homozygous flies are viable. The nosGAL4 transgene used is from the w[*]; PBac{w[+mW.hs]=GreenEye.nosGAL4}Dmel6 line (FBti0131635). Modified miRNA against moonshiner (lines PA61 and PA62) were a kind gift from Julius Brennecke (Andersen et al., 2017). Additional information about stocks are available at Flybase: ‘http://flybase.bio.indiana.edu/".
Thermic and osmotic treatments
Request a detailed protocolSince maintaining flies at high temperature (29°C) decreases viability, we used the following procedure at each generation: fertilized adult females (G0) were allowed to lay eggs for three days at 25°C on standard cornmeal medium. Adults were then discarded or tested for ovarian ß-Galactosidase expression. Vials containing progeny were transferred at 29°C for the rest of the development until complete emergence of G1 adults. Young adults were then transferred into a new vial where they were allowed to lay eggs for three days at 25°C. For heat-shock treatment, embryos (0–2 hr) were incubated at 37°C for 1 hr as described in Seong et al. (2011) and then transferred at 25°C until adult emergence. For osmotic treatments, culturing flies on 300 mM NaCl, as described in Seong et al. (2011), leads to either a large increased time of development or lethality and did not allow us to perform conversion measurements. Accordingly, flies were incubated on 150 mM NaCl for one generation before dissection and ß-Galactosidase staining.
ß-Galactosidase staining
Request a detailed protocolOvarian lacZ expression assays were carried out using X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) overnight staining at 37°C as previously described (Lemaitre et al., 1993), except that ovaries were fixed afterwards for 10 min. After mounting in glycerol/ethanol (50/50), the germline lacZ repression was then calculated by dividing the number of repressed egg chambers by the total number of egg chambers. Most of the time, the total number of egg chambers was estimated by multiplying the number of mounted ovaries by 60, corresponding to an average of three to four egg chambers per ovariole and 16 to 18 ovarioles per ovary. Images were acquired with an Axio-ApoTome (Zeiss) and ZEN2 software.
Fly dissection and RNA extraction
Request a detailed protocolFor each genotype tested, 20 pairs of ovaries were manually dissected in 1X PBS. For small RNA sequencing, total RNA was extracted using TRIzol (Life Technologies) as described in the reagent manual (http://tools.lifetechnologies.com/content/sfs/manuals/trizol_reagent.pdf). For the RNA precipitation step, 100% ethanol was used instead of isopropanol. For RT-qPCR experiments, total RNA was extracted using TRIzol for BX2 and w1118 females or RNeasy kit (Qiagen) for P(TARGET) females. Up to six biological replicates were used for each genotype.
Small RNA sequencing analyses
Request a detailed protocolA small RNA fraction of 18 nt to 30 nt in length was obtained following separation of total RNA extracted from dissected ovaries on a denaturing polyacrylamide gel. This fraction was used to generate multiplexed libraries with Illumina TruSeq Small RNA library preparation kits (RS-200–0012, RS200-0024, RS-200–036 or RS-200–048) at Fasteris (http://www.fasteris.com). A house protocol based on TruSeq, which reduces 2S RNA (30 nt) contamination in the final library, was performed. Libraries were sequenced using Illumina HiSeq 2000 and 2500. Sequence reads in fastq format were trimmed from the adapter sequence 5’-TGGAATTCTCGGGTGCCAAG-3’ and matched to the D. melanogaster genome release 5.49 using Bowtie (Langmead et al., 2009). Only 18–29 nt reads matching the reference sequences with 0 or one mismatch were retained for subsequent analyses. For global annotation of the libraries (Supplementary files 5 and 7), we used the release 5.49 of fasta reference files available in Flybase, including transposon sequences (dmel-all-transposon_r5.49.fasta) and the release 20 of miRNA sequences from miRBase (http://www.mirbase.org/).
Sequence length distributions, small RNA mapping and small RNA overlap signatures were generated from bowtie alignments using Python and R (http://www.r- project.org/) scripts, which were wrapped and run in Galaxy instance publicly from ARTbio platform available at http://mississippi.fr. Tools and workflows used in this study may be downloaded from this Galaxy instance. For library comparisons, read counts were normalized to one million miRNA (Supplementary files 5 and 7). A second normalization, performed using the total number of small RNAs matching the D. melanogaster genome (release 5.49), gave similar results (Supplementary files 5 and 7). For small RNA mapping (Figures 2 and 4, Figure 2—figure supplements 1 and 2), we took into account only 23–29 nt RNA reads that uniquely aligned to reference sequences. Logos were calculated using Weblogo (Crooks et al., 2004) from 3' trimmed reads (23 nt long) matching either P(lacW) (Figure 2B) or 42AB (Figure 2—figure supplement 1B). The percentage of reads containing a ‘U’ at the first position was calculated with all 23–29 nt RNA matching the reference sequence (BX2 transgene in Figure 2B and 42AB in Figure 2—figure supplement 1B). Distributions of piRNA overlaps (ping-pong signatures, Figure 2C and Figure 2—figure supplement 1C) were computed as first described in Klattenhoff et al. (2009) and detailed in Antoniewski (2014). Thus, for each sequencing dataset, we collected all of the 23–29 nt RNA reads matching P(lacW) or the 42AB locus whose 5’ ends overlapped with another 23–29 nt RNA read on the opposite strand. Then, for each possible overlap of 1 to 29 nt, the number of read pairs was counted. To plot the overlap signatures, a z-score was calculated by computing, for each overlap of 1 to i nucleotides, the number O(i) of read pairs and converting the value using the formula z(i) = (O(i)-mean(O))/standard deviation (O). The percentage of reads containing a ‘A’ at the tenth position was calculated within the paired 23–29 nt RNA matching the reference sequence as described in de Vanssay et al. (2012) (BX2 transgene in Figure 2C and 42AB in Figure 2—figure supplement 1B). GRH49 (BX2*) was previously analyzed in Hermant et al. (2015). Small RNA sequences and project have been deposited at the GEO under accession number GSE116122.
ChIP experiments
Request a detailed protocol100 ovaries were dissected in Schneider medium supplemented with insulin at room temperature. Cross-linking was performed for 10 min at room temperature in 1X PBS 1% formaldehyde (Sigma). The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 mM in PBS 0.1% Triton and incubating 5 min on ice. The cross-linked ovaries were washed with 1 ml of PBS 0.1% Triton and crushed in a dounce A potter 20 times. Then a centrifugation at 400 g for 1 min was performed. The pellet was suspended with 1 ml of cell lysis buffer (KCl 0.085 M, HEPES 5 mM, IGEPAL 0.5%, DTT 0.5 mM, Na butyrate 10 mM, 0.01 M EDTA free protease inhibitor cocktail Roche) and crushed in a dounce B potter 20 times, then 2 mL of cell lysis buffer were added. Centrifugation at 2000 g for 5 min was performed and the pellet was resuspended in 0.5 mL nucleus buffer (HEPES 0.05 M, EDTA 0.01 M, N lauryl Sarcosyl 0.5%, Na butyrate 0.01 M EDTA free protease inhibitor cocktail Roche) and incubated 15 min in a cold room on a rotator. Sonication was performed with Bioruptor (Diagenode) set to high power for 10 cycles (15 s on and 15 s off). A centrifugation was performed 15 min at 16000 g at 4°C. Five µg of chromatin was used for each immunoprecipitation. A preclear of 4 hr was performed with 25 µL of dynabead Protein A. The immunoprecipitation reaction was performed with 50 µL of dynabead Protein A coated with 5 µg of antibodies (H3K9me3 polyclonal antibody C1540030 diagenode or Normal Mouse IgG polyclonal antibody 12–371 Millipore), or 20 µL of serum for the Rhino antibody (kindly provided by Dr W. Theurkauf) over night in the cold room on a rotator. Three washes of 10 min in a high salt buffer (Tris HCl pH 7.5 0.05 M, NaCl 0.5 M, Triton 0.25%, IGEPAL 0.5%, BSA 0.5%, EDTA 5 mM) were performed and the elution of chromatin was performed 30 min with 500 µL of elution buffer (Tris pH 7.5 0.05 M, NaCl 0.05 M, EDTA 5 mM, SDS 1%); RNase treatment was omitted; H3K9me3 and Rhino ChIP were respectively done on 5 and 4 independent biological samples followed by qPCR (Roche light Cycler) on each sample. Values were normalized to respective inputs and to two genomic regions known to be enriched in H3K9me3 and Rhino (42AB): region 1 (chr2R: 6449409–6449518) and region 2 (chr2R: 6288809–6288940). An unpaired t-test was used to calculate significance of the differences (p<0.05). Error bars represent the standard deviation.
RT-qPCR experiments
Request a detailed protocolFor each sample, 10 µg of total RNA was treated with DNase (Fermentas). For classical RT-qPCR experiments, 1 µg of DNase-treated RNA was used for reverse transcription using random hexamer primers (Fermentas). Real-time qPCR was performed on triplicates of each sample. RpL32 was used as reference. The same series of dilution of a mix of different RT preparations was used to normalize the quantity of transcripts in all RT preparations leading to standard quantity (Sq) values. Variations between technical triplicates was very low when compared to variations between biological replicates. The mean of the three technical replicates was then systematically used (meanSq). For each biological sample, we calculated the ratio meanSq(gene)/meanSq(RpL32) to normalize the transcript quantity. Then, the mean of each sample ratio was used to compare the two conditions. For sense-specific RT-qPCR experiments, three reverse transcription were performed using 1 μg of DNase-treated RNA (Fermentas): first without primer (control RT), second with a lacZ sense primer (antisense transcript specific RT) and third with a lacZ antisense primer (sense transcript specific RT). qPCR was then performed on technical triplicates of each RT using a primer pair specific for lacZ sequence. A series of dilutions - ranging from 50 × 10−15 g.µl−1 to 0.08 × 10−15 g.µl−1 - of a plasmid containing the P(lacW) transgene was used as reference to normalize the quantity of lacZ transcripts (Sq values). The number of molecules was estimated by considering that P(lacW) is 11191 bp long and that the average weight of a base pair is 650 g/mol. Using Avogadro's number, the number of copies was estimated as equal to the dsDNA amount (in g) times 6.022 × 1023 divided by the dsDNA length times 650. For example, 50 × 10−15 g corresponds to approximately 4139 molecules. Variations between technical triplicates were very low when compared to variations between biological replicates. The mean of the three technical replicates was then systematically used (meanSq). The measure of the quantity of transcripts (sense or antisense) for a biological sample was then calculated as the (meanSq(sense or antisense specific) - meanSq(control)). This allowed us to eliminate background noise due to unspecific RT amplification for both sense or antisense without specific primer. The mean of each sample ratio was used to compare the two conditions.
Primer sequences
Request a detailed protocolFor classical RT-qPCR experiments, primers used were for w (ChIP experiment): 5'-GTCAATGTCCGCCTTCAGTT-3' and 5'-GGAGTTTTGGCACAGCACTT-3', these primers are specific of the P(lacW) transgene in a w1118 background; for 42AB regions, 5'-TGGAGTTTGGTGCAGAAGC-3' and 5'-AGCCGTGCTTTATGCTTTACT-3' (region 1) and 5'-AAGACCCAATTTTTGCGTCGC-3' and 5'-CAAGGATAGGGATTTGGTCC-3' (region 2); for RpL32: 5’-CCGCTTCAAGGGACAGTATCTG-3’ and 5’-ATCTCGCCGCAGTAAACGC-3’; for lacZ: 5’-GAGAATCCGACGGGTTGTTA-3’ and 5’-AAATTCAGACGGCAAACGAC-3’; for eEF5: 5’-TAACATGGATGTGCCCAATG-3’ and 5’-AACGCAATTGTTCACCCAAT-3’; for AGO1, primers have been chosen in order to detect spliced forms of transcripts coming upstream of the insertion point of BX2 and encoding AGO1-RA, -RD and -RC isoforms (Figure 4C): 5’-GGATCTCCAGATGACCTCCA-3’ and 5’-GGACACTTGTCCGGCTGTAT-3’. For detecting all AGO1 transcripts isoforms, including the AGO1-RB isoform that originates from a promoter located downstream the BX2 insertion point: 5'-ATGAGCCGGTCATCTTTTTG-3' and 5'-GGCAATCGATGGTTTCTTGT-3'. For sense-specific RT-qPCR experiments, we used specific primers during the reverse transcription step: 5’-AGTACGAAATGCGTCGTTTAGAGC-3’ for detection of antisense lacZ transcripts and 5’-AATGCGCTCAGGTCAAATTC-3’ for detection of sense lacZ transcripts.
Data availability
Small RNA sequences and project have been deposited at the GEO under accession number GSE116122.
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NCBI Gene Expression OmnibusID GSE116122. Environmentally-induced epigenetic conversion of a piRNA cluster.
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Decision letter
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Timothy W NilsenReviewing Editor; Case Western Reserve University, United States
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James L ManleySenior Editor; Columbia University, United States
In the interests of transparency, eLife includes the editorial decision letter, peer reviews, and accompanying author responses.
[Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed.]
Thank you for submitting your article "Environmentally-induced epigenetic conversion of a piRNA cluster" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The reviewers have opted to remain anonymous.
We have included the separate reviews below for your consideration. We would recommend that you first respond to outline your replies to the reviewers and what revisions you intend to make, so that we can offer further guidance. We note that all the reviewers felt that more mechanistic insight would be desirable. If you have any questions, please do not hesitate to contact us.
Separate reviews (please respond to each point):
Reviewer #1:
The authors report how high temperature (environment) can induce a DNA stretch to become a piRNA-producing locus in female flies.
Findings
1) Some (25% of the females studied) flies within a population are randomly induced to turn an inactive piece of DNA into an active locus that generates piRNAs, when grown at higher temperature (29oC instead of normal 25oC). This is monitored by the clever use of a b-gal reporter assay. This induction increases with generation time.
2) Such temperature-activated clusters are identical to those activated by maternally-inherited piRNAs. They present similar piRNAs produced and are decorated with the expected chromatin and piRNA-factor marks.
3) The higher temperature increased antisense RNA production from the previously-silent locus and this was channelled into the siRNA pathway and did not lead to increased piRNA production.
4) The presence of the complementary target RNA (expressed from a euchromatic locus) was essential for the high temperature-triggered piRNA generation.
Concerns
1) Abstract, Second sentence: There is no data to show that piRNA clusters are maternally defined except in flies. In mice germ cells are induced from the somatic lineage at embryonic day 7.5 and there is no possibility of maternal inheritance playing a role. If the authors want to keep this sentence, specify it is for flies.
2) Abstract, third sentence: The sentence sounds very dramatic as it means that higher temperature alone was sufficient to convert the P-transgene locus into a piRNA cluster. Perhaps the features of the genomic region (having P-transgenes), location, chromatin status etc are responsible. I would rephrase it. It is misleading as although temperature might be the trigger, this locus might already be primed and ready to go.
3) The authors make a very striking finding regarding high temperature-induced piRNA production from a locus, and link it to presence of both sense and antisense transcripts are necessary for this. This still leaves open the question how the locus was made into piRNAs. There is no explanation for this.
4) It would be good to have the actual sequence information for the different transgenes and the b-gal reporter, and the actual transcripts present in these flies. One is left in the dark as to the level of complementarity that exists between them when they are referred to as sense and antisense transcripts.
In conclusion, the observations made are striking and extremely interesting, but perhaps a bit more of molecular insights might be useful to appreciate the observations made.
Reviewer #2:
Piwi-interacting RNAs (piRNAs) produced from piRNA clusters repress transposable elements in animal gonads. It has been shown that use of piRNA clusters is determined by maternally-deposited piRNAs. In this study, Casier et al. however showed that raising flies at high temperature (29oC) resulted in ectopic expression of piRNAs from the BX2 transgene locus in flies that had no maternally-deposited, BX2-derived piRNAs. The BX2 transgene consists of P5’, lacZ, white, a part of pBR322 (pBR) and P3’, and does not express piRNAs at normal temperature (25oC). The authors claimed that this was the first case showing that heat treatment can activate de novo piRNA production from the BX2 locus (i.e., non-piRNA cluster) in a manner independent of maternally-deposited piRNAs.
The authors' findings also include:
1) Activation of de novo piRNA production by heat treatment was restricted to the BX2 locus.
2) BX2-derived piRNAs produced upon heat treatment showed molecular properties similar to those of BX2-derived piRNAs expressed in a manner dependent of maternally-deposited piRNAs.
3) Ectopically expressed lacZ-piRNAs from the BX2 locus were capable of silencing βGal over generations.
4) The βGal silencing effect was low at the first generation but gradually increased over generations.
5) Rhino and H3K9me3 accumulated at the BX2 locus upon heat treatment. However, this may not be the cause to ectopic expression of BX2-derived piRNAs, as the authors claimed that BX2OFF and BX2ON produced anyway similar amounts of sense and antisense transcripts (subsection “Epigenetic conversion at 29°C occurs at a low rate from the first generation”).
6) An increase of transcription from the AGO1 promoter at 29oC could lead to an increase of BX2 antisense RNA transcription. The BX2 transgene was inserted in the AGO1 gene in a convergent transcription manner.
7) Activation of BX2-derived piRNA production by heat treatment required a homologous sequence.
I found the authors' finding that heat treatment induces de novo piRNA production from non-piRNA clusters potentially interesting. However, the mechanistic insights remain vague. The authors claimed that homologous sequence was required for the induction. However, to claim this, much more supportive data should be provided. The authors also found that H3K9me3 and Rhino started to accumulate at the BX2 locus upon heat treatment. However, the meaning of the accumulation remains unclear. My other concerns were indicated below. I hope that this reviewer's concerns might be helpful for the authors to revise the manuscript.
Concerns and suggestions from this reviewer:
1) The authors proposed that the interaction between the excess of BX2 antisense transcripts and the sense P(TARGET) transcripts is a prerequisite for the production of de novo piRNAs (subsection “Heat conversion requires a homologous sequence in trans”). To confirm this, I recommend the authors conducting experiments using heat treated BX2OFF that do not have lacZ at the BX2 loci but contains P(TARGET).
2) The level of AGO1 RNA transcripts should be examined in BX2OFF line containing P(TARGET). I do not understand why the authors used the line without P(TARGET) in this particular experiment.
3) Figure 4B: Upon heat treatment, the expression level of BX2 antisense transcripts was raised. Was this phenomenon related to Rhino and H3K9me3 accumulation observed in Figure 2DE? Experiments in Figure 2 and Figure 4 should be performed using the same fly lines.
4) Is the BX2 transgene in the BX2OFF line containing P(TARGET) also inserted in the AGO1 gene? This should be examined. If this were the case, the authors better examine whether piRNAs were derived from the AGO1 gene.
5) The authors should test experimentally whether BX2-derived piRNAs are loaded into PIWI proteins. This is very important.
6) Figure 2B: Explain why BX2-derived piRNAs did not show 10A bias. The data shown in Figure 2C supported the idea that BX2-derived piRNAs were products of the ping-pong pathway. Then, they should have shown 10A bias, but in reality they did not.
7) Figure 2DE: The authors should examine genome-widely where in the genome H3K9me3 and Rhino accumulated upon heat treatment. It is hard to imagine that H3K9me3 and Rhino accumulation only occurred at the BX2 locus upon heat treatment.
8) The authors should examine whether H3K9me3 and Rhino accumulated at the BX2 locus in BX2OFF line without P(TARGET) upon heat treatment.
9) This manuscript should be edited thoroughly by a native English speaker.
10) Table 1 should be removed from the main text.
Reviewer #3:
This work takes advantage of a clever system using suppression of a lacZ reporter gene as a read out indicating the production of piRNAs from the BX2 locus. Use of this system has resulted in several novel and interesting results, most notably the paramutation phenomenon. In this work, Casier et al. show that the BX2 locus is silent at 25oC, but produces piRNAs at 29 oC in the presence of homologous sequence in the background.
While I have no problem with the experiments, but did wish for more insight into the mechanism, and more caution in the interpretation. In particular, entirely environmental specification of piRNA clusters implied in the Abstract is a strong claim, and as the authors show themselves, is an oversimplification. The induction they find is quite specific to their system. Specifically, there are higher levels of homologous sense and anti-sense transcript at 29 than at 25, and temperature alone doesn't change other piRNA production. Similarly, it would be useful to know if the expression of the sense transcript from P-Target is also higher at 29 that at 25. (I think this construct has a heat-shock promoter, as well as that of the P-element, which shows temperature sensitive effects.)
I wasn't entirely convinced by the suggestion that expression of natural piRNA clusters is higher at 29 oC. Many piRNAs are generated as a secondary byproduct of transposable element message, and many transposable elements seem to have temperature sensitive expression (possibly as a byproduct of changes in chromatin.) The piRNA dependent splicing suppression of the P-element is also, apparently, not temperature sensitive (Teixiera et al., 2017).
Minor points. It does seem important (and quick) to eliminate the possibility that there's any active P-elements in these lines. While w1118 is a classic M-type background, there are some cases of sublines of other M-types harboring P-elements via contamination (Rahman et al. Nucleic Acids Research, Volume 43, Issue 22, 15 December 2015, Pages 10655-10672). If there is active P-element, I think it is possible that transgenes (which has the TIRs necessary for P-element insertion) has been picked up and put in another piRNA cluster. It's a simple matter of a few PCRs to exclude this possibility. The Cy and CyRoi backgrounds should be tested as well, to show that they are devoid of any plus-strand transcript that could be an ongoing trigger for piRNA production.
Subsection “Epigenetic conversion at 29°C occurs at a low rate from the first generation” paragraph two. The model is entirely sensible, but the data are quite noisy, and seem like they would be consistent with any model predicting an increase over generations. Can they compare this model to, for example, a model without the “c” parameter using Akaike Information Criteria or a log-likelihood test? This would make the analysis more informative.
Subsection “Heat conversion requires a homologous sequence in trans”: The chi-square statistic and degrees of freedom should be reported as well as the p-value. (And this is very minor, but the Greek letter is usually used in place of “chi”.)
[Editors' note: further revisions were suggested, as described below.]
Thank you for resubmitting your work entitled "Environmentally-induced epigenetic conversion of a piRNA cluster" for further consideration at eLife. Your revised article has been evaluated by James Manley (Senior Editor), a Reviewing Editor, and two reviewers.
The manuscript has been improved but the referees still have significant concerns. If you decide to proceed with publication of the present version, our editorial rating, which will be displayed immediately below the Abstract, would be that "major issues remain unresolved."
The reviewer comments (on the original version and revised version) would also be published, along with your responses.
Alternatively, you might decide to undertake significant extra work to try to address the concerns. Or, you might decide to shorten the paper (highlighting the most interesting findings) and leaving extensive additional work for a future paper. You also have the option to withdraw the present paper from consideration.
The reviews follow below, and we will look forward to hearing how you propose to proceed.
Reviewer #1:
In the revised version of the manuscript the authors find an involvement of sense-antisense transcripts and Dicer-2 in piRNA biogenesis. The authors may remember that one of the early findings celebrated as a major discovery was the lack of a role for double-stranded RNAs and the double-stranded RNA cleaving enzyme Dicer, in piRNA biogenesis. If now, the authors find a role for these in piRNA biogenesis, it has to be backed with strong data.
1) Can the authors demonstrate loading of the 21 nt RNAs into Piwi proteins?
2) If its role is epigenetic conversion, then ideally it has be loaded into the nuclear Piwi.
3) Are there experiments linking Dicer-2 to loading into a Piwi protein? Are they ever found in the same complexes?
I agree that there is a strong functional/genetic data that is mysterious, but very interesting. The heat-activated piRNAs are functional as they can silence a LacZ transgene. Since this manuscript will be published in some form, I am happy to support description of this part in a revised manuscript, but without wild speculation of Dicer-2 in piRNA biogenesis (even if they examine a Dicer mutant). These attempts at explaining the molecular mechanism may be toned down. I am worried that any prominent mention of Dicer-2 in this context may only serve to foster suspicion of the interesting genetic observation.
Reviewer #3:
I still find this study clever and interesting, and think the manuscript shows some specific improvements:
The “primed” nature of the BX2 locus is clearer, and the claim in the paper is more measured and reasonably justified.
The experiment showing the Dcr2 dependence of the conversion of the BX2 from off to on goes a little way toward understanding the mechanism of piRNA cluster formation, implying that it is dependent on siRNAs.
The addition of the figure showing the model for the mechanism. The weak point of the proposed mechanism, as I see it, is the lack of evidence that the siRNAs are loaded onto PIWI, any evidence the authors can provide for this mechanism would greatly improve the paper. I can't make any suggestions that seem technically feasible, however, in light of the low conversion rate in this system.
Further clarification would also be helpful for these aspects:
Regarding the model in Figure 1—figure supplement 3: I previously suggested a statistical analysis of this model. Rather than the more detailed analysis I suggested previously, I think a reasonable compromise would be to show that there is a significant increase over generations. That is the important point here (and, in fact, it's hard to imagine a scenario where the “memory” of conversion does not play a role in the increase over generations). But, while there appears to be a trend, the data are sufficiently noisy that it would be useful just to see that the increase is significant (perhaps a linear regression on transformed data?).
Discussion opening paragraph: Please explain what you mean by the “specific spliced transcript” comment; it wasn't clear.
e.g., supplementary file 11: It would be nice to more cautious in the interpretation where there are large differences in the number of flies examined. In Figure 3, for example, I think the numbers show that 41 of 1447 females show partial or complete repression; the comparable (?) numbers is Supplementary file 11 show 0 of 32 (?) females show any repression. This is not a significant difference via Fisher's exact test.
Additional data files and statistical comments:
Please see major comments for suggestions regarding statistical analyses, particularly in cases where conclusions are drawn about the absence of repression in small sample sizes.
https://doi.org/10.7554/eLife.39842.032Author response
Reviewer #1:
[…] Concerns
1) Abstract, Second sentence: There is no data to show that piRNA clusters are maternally defined except in flies. In mice germ cells are induced from the somatic lineage at embryonic day 7.5 and there is no possibility of maternal inheritance playing a role. If the authors want to keep this sentence, specify it is for flies.
We have specified that the hypothesis of the determination of piRNA cluster by maternal inheritance of homologous piRNA is for flies.
2) Abstract, third sentence: The sentence sounds very dramatic as it means that higher temperature alone was sufficient to convert the P-transgene locus into a piRNA cluster. Perhaps the features of the genomic region (having P-transgenes), location, chromatin status etc are responsible. I would rephrase it. It is misleading as although temperature might be the trigger, this locus might already be primed and ready to go.
The reviewer #1 is completely right, our results show that the locus is ready to become a piRNA cluster and the temperature is the trigger for the activation. We have rephrased the sentence.
3) The authors make a very striking finding regarding high temperature-induced piRNA production from a locus, and link it to presence of both sense and antisense transcripts are necessary for this. This still leaves open the question how the locus was made into piRNAs. There is no explanation for this.
This is the major point raised by the three reviewers. The major concern in our system is the low occurrence of the epigenetic conversion (2%). It was possible to identify and quantify it through generations because of the use of the high reliability of the ßGalactosidase staining allowing us to (i) visualize the status of each eggs chambers individually at each generation, (2) study a large number of flies (Figure 1 and 3, for instance), (3) do statistics. However, observing 2% difference during the process of activation by molecular analysis appeared impossible (for example, the observation of 2% increase of piRNAs or chromatin modification through ChIP analyses is not statistically possible). Therefore, molecular analyses were performed on stable epigenetic states (BX2OFF or BX2ON converted by maternally inherited piRNAs or by high temperature) and after being transferred at 25°C.
In order to answer to this major point, we performed additional experiments to test if double stranded RNAs should be formed prior piRNA biogenesis to activate BX2. So, we tested whether Dcr-2 might affect the activation of BX2 by temperature. We generated flies containing BX2, Dcr-2 mutant allele and a P(TARGET) tested the BX2 activation at each generation by ßGalactosidase staining. We present results showing that Dcr-2 mutation impairs the activation of BX2 by high temperature (Figure 3—figure supplement 3). This result is consistent with the requirement of double strand RNA intermediates for BX2 conversion into a piRNA cluster. We have added these experiments at the end of the result section as well as an additional figure (Figure 5) containing a cartoon putting together all of the results in a model. We hope that these new data will satisfy the reviewer concerns on the mechanism of activation.
4) It would be good to have the actual sequence information for the different transgenes and the b-gal reporter, and the actual transcripts present in these flies. One is left in the dark as to the level of complementarity that exists between them when they are referred to as sense and antisense transcripts.
We have added the precise structure of all transgenes used in this study showing that the complementarity between the BX2 and the P(TARGET) transgenes is restricted to the lacZ gene and the 5' and 3' region of the P element required for transgenesis (new Figure 1—figure supplement 1).We have specified what are sense and antisense BX2 transcripts (they correspond to sense and antisense lacZ transcripts, respectively) in the text and in the legend of the Figure 4.
In conclusion, the observations made are striking and extremely interesting, but perhaps a bit more of molecular insights might be useful to appreciate the observations made.
We appreciate the interest of this reviewer to our work and hope that the additional genetic results obtained with others euchromatic transgenes (see below) and with Dcr-2 sufficiently improve the molecular understanding of the phenomenon we are describing.
Reviewer #2:
[…] I found the authors' finding that heat treatment induces de novo piRNA production from non-piRNA clusters potentially interesting. However, the mechanistic insights remain vague.
As previously described, we hope that the experiments with others transgenes (see below) and with Dcr-2 give more insight on the mechanistic part of the phenomenon. Based on the new results presented in this revised version, we have added a new figure presenting a model of mechanism (Figure 5).
The authors claimed that homologous sequence was required for the induction. However, to claim this, much more supportive data should be provided.
We have performed additional experiments using another target, a P-lacZ-rosy reporter transgene inserted in euchromatin and expressed in the germline (P(A92), BQ16, FBti0003435). In the presence of this transgene, BX2 was converted at 29°C at the same rate than with P(TARGET) showing that BX2 conversion of BX2 by temperature cannot be attributed to any specificity linked to the P(TARGET) insertion. We have added these results in the main text and in a supplementary table (Supplementary file 10).Moreover, the potential effect of the background has been now tested over 30 generations and no conversion event has been observed, reinforcing the specific role of the germline expressed target in the activation of BX2. The Supplementary file 9 has been completed. We also performed a new experiment using a P(A92) transgene that is expressed in the somatic follicle cells but not in the germline nurse cells. With this transgene, no conversion was observed, showing that the euchromatic homologous transgene need to be transcribed (Figure 3—figure supplement 2). This reinforces the role of the "sense" transcripts brought by the euchromatic transgene in the conversion process.
The authors also found that H3K9me3 and Rhino started to accumulate at the BX2 locus upon heat treatment. However, the meaning of the accumulation remains unclear. My other concerns were indicated below. I hope that this reviewer's concerns might be helpful for the authors to revise the manuscript.
As a matter of fact, the enrichment in H3K9me3 and Rhino on the BX2 locus can be interpreted by a progressive accumulation of all the loci present in egg chambers studied or by a progressive increase number of egg chambers activated through generation (each one of them being fully activated or not activated at all, ON/OFF conversion). Based on our results obtained by lacZ staining showing exclusively egg chambers converted or not converted with low occurrence, we favor an on/off conversion per egg chamber induced by maternal inheritance of homologous piRNA or by temperature.
Concerns and suggestions from this reviewer:
1) The authors proposed that the interaction between the excess of BX2 antisense transcripts and the sense P(TARGET) transcripts is a prerequisite for the production of de novo piRNAs (subsection “Heat conversion requires a homologous sequence in trans”). To confirm this, I recommend the authors conducting experiments using heat treated BX2OFF that do not have lacZ at the BX2 loci but contains P(TARGET).
This could be a good idea but we cannot modify at will the BX2 locus that had been obtained through several rounds of transposition followed by thousand of flies selection (Dorer and Henikoff 1994). P-transgenes have the capacity to inserted in the genome without hot spots (except gene regions rich), therefore it seems to be very difficult to recover 7 insertions within the same location. Moreover, if 7 P(lacW) was identified in another locus, it will not have necessary the same biological properties than the BX2 locus.
2) The level of AGO1 RNA transcripts should be examined in BX2OFF line containing P(TARGET). I do not understand why the authors used the line without P(TARGET) in this particular experiment.
The presence of the BX2 cluster may affect specific transcripts of one allele of the AGO1 gene and to be sure to measure the effect of temperature on AGO1 transcripts, we performed experiments without BX2. However, we have reproduced the measurement of AGO1 steady-state RNA level by qRT-PCR experiment in a BX2 context, with and without P(TARGET) and confirmed that the amount of AGO1 transcripts is higher at 29°C compared to 25°C. These results have been added in the Figure 2—figure supplement 2.
3) Figure 4B: Upon heat treatment, the expression level of BX2 antisense transcripts was raised. Was this phenomenon related to Rhino and H3K9me3 accumulation observed in Figure 2DE?
The two studies were addressing different questions:
The Figure 2D-E was studying the molecular changes of a stable epigenetic conversion: that is to say the BX2 locus was activated by heat treatment after several generations at 29°C and this activation is stable because it is maintained even when flies are incubated at 25°C (the classical temperature). In this context, we compared a stable OFF line and stable ON lines (converted by maternally inherited piRNAs or by temperature) for differences of chromatin marks. Statistical but weak differences were identified (Figure 2).
In the Figure 4, we were addressing the mechanism of the triggering, that is to say what is happening after one generation at 29°C. Therefore, in this experiment, we were not expecting to be able to identify chromatin differences with a 2% conversion rate.
Experiments in Figure 2 and Figure 4 should be performed using the same fly lines.
In Figure 2D-E, ChIP experiments were performed on flies stably activated by temperature. They hold the P(TARGET) since it is required for their activation and used as internal reporter of the activated state. In Figure 4, measurement of antisense lacZ transcripts from BX2 implies that all lacZ RNA should come from BX2 locus. The presence of P(TARGET) which shares lacZ sequence would impair the specific measurement of lacZ antisense coming from BX2 locus.
4) Is the BX2 transgene in the BX2OFF line containing P(TARGET) also inserted in the AGO1 gene? This should be examined. If this were the case, the authors better examine whether piRNAs were derived from the AGO1 gene.
All BX2 transgenes, whether ON or OFF, are inserted into the AGO1 gene. We have analyzed the small RNA produced from AGO1 gene region in the BX2OFF and BX2ON strains: no significant piRNA amount was produced in all epigenetic contexts. This shows that AGO1 is not a natural piRNA producing locus. Later, looking for piRNAs from AGO1 gene region at 25°C and 29°C in BX2OFF strains confirm that the AGO1 gene is not a piRNA cluster and that 29°C does not induce de novo production of small RNAs from the AGO1 gene region. These data have been added in a new Figure 2—figure supplement 2 and in Figure 4J and are discussed in the text. Figure legends have been modified as well.
5) The authors should test experimentally whether BX2-derived piRNAs are loaded into PIWI proteins. This is very important.
From the first description of piRNAs and piRNA clusters by Brennecke et al., 2007, it is known that piRNAs are loaded by the PIWI proteins allowing their biogenesis. Through these loadings, piRNAs have four main signatures that defined piRNAs: 23-29 nt, a 1U bias, a 10A bias and a 10 nt overlap between pairs of 23-29 nt small RNAs (ping-pong signature). Since this first description, several other studies used these properties in numerous organisms (Nematostella, Bombyx mori, D. simulans, marmoset, ….) and identified piRNAs. The data of Figure 2 shows these characteristics that convince us that bona fide piRNAs were produced by the new activated BX2 locus. On top of that, the strength of the transgene system used in this study allows also to provide the ultimate proof: the converted BX2theta locus is producing small RNA that are functionally active for germline lacZ repression.
6) Figure 2B: Explain why BX2-derived piRNAs did not show 10A bias. The data shown in Figure 2C supported the idea that BX2-derived piRNAs were products of the ping-pong pathway. Then, they should have shown 10A bias, but in reality they did not.
The 10A bias is only observed in the fraction of AGO3-loaded piRNA (Brennecke et al., 2007) or can be detected in the fraction of piRNA that paired each other (De Vanssay et al., 2012). We have performed this last analysis and the results are now shown on Figure 2C and Figure 2—figure supplement 1C. The figure legends and the Materials and methods section have been completed as well.
7) Figure 2DE: The authors should examine genome-widely where in the genome H3K9me3 and Rhino accumulated upon heat treatment. It is hard to imagine that H3K9me3 and Rhino accumulation only occurred at the BX2 locus upon heat treatment.
We agree that studying whole genome effects of heat treatment could be an interesting question. However, we did not detect production of new piRNA from other loci (Figure 2—figure supplement 3). Therefore, even if modification of chromatin marks were detected elsewhere in the genome, they are not followed by a stable new piRNA production, that is the question addressed in this study. Once again, molecular analyses were performed on flies that have been activated at 29°C and then put back at 25°C. Only stable modifications can be observed. Our results showed that only BX2 is metastable for piRNA production, all other potential piRNA producing loci were already activated before heat treatment, and even if others loci are stably modified in their chromatin marks (H3K9me3 or Rhino), they did not become piRNA producing loci.
8) The authors should examine whether H3K9me3 and Rhino accumulated at the BX2 locus in BX2OFF line without P(TARGET) upon heat treatment.
The low occurrence of epigenetic conversion (2%) does not allow enough dynamics to analyze chromatin modification. Without P(TARGET), no conversion is observed and ChIP experiment on BX2OFF has already been performed and show less H3K9me3 or Rhino enrichment compared to BX2* or BX2theta.
9) This manuscript should be edited thoroughly by a native English speaker.
The manuscript has been read and corrected by a native speaker (Dr Lori Pile).
10) Table 1 should be removed from the main text.
It has been done. Table 1 has been renamed Supplementary file 8
Reviewer #3:
[…] While I have no problem with the experiments, but did wish for more insight into the mechanism, and more caution in the interpretation.
As previously described, we believe that the experiment with additional transgenes and with Dcr-2 provide more insight related to the mechanistic part of the phenomenon that is illustrated by a model presented in Figure 5.
In particular, entirely enviromental specification of piRNA clusters implied in the Abstract is a strong claim, and as the authors show themselves, is an oversimplification. The induction they find is quite specific to their system. Specifically, there are higher levels of homologous sense and anti-sense transcript at 29 than at 25, and temperature alone doesn't change other piRNA production.
We agree that we oversimplified the message in the Abstract and as the first reviewer suggested, we now precisely state the role of different parameters required for the activation of BX2.
Similarly, it would be useful to know if the expression of the sense transcript from P-Target is also higher at 29 that at 25. (I think this construct has a heat-shock promoter, as well as that of the P-element, which shows temperature sensitive effects.)
We performed this experiment and the result is that P(TARGET) is less expressed at 29°C. This result has been added in the text and in a new supplementary figure (Figure 4—figure supplement 1C). As mentioned in the Materials and Methods, this transgene does not carry a heat shock promoter, it is an enhancer trap transgene inserted in the eEF5 gene, an ubiquitous translation elongation factor.
I wasn't entirely convinced by the suggestion that expression of natural piRNA clusters is higher at 29C. Many piRNAs are generated as a secondary byproduct of transposable element message, and many transposable elements seem to have temperature sensitive expression (possibly as a byproduct of changes in chromatin.)
We do not suggest that expression of natural piRNA clusters is higher at 29°C. Actually, the analysis of the major 42AB piRNA cluster shows a slight decrease of piRNA at 29°C compare to 25°C (Figure 4E). We totally agree that variations in piRNA production from natural piRNA clusters may result from several parameters including ping-pong amplification and variation in the expression of piRNA genes (as suggested by Fast et al., 2017). We have rephrased this part to clarify this point.
The piRNA dependent splicing suppression of the P-element is also, apparently, not temperature sensitive (Teixiera et al., 2017).
We note that the intron 2-3 of the P element, whose splicing is dependent of piRNA, is absent from all transgenes that have been used in this study: the P(lacW) transgene that constitutes the BX2 locus, P(TARGET) and P(A92), the two P-lacZ-rosy used as reporter transgenes.
Minor points. It does seem important (and quick) to eliminate the possibility that there's any active P-elements in these lines. While w1118 is a classic M-type background, there are some cases of sublines of other M-types harboring P-elements via contamination (Rahman et al. Nucleic Acids Research, Volume 43, Issue 22, 15 December 2015, Pages 10655-10672). If there is active P-element, I think it is possible that transgenes (which has the TIRs necessary for P-element insertion) has been picked up and put in another piRNA cluster. It's a simple matter of a few PCRs to exclude this possibility. The Cy and CyRoi backgrounds should be tested as well, to show that they are devoid of any plus-strand transcript that could be an ongoing trigger for piRNA production.
Several years ago we tested by PCR the absence of P element in the strains we used for our studies, especially in the balanced stocks commonly used. We checked again by PCR (using the primer 5'-TGATGAAATAACATAAGGTGGTCCCGTCG-3' known as P3-31 that recognized the P inverted repeated sequences) all of the stocks that we used in this study and all of the strains are devoid of natural P sequences. We propose to not add this result in the paper since it is a very specialized point but our results are available if needed. Another point to this remark is that we have established recombined lines from BX2, P(TARGET) and, in the absence of P(TARGET), they did not show activation of the BX2 locus at 29°C. If uncontrolled, natural P elements were present in our strain and responsible of the conversion, they should not all be eliminated by recombination and control lines could still show BX2 activation. Additionally, we have published that piRNA produced by P sequences only are not able to activate BX2 by paramutation (Hermant et al., 2015) demonstrating that the homology length conferred by P sequences is not sufficient to trigger BX2 conversion, even by piRNA maternal inheritance.
Subsection “Epigenetic conversion at 29°C occurs at a low rate from the first generation” paragraph two. The model is entirely sensible, but the data are quite noisy, and seem like they would be consistent with any model predicting an increase over generations. Can they compare this model to, for example, a model without the “c” parameter using Akaike Information Criteria or a log-likelihood test? This would make the analysis more informative.
We agree that the data are noisy. This is due to the high variation of occurrence of the epigenetic conversion and its sampling through generations and among replicates. The first rough observations of the phenomenon reported complete repression in ovaries after several generations. We tried to understand this effect by analyzing thousands of flies in one generation rather that dozens of flies through multiple generations and observed a small conversion frequency (≈2%, Figure 3). Then, we searched for a simple model based on the conversion frequency we observed in one generation and the fact that the ON state is transmitted to the next generation (De Vanssay et al., 2012 and Figure 3). The major problem we encountered is related to the sampling that occurred at each generation: among all egg chambers that are produced, only dozens of flies were analyzed. We think that the sampling size generates the variation we observed among replicates with a 2% conversion phenomenon. We agree with the reviewer that our data would fit with a number of models describing an increase over generations. We can remove this part if it is still considered non informative.
Subsection “Heat conversion requires a homologous sequence in trans”: The chi-square statistic and degrees of freedom should be reported as well as the p-value. (And this is very minor, but the Greek letter is usually used in place of “chi”.)
We have added in the new version the χ2 value (23.35) and the degrees of freedom (2) and replaced “chi2” by “χ2” each time we used the test.
[Editors' note: further revisions were suggested, as described below.]
The reviews follow below, and we will look forward to hearing how you propose to proceed.
Reviewer #1:
In the revised version of the manuscript the authors find an involvement of sense-antisense transcripts and Dicer-2 in piRNA biogenesis. The authors may remember that one of the early findings celebrated as a major discovery was the lack of a role for double-stranded RNAs and the double-stranded RNA cleaving enzyme Dicer, in piRNA biogenesis. If now, the authors find a role for these in piRNA biogenesis, it has to be backed with strong data.
1) Can the authors demonstrate loading of the 21 nt RNAs into Piwi proteins?
2) If its role is epigenetic conversion, then ideally it has be loaded into the nuclear Piwi.
3) Are there experiments linking Dicer-2 to loading into a Piwi protein? Are they ever found in the same complexes?
I agree that there is a strong functional/genetic data that is mysterious, but very interesting. The heat-activated piRNAs are functional as they can silence a LacZ transgene. Since this manuscript will be published in some form, I am happy to support description of this part in a revised manuscript, but without wild speculation of Dicer-2 in piRNA biogenesis (even if they examine a Dicer mutant). These attempts at explaining the molecular mechanism may be toned down. I am worried that any prominent mention of Dicer-2 in this context may only serve to foster suspicion of the interesting genetic observation.
We agree with the first reviewer that implications of Dcr-2 in piRNA biology suggest a major breakthrough that our experiments do not definitively prove. We would like to clarify, however, that the role of Dcr-2 we proposed was not in the maintenance of piRNA production, but rather, a more opportunistic way to accidentally produce some new piRNAs. In our system, where the BX2 cluster is a peculiar genomic structure, this de novo piRNAs production would be sufficient to convert BX2 into an active piRNA cluster. Once activated, Dcr-2 is no longer required for piRNA production from BX2 as we published in Hermant et al., 2015. We agree that the biochemical experiments proposed by the first reviewer are well adapted in theory. We think, however, that the 2% conversion rate is too low to be able to observe such molecular details. For these reasons, we agree to your proposal to remove the results obtained with Dcr-2 and tone down our model presented in Figure 5. We propose to consider the hypothesis of somehow making double strand RNAs as a starting point to accidentally produce the first new piRNAs, but clearly state that much of the mechanistic aspects remain unknown, as asked by the editors.
Reviewer #3:
I still find this study clever and interesting, and think the manuscript shows some specific improvements:
The “primed” nature of the BX2 locus is clearer, and the claim in the paper is more measured and reasonably justified.
The experiment showing the Dcr2 dependence of the conversion of the BX2 from off to on goes a little way toward understanding the mechanism of piRNA cluster formation, implying that it is dependent on siRNAs.
The addition of the figure showing the model for the mechanism. The weak point of the proposed mechanism, as I see it, is the lack of evidence that the siRNAs are loaded onto PIWI, any evidence the authors can provide for this mechanism would greatly improve the paper. I can't make any suggestions that seem technically feasible, however, in light of the low conversion rate in this system.
As stated above, we agree that the biochemical experiments proposed by the reviewer are well adapted in theory but that the 2% conversion rate is too low to be able to observe such molecular details. For these reasons, we agree to your proposal to remove the results obtained with Dcr-2 and tone down our model presented in Figure 5.
Further clarification would also be helpful for these aspects:
Regarding the model in Figure 1—figure supplement 3: I previously suggested a statistical analysis of this model. Rather than the more detailed analysis I suggested previously, I think a reasonable compromise would be to show that there is a significant increase over generations. That is the important point here (and, in fact, it's hard to imagine a scenario where the “memory” of conversion does not play a role in the increase over generations). But, while there appears to be a trend, the data are sufficiently noisy that it would be useful just to see that the increase is significant (perhaps a linear regression on transformed data?).
We removed the mathematical model of progression of conversion during generation and propose a simpler description of the variability observed among lines. Mean and 95% confidence interval per generation were calculated on modified data (arcsine square root). The text (subsection “Epigenetic conversion at 29°C occurs at a low rate from the first generation”), the Figure 1—figure supplement 3B and its legend have been modified accordingly.
Discussion opening paragraph: Please explain what you mean by the “specific spliced transcript” comment; it wasn't clear.
By “specific spliced transcript”, we mean that we measured AGO1 transcripts that overlap the BX2 insertion point and that come from a distant promoter. We have added this specific description in the legend of Figure 4. There is another promoter for AGO1 that is located after the BX2 insertion point. AGO1 transcripts produced from this promoter are only slightly affected by temperature (not significantly), allowing us to conclude that it could explain the apparent discrepancy with the results published in Fast et al., 2017. We have added this analysis in Figure 4—figure supplement 1D and modified the discussion and the legend, accordingly.
e.g., supplementary file 11: It would be nice to more cautious in the interpretation where there are large differences in the number of flies examined. In Figure 3, for example, I think the numbers show that 41 of 1447 females show partial or complete repression; the comparable (?) numbers is Supplementary file 11 show 0 of 32 (?) females show any repression. This is not a significant difference via Fisher's exact test.
We would like to clarify that the results obtained with NaCl and heat shock have to be compared to those obtained at 29°C in G1 (showed in Figure 3), considering the proportion of converted egg chambers and not flies (that are analyzed in G2). For the heat shock experiment, the Χ2 test was 106.03 and the p-value was 7.25 x 10-25 (heat shock 0/3840 versus 29°C 586/21720) and for the NaCl experiment, the Χ2 test was 115.93 with a p-value of 4.91 x 10-27 (NaCl 0/4200 versus 29°C 586/21720). We have described this analysis in a clearer way in the legend of supplementary file 11 and by adding the results obtained at 29°C (from Figure 3) in the table of the revised version.
Finally, we renamed the P(TARGET) transgenes used in this study as asked by editors and specified their domain of expression to be as clear as possible for a large audience.
https://doi.org/10.7554/eLife.39842.033Article and author information
Author details
Funding
Fondation ARC pour la Recherche sur le Cancer (SFI20131200470)
- Stéphane Ronsseray
Fondation pour la Recherche Médicale (DEP20131128532)
- Stéphane Ronsseray
Agence Nationale de la Recherche (plastisipi)
- Stéphane Ronsseray
Pierre and Marie Curie University (EME1223)
- Laure Teysset
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements
We thank Doug Dorer, Steve Henikoff, Julius Brennecke and the Bloomington Stock Center for providing stocks. We thank Bill Theurkauf for providing antibodies. We thank flybase.org for providing databases. We thank Ritha Zamy for technical assistance. We thank Clément Carré, Lori Pile, Ana Maria Vallès and Jean-René Huynh for critical reading of the manuscript. We thank Christophe Antoniewski for helpful advices and development of the ARTbio platform (http://artbio.fr/).
Senior Editor
- James L Manley, Columbia University, United States
Reviewing Editor
- Timothy W Nilsen, Case Western Reserve University, United States
Version history
- Received: July 4, 2018
- Accepted: March 6, 2019
- Version of Record published: March 15, 2019 (version 1)
Copyright
© 2019, Casier et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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