Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes

In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes.


Introduction
In recent years it has become increasingly evident that the expression of eukaryotic genomes is far more complex than it had been previously explored. An emerging body of evidence, coming from next generation sequencing approaches, is showing that the genomes of all studied eukaryotes are almost entirely transcribed, generating an enormous number of non-coding RNAs (ncRNAs) [1]. The ENCODE project showed that at least 90% of analyzed eukaryote genome is transcribed in different cell types, indicating that there is a huge reservoir of RNA molecules with potentially unexplored biological function [2,3,4]. These RNAs are remarkably different in their number, size, subcellular localization, and mechanisms of action, and many are essential to finely control gene expression as well as genomic plasticity [5].
It is becoming increasingly clear that some ncRNAs are part of epigenetic regulatory networks with striking evolutionary conservation [6,7]. For example, dynamic changes in chromatin function are frequently transacted by nuclear RNA signaling pathways. Although the evolutionarily conservation and precise molecular mechanisms are poorly understood, a differential recruitment of a hierarchy of chromatin modifying complexes to specific loci by RNAs sets precise transcriptional states leading to differentiation [8,9,10,11,12]. Moreover, the unusual epigenetic phenomena of paramutation [13,14,15], trans-induction [16] and transvection [17,18] observed in a variety of higher eukaryotes involve the activity of ncRNAs that 'rewrite' the transcriptional state of an allele, in processes that apparently escape classic Mendel's laws of genetic inheritance. These epigenetic phenomena are clear examples of ncRNAdirected regulatory processes that transfer epigenetic information both across cells, between tissues and across generations, though the mechanisms underlying these phenomena still remain elusive.
In order to unveil the role played by cellular RNA pools produced by homologous genomic loci in changing the transcriptional state of a silenced gene, we used classic Position Effect Variegation (PEV) assays in the model system D. melanogaster [19,20] and tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin (w m4h ). Surprisingly, we found that several non-functional white alleles, unable to produce the main wildtype white coding transcript but with the potential of transcribing other RNA's from the white locus, trans-reactivate the variegating w m4h line thus increasing eye pigmentation. Strikingly, the presence of non-functional white alleles cause an increase in the w m4h gene transcript as well as an opening in the chromatin structure at the w m4h locus. Remarkably, this new epigenetic phenomenon is heritable, relies on the presence of diffusible homologous RNA's, and is affected by mutations in genes involved in post-transcriptional gene silencing. Overall, our data strongly indicate that trans-reactivation is a new epigenetic phenomenon that positively control gene expression in the context of heterochromatin through homologous RNA molecules.

Alleles of white can suppress w m4h eye color variegation
The white (w) gene encodes an ABC transporter essential for the red pigment transportation in the compound Drosophila eye. The In(1)w m4h X chromosome inversion places the fully functional wildtype euchromatic w gene adjacent to a region of pericentromeric heterochromatin, creating the variegated white-mottled 4 (w m4h ) allele that is characterized by the random inactivation of w by the heterochromatin spreading from the inversion chromosome breakpoint ( Fig  1A, upper panel). This cell autonomous transcriptional inactivation once established is clonally inherited, and it is responsible for an eye with a variegated expression of the red pigment,  (1)w m4h inversion juxtaposing the w wildtype gene in the proximity of X chromosome pericentric heterochromatin, resulting in the generation of the w m4h allele with silenced w expression in most eye cells and leading to a variegated eye color. Lower panel; parental (P) variegating w m4h females were crossed with males carrying a hypomorphic or loss-offunction allele of the white gene (w*). The recovered progeny (F1) was scored for increased, decreased or unchanged eye color pigmentation. Number of alleles recovered (n) and corresponding percentage (%) of constituting an example of the genetic phenomenon known as Position Effect Variegation (PEV) [20].
Genetic screens have shown that a large number of mutations alter PEV phenotypes, resulting in the isolation of enhancers E(var)s or suppressors Su(var)s of variegating phenotypes. The vast majority of these modifiers were originally isolated in Drosophila as dominant mutations that suppressed or enhanced eye color variegation caused by the w m4h allele [21]. The molecular characterization of those mutants have shown that the products of E(var)'s and Su (var)'s are structural components of chromatin, or enzymes that covalently modify chromatin proteins [19]. However, to date the effect exerted by non-functional w alleles on the w m4h chromatin-dependent variegation is unknown. Therefore, we took advantage of the classic w m4h PEV assay to analyze if non-functional alleles of the w gene were able to modify eye color variegation when in trans-heterozygosis with the w m4h allele (Figs 1A, lower panel, and S1A and S1C).
We screened 19 loss-of-function or hypomorphic w alleles (w Ã ) for genetic interaction with w m4h , and measured eye red pigment to quantify the strength of the genetic interaction [22]. The pilot screen resulted in the isolation of 14 suppressors (74% of total w Ã alleles screened) able to increase w m4h eye color pigmentation (Fig 1A and S1 Table). Among the tested hypomorphic alleles that behaved as robust suppressors of w m4h variegation, the w sey allele interacted more strongly (Figs 1B and S2). Moreover, loss-of-function alleles of w, including the widely used w 1118 allele, showed a weak but highly reproducible suppression effect (Figs 1C and S3). Notably, F1 w m4h /Y males derived from the crosses did not show any increase in eye color pigmentation (Fig 1B and 1C), indicating that the genetic locus responsible for the increased eye color pigmentation in the w m4h /w Ã trans-heterozygous females is carried by the w Ã bearing X chromosome. Reciprocal crosses using multiple w m4h balanced lines (S1B, S1D, S1E and S1F Fig) and classic recombination mapping (S1G and S1H Fig) confirmed the effect of the w Ã loci tested in increasing eye color pigmentation of w m4h . Interestingly, deletions spanning the w locus (Δw) did not show any modification of the w m4h variegation (Fig 1D and S1  Table). Moreover, none of the w Ã alleles tested was able to reduce the red pigmentation of w m4h , while 5 of them (26%) had no effect ( Fig 1A, lower panel, and S1 Table). The pilot screen we conducted suggested that the presence of white genomic homologous sequences of nonfunctional w Ã alleles could reactivate in trans-heterozygosis the w m4h heterochromatic silenced locus.
The suppression of w m4h variegation by w* alleles is a robust effect, unrelated to classic Su(var) function and independent from chromosome pairing To further characterize the effect of w sey and w 1118 alleles (from now on used as representative examples of w Ã alleles giving a strong and weak interaction with w m4h , respectively), we first decided to measure the strength of their suppression on the w m4h variegation. Interestingly, in the presence of a strong E(var) mutation encoding for a factor contributing to the opening of chromatin [23], both w 1118 and w sey retain their ability to increase eye pigmentation of w m4h , though w 1118 with a weaker strength (Figs 2A and S4A). This data indicate that the suppressing total alleles screened are reported. Eye pigment quantification of parental stocks (P) and of the resulting trans-heterozygous female (w m4h /w*) and control male (w m4h /Y) progeny (F1) for the w sey (B), w 1118 (C) alleles and Δw (D), are shown together with representative eye pictures for each genotype tested. P and F1 eye pigment quantification graphs have different scale.
doi:10.1371/journal.pgen.1005444.g001 w Ã alleles are able to increase eye pigmentation even when the w m4h allele is in a strongly silenced heterochromatin context.
Next we wanted to test the ability of w Ã alleles to behave as suppressors in other PEV assays, a feature that is shared by classic Su(var)s encoding for factors responsible for the establishment and maintenance of inactive chromatin [24]. We choose the Sb v PEV assay, characterized by the T(2;3)Sb v translocation that juxtaposes the dominant Sb mutation to centric heterochromatin, resulting in mosaic flies with both short (Sb) and normal bristles ( Fig 2B). As expected, when the Sb v stock was crossed with a strong Su(var) [25] a significant suppression was observed in the frequency of Sb bristles (Figs 2B, upper graph, and S4B). However, both w 1118 and w sey as well as the w locus deletion (Δw) were not able to increase the number of short bristles when crossed with Sb v . These results suggest that the reactivating w Ã alleles do not likely encode for a factor that, beyond its role in eye pigmentation, it is also responsible for chromatin structure organization. Thus, our data strongly indicate that the mechanism underlying w m4h suppression by w Ã alleles is very likely unrelated to classic Su(var)'s function.
The suppression effect of w Ã alleles we observed over w m4h is highly reminiscent of transvection, a well-studied epigenetic phenomenon that results from the interaction between an allele on one chromosome and the corresponding allele on the homologous chromosome. Transvection may lead to both gene activation or repression, and is strongly dependent upon chromosome pairing [18]. However, because of the large In(1)w m4h inversion, the w m4h bearing chromosome is never paired with the w Ã containing sister chromatid in the suppressed transheterozygous females (Fig 1A), thus suggesting that the effect we observed could not be assimilated to classic transvection. However, to exclude intra-chromosomal long distance pairing or X chromosome specific effects, we tested the ability of w Ã alleles to de-repress variegated wildtype w insertions in pericentric and telomeric autosomal chromosome heterochromatin [26,27,28]. In all lines tested the strong w sey and in some cases also the weak w 1118 allele, but not the Δw deletion, consistently increased eye pigmentation of variegated wildtype w pericentric or telomeric autosomal insertions (Figs 2C-2F and S4C). Our data strongly suggest that the increase in eye pigmentation exerted by w Ã alleles over w m4h is independent from the type of heterochromatin tested, it does not rely on intra-chromosomal pairing and it is chromosome independent. w* alleles can increase w m4h eye pigmentation by opening chromatin at the w m4h locus and increasing the levels of wild type white coding transcripts To explore the correlation of increased eye pigmentation in trans-heterozygous interacting females (w m4h /w Ã ) and transcriptional activation of the w gene, we conducted semi-quantitative RT-PCR on total RNA extracted from adult heads. To measure the amount of full-length wildtype white coding mRNA we designed primers against the 3' of the transcript that are able to amplify the fully transcribed spliced and unspliced mRNA products ( Fig 3A). As expected, Remarkably, when w 1118 or w sey are introduced in trans with w m4h , we observed a highly reproducible and robust amplification of white transcripts (Fig 3, lanes 5 and 6). These data strongly correlate the levels of eye pigmentation, we observed in the trans-heterozygous w Ã /w m4h adults, with the levels of expressed coding white transcripts.
However, an increased white transcript stability in w Ã /w m4h trans-heterozygous could in theory also explain the observed high levels of amplification of coding white transcripts. Therefore, we tested the ability of mutants in genes known to be involved in maternal and zygotic fly mRNA stabilization for their ability to modify the levels of eye pigmentation normally scored in w Ã /w m4h adults. However, none of the mutants tested, including pumilio (pum) and smaug (smg), were able to significantly change eye pigmentation in our PEV assay (S5A Fig). These data strongly suggest that the increased levels of eye pigmentation observed in w Ã /w m4h transheterozygous females, cannot be directly correlated with general changes in white mRNA stability (S5A Fig). Structure of the white gene shows the 3' region used to design the primers (arrows) for semi-quantitative RT-PCR conducted on total RNA, derived from adult heads dissected from the different genomic combinations shown. The graph shows the levels of amplified w transcripts. Primers designed against the Act5C transcript have been used as internal control. The two amplified bands represent unspliced and spliced forms of the white transcript. Polytene chromosome FISH using genomic probes covering the entire w gene (with the exception of the first intron) and coding sequences for the hsp70 gene (mapping chromosome 3R) on homozygous w m4h / w m4h (B), w 1118 /w 1118 (C), w sey /w sey (D) and trans-heterozygous w m4h /w 1118 (E) and w m4h /w sey (F) allelic combinations. FISH signals for white (red) and hsp70 (green) genomic sequences are indicated by arrowheads. The asterisks indicate the region of pericentric heterochromatin. The X indicates the chromosome where the w gene maps. FISH signals for the w locus are detected in the w 1118 /w 1118 and w sey /w sey chromosomes because in these genotypes the w genomic sequences reside on euchromatic accessible region. doi:10.1371/journal.pgen.1005444.g003

trans-Reactivation
While, for all the interacting loss-of-function w Ã alleles, we could be sure that any white coding transcript we detect could only be produced from the wildtype w m4h locus, the same conclusion cannot be drawn for the interacting hypomorphic w Ã alleles. Therefore, we decided to use genomic probes from the white genomic locus to conduct fluorescent in situ hybridization (FISH) analyses on polytene chromosomes, for monitoring the level of accessibility of the interacting w Ã and w m4h chromatin loci. Polytene chromosomes represent a special structural organization of Drosophila salivary glands, consisting of polyploid interphase nuclei, which originate by repeated rounds of DNA replication without cell division. Drosophila polytene chromosomes have proven to be an invaluable cytogenetic tool to examine chromosome structure. Moreover, DNA FISH probes hybridization is dependent on the level of replication of specific genomic regions on polytene chromosomes, which in turn depends on the overall level of transcriptional activity present on that locus [24].
Exploiting this special feature of polytene chromosomes, we designed genomic probes covering both the w locus and the coding sequences of the hsp70 gene as internal positive control for FISH probe chromatin accessibility. As expected, w m4h homozygous female chromosomes gave a FISH signal for the hsp70 gene but failed to show a detectable band from the w m4h pericentric chromatin region because this heterochromatic locus is under-replicated [24] (See arrowhead and asterisk in Fig 3B). On the other hand, homozygous chromosomes for the w 1118 and w sey alleles or wildtype flies gave detectable FISH signals, for both the w and hsp70 loci (see arrowheads in Figs 3C and 3D and S5B). Remarkably, w m4h /w 1118 and w m4h /w sey but not w m4h / Δw trans-heterozygous female chromosomes, on top of the expected signals coming from the w and hsp70 loci, gave a highly reproducible FISH band originating from the pericentric w m4h chromatin locus (see arrowhead in the vicinity of the asterisk in Figs 3E and 3F and S5B). This analysis strongly suggests that FISH signals detected from the w m4h locus in trans-heterozygous w Ã /w m4h chromosomes are the result of increased DNA replication, consistent with an increase in transcription at the w m4h locus, as also found for other classic Su(var)s [24]. In conclusion, our data strongly indicate that the increase in eye pigmentation, observed in the w m4h /w Ã trans-heterozygous combinations, is strongly correlated with a reopening of wildtype heterochromatic w m4h locus by the presence of homologous genomic sequences present in the interacting loss of function and hypomorphic w Ã alleles. We called the ability of w Ã alleles to rereactivate wild type white at the w m4h locus: trans-reactivation.

trans-reactivation is heritable
The epigenetic interaction between the w Ã and w m4h alleles, leading to an increase in white transcription as a consequence of chromatin reopening at the silenced w m4h locus, is a phenomenon highly reminiscent of the epigenetic switch occurring in paramutation. In this epigenetic phenomenon, identified in plants, mice, and recently also in flies, the paramutating allele has the ability to change the activity state of its partner allele on the homologous chromosome (usually to a silent state) [14,15,29]. This effect is dependent on homologous sequences present in the two interacting alleles, is heritable and once established, is independent from the presence of the paramutating allele. To investigate if the reactivation of the w m4h locus was heritable through meiosis, we crossed F1 trans-reactivated w m4h /w Ã females with w Ã /Y males to look for trans-generational inheritance of trans-reactivation in the F2 progeny (Figs 4A and S6A).
In the F2 progeny we expect to score w m4h /w Ã females (that should continue to show increased levels of eye pigmentation), w Ã /Y males (deficient of red eye pigment), and finally w m4h /Y males where the F1 trans-reactivated w m4h allele, inherited from the F1 mothers has segregated from the w Ã trans-reactivating allele. If F2 w m4h /Y males have normal variegated eyes, the F1 trans-reactivated w m4h allele had lost its trans-reactivating potential during trans-reactivation of w* over w m4h is heritable. (A) Cross scheme used to test trans-generational inheritance of w m4h trans-reactivation. Parental (P) variegating w m4h /w m4h homozygous females were crossed with males carrying the trans-reactivating w* allele. The (F1) trans-reactivated w m4h /w m4h meiosis. On the other hand, if F2 w m4h /Y males showed increased eye pigmentation, this would be a strong indication that the trans-reactivated state of the F1 w m4h allele is transmitted in the germline and inherited in the F2 generation ( Fig 4A).
Remarkably, both w 1118 and w sey trans-reactivating alleles when segregated away from the F1 activated w m4h allele were able to generate F2 w m4h /Y males with strong eye pigmentation (Fig 4B and 4C). However, when the same F1 trans-heterozygous w m4h /w Ã females were crossed with Δw /Y males not bearing any homologous genomic w sequence (Figs 4D and S6B), the resulting F2 w m4h /Y males and w m4h /Δw females failed to trans-reactivate showing levels of eye variegation indistinguishable from parental (P) w m4h stocks (Fig 4E and 4F). Remarkably, when wild type w+ carrying flies are tested for their ability to trans-reactivate in F2 w m4h males, we did not observe any trans-reactivation (S6C Fig). However, when we crossed homozygous trans-reactivated w m4h /w m4h female with trans-reactivated w m4h /Y male, where the trans-reactivating w Ã allele was no longer present in both lines, we could reproducible score only trans-reactivated progeny up to the F5 (Fig 4G and 4H).
Although, trans-reactivation also works in reciprocal crosses where the trans-reactivating w Ã allele is carried by the mother (S1E and S1F Fig), our data show that w m4h trans-reactivation is dependent upon the presence of non-functional w Ã homologous genomic sequences present in at least one gamete (the sperm, in this set of experiment). Moreover, since fully functional coding wild type w + alleles are unable to trans-reactivate, our data also indicate that the w Ã reactivating alleles likely encode trans-reactivating factors able to maintain the trans-reactivated state of w m4h coming from the other gamete, a feature that does not meet the classic definition of paramutation. Indeed, paramutated alleles maintain their state independently from the presence of the paramutating allele. However, crosses in which both parents have exclusively w m4h trans-reactivated alleles strongly maintain the trans-generational inheritance of trans-reactivated w m4h , making trans-reactivation a new epigenetic phenomenon, distinct from paramutation, causing the trans-generational inheritance of trans-reactivated w m4h .
A diffusible factor carrying genetic information is responsible for transreactivation A long-standing question in the understanding of the mechanisms underlying epigenetic memory inheritance is whether a genetic information could be transferred from one allele to another through physical pairing of homologous chromosomes, or via a diffusible intermediate carrying genetic information. Although, our data indicate that trans-reactivation of w Ã over w m4h is independent from classic chromosome pairing (Fig 2C-2F), some of the features of w Ã transreactivation are highly reminiscent of the process known as trans-inactivation observed for the brown (bw) locus in flies, and of trans-induction occurring at the globin gene cluster in mammals, two epigenetic phenomena opposite in their final transcriptional outcome, but highly dependent on long-range chromosome physical interaction [16,30].
female was back crossed with w*/Y males carrying the trans-reactivating w* allele. Finally, the F2 progeny was scored for eye color pigmentation in control w m4h /w* females, w*/Y males and the experimental w m4h /Y males progeny, where the w* trans-reactivating allele had segregated. The possible F2 w m4h /Y males eye phenotypes coming from the cross are boxed. Eye pigment quantification of the P and F1 stocks, together with the resulting F2 control transheterozygous female (w m4h /w*), control male (w m4h /Y) and experimental segregating w m4h /Y male progeny for the w 1118 (B) and w sey (C) alleles, together with representative eye pictures for each genotype tested are shown. (D) Cross scheme used to test if the Δw/Y males stock, carrying a deletion of the entire w locus, was able to induce the inheritance of w m4h trans-reactivation in F2 w m4h /Δw females and w m4h /Y male progeny derived from segregating w m4h /w 1118 (E) and w m4h /w sey (F) F1 trans-heterozygous. (G) Cross scheme showing the possible F1 progeny arising from an homozygous trans-reactivated w m4h /w m4h female crossed with a trans-reactivated w m4h /Y male, (H) and the corresponding eye pigment quantification of the P and F1 progeny obtained with crosses with stably trans-reactivated parental w m4h /w m4h females and w m4h /Y males (produced with the trans-reactivating w sey allele). doi:10.1371/journal.pgen.1005444.g004

trans-Reactivation
Indeed, in trans-inactivation when two bw alleles are paired, heterochromatin insertion in one causes both alleles to associate with the heterochromatic centromere, suppressing brown expression [30]. On the other hand, in trans-induction intergenic transcription of the globin cluster specific for the erythroid cells can be induced in non-erythroid cells by the expression of transiently transfected globin genes [16]. Physical interaction of homologous chromatin region of the affected alleles appears to inactivate, in the case of trans-inactivation, or activate, in the case of trans-induction, chromosomal transcription. Indeed, the activating effect of w Ã alleles over w m4h could be explained by the two homologous inverted loci engaging on a longrange physical interaction. Under this model, the silent w m4h locus could dilute out heterochromatic factors over the non-functional w Ã homologous genomic sequences, causing a partial derepression of w m4h , thus explaining the opening of the chromatin locus, as well as the increased white transcription and eye pigmentation.
In order to determine if w Ã trans-reactivation is mediated by a long range physical interaction between the homologous w Ã and w m4h loci or instead through diffusible factors produced by the homologous genomic sequences present in the w Ã alleles, we made use of the unique reproductive features of flies able to generate adults through the process of gynogenesis [31,32]. Gynogenesis is like parthenogenesis in that diploid zygotes inheriting all chromosomes from their mother can develop without a genetic contribution from fathers. However, even though gynogenetic diploid eggs do not require paternal chromosomes, they do require the physical penetration of the sperm into the mature egg and the contribution of paternal diffusible nuclear and cytoplasmic factors in order to initiate zygotic development (Fig 5A). In particular, when crossing the paternal effect lethal ms(3) K81 males with gynogenetic gyn 2 , gyn 3 females, gynogenesis occurs and the only possible progeny resulting from this cross are gyn 2 , gyn 3 females (Fig 5A).
Therefore, we asked what happened to eye color pigmentation in the progeny resulting from w m4h gynogenetic gyn 2 ; gyn 3 eggs fertilized by ms(3) K81 males carrying the strong transreactivating w sey allele (Fig 5B). If a long range physical interaction between the male-carrying w sey and female-carrying w m4h chromosomes is required for trans-reactivation we should observe no increase in eye pigmentation in the w m4h ; gyn 2 ; gyn 3 F1 female progeny, because the father chromosomes do not contribute to the gynogenetic zygote ( Fig 5B). However, if diffusible factors carrying epigenetic information are present in the ms(3) K81 males encoding the trans-reactivating w sey allele, then diploid w m4h ; gyn 2 ; gyn 3 F1 female progeny should show trans-reactivated eyes ( Fig 5B). Remarkably, F1 w m4h ; gyn 2 ; gyn 3 females, not carrying any genomic contribution from the father, show strong trans-reactivated eyes when fertilized by ms (3) K81 males carrying the trans-reactivating w sey allele (Figs 5C and S7A). However, no effect on eye pigmentation was observed in the F1 progeny of gynogenetic females crossed with ms (3) K81 males carrying the Δw deletion (Figs 5D and S7B).
The data resulting from the gynogenesis experiment strongly rule out the possibility that trans-reactivation is due to any type of long-range physical genomic interactions between the w m4h and w Ã alleles. Instead, our data strongly indicates that diffusible factors, present in the gamete carrying the homologous trans-reactivating w Ã allele, are responsible for the phenomenon of trans-reactivation of w m4h .

Mutations in genes encoding for factors involved in the generation, modification, and processing of ncRNA influence trans-reactivation
The diffusible factors able to mediate trans-reactivation should carry a genetic information coming from the w Ã allele, epigenetically transfer this information to the w m4h locus, and amplify this information in the zygote in order to exert their effect later in the fully differentiated adult eye tissue. The high diffusibility, self-amplification property, and capacity to epigenetically transfer information make RNA a great candidate for such a trans-reactivating diffusible factor. We therefore looked at the effect of known mutations in genes involved in the generation, modification and processing of a variety of family of small ncRNA in the process of trans-reactivation (S7C and S7D Fig).
With the single exception of spd-E allele, mutations in genes encoding for factors involved in piRNA (armi, piwi, aub, hsp83, and ago2) and siRNA (dcr2 and r2d2) biogenesis enhanced the trans-reactivating effect of w Ã over w m4h (Fig 6A). On the other hand, mutations in genes process of gynogenesis in which a viable adult fly that does not carry any genetic contribution from the father is generated through the fertilization of a gynogenetic gyn 2 , gyn 3 egg (where diploidy is restored by the fusion of two non-sister nuclei out of the four egg pronuclei which result from the second meiotic division) by a ms(3) K81 sperm (one of a rare class of fly male sterile mutations in which sterility is caused by the elimination of male pronucleus after sperm entry into the egg). Despite, the male pronucleus (blue) does not contribute to the zygote, the male nucleoplasm (light blue) contributes together with the two female pronuclei (pink) to the development of a gynogenetic adult fly. (B) Cross scheme used to test if nucleoplasmic factors devoid of any male genomic contribution coming from the w* trans-reactivating allele can trans-reactivate w m4h in female gynogenetic adult flies. Possible F1 eye phenotypes coming from the cross are boxed. Eye pigment measurements of parental and gynogenetic progeny were conducted to score the presence of a trans-reactivated w m4h in (C) w m4h ; gyn 2 , gyn 3 female, w sey ; ms(3) K81 male parental stocks, and F1 gynogenetic w m4h ; gyn 2 , gyn 3 progeny, or in control (D) w m4h ; gyn 2 , gyn 3 female, Δw; ms(3) K81 male parental stocks, and F1 gynogenetic w m4h ; gyn 2 , gyn 3 progeny. Eye pigment quantification graphs have a different scale. , aubergine (aub QC42 ), hsp83 (hsp83 scratch ), spn-E (spn-E 1 ), argonaute2 (ago2), argonaute1 (ago1 04845 ), dicer1 (dcr1), dicer2 (dcr2), r2d2 (r2d2 1 ), and dnmt2 (dnmt2), were tested in heterozygosis for their ability to modify the levels of eye pigmentation we scored in w m4h /w* trans-reactivation. The Log 2 ratio of w m4h /w* trans-reactivation in the mutant background over the normal levels of w m4h /w* trans-reactivation was calculated encoding factors involved in the production of miRNA (ago1 and dcr1) suppress trans-reactivation, with the exception of ago2 that is also involved in the piRNA pathway ( Fig 6A). Recently, a non-coding RNA expressed from a human pseudogene was reported to regulate the corresponding protein-coding mRNA by acting as a decoy for microRNAs [33]. This study raised the questions about the potential ability of non-coding transcripts to act as 'sponges' to attract miRNAs, thus boosting the expression of miRNA target genes. If the trans-reactivating w Ã alleles produced miRNAs sponge RNA's we should expect that mutations in genes affecting miRNA biogenesis should enhance the effect of the trans-reactivating w Ã alleles over w m4h . However, our data clearly show that mutations in components of the miRNA processing machinery suppress trans-reactivation (Fig 6A and 6B), making it very unlikely that trans-reactivation could be explained by a miRNAs sponge effect. Remarkably, mutation in dnmt2, an RNA-dependent Methyl Transferase (RdMT) very recently shown to be involved in the phenomenon of paramutation in mice [34,35], has also strong suppressing effect on trans-reactivation ( Fig 6A). Overall, our genetic interaction data strongly indicate that altering the biogenesis, the post-translational modification and processing of a variety of small ncRNA families interfere at various levels the efficiency of trans-reactivation (Fig 6B), strongly supporting the involvement of RNA molecules in the onset and maintenance of this new epigenetic phenomenon.
In order to identify unique specific RNA species produced by the w Ã alleles during the process of trans-reactivation of w m4h we conducted high throughput RNA sequencing analysis of total as well as small RNA enriched fractions from larval Malpighian tubules, an abundant fly larval tissue that express high levels of the w gene. However, the analysis of short RNAs did not result in the identification of specific or enriched RNA species in the trans-reactivated w sey / w m4h line when compared to the w sey or w m4h lines alone (lower panel Fig 6C). On the other hand, the analysis of long RNA species clearly showed that the trans-reactivated w sey /w m4h line overall produces more coding RNA from the w locus than the single w sey or w m4h lines alone (upper panel Fig 6C and 6D), in line with our previous semi quantitative RT-PCR analysis ( Fig  3A). In conclusion, our RNA sequencing data did not allow us to identify specific or discrete RNA species present in the w sey trans-reactivating allele that could explain the onset of transreactivation.
However, if RNA species produced at the homologous w Ã alleles could influence in trans the transcription of the silenced w m4h locus, we expect that injection of w m4h eggs with total RNA extracted from w Ã individuals should result in trans-reactivated adult flies. Remarkably, when we injected w m4h eggs with total RNA extracted from w sey testis (Fig 6E) we obtained adults with trans-reactivated eyes. However, w m4h eggs injected with RNA derived from Δw testis had no effect (Fig 6E), strongly indicating that RNA molecules produced by the homologous w sey allele are responsible for the trans-reactivation of the w m4h locus. In conclusion, we propose that the RNA produced by the w Ã alleles from the w locus can induce in trans the activation of the silenced w m4h locus through a yet to be defined mechanism in which post transcriptional gene silencing and RNA methylation factors are involved. and plotted (see also S7D Fig). A positive value indicates that the mutation enhances trans-reactivation (dark orange bars), while a negative value is indicative of a suppression of trans-reactivation (pale orange bars). The strong trans-reactivating w sey allele was used in this assay. (B) A schematic representation of the biological significance of the data obtained shows that mutations in genes involved in the biogenesis of siRNA and piRNA counteract trans-reactivation (red bars). On the other hand, mutations in genes involved in the processing of miRNA and in RNA methylation (RdMT; RNA-dependent Methyl Transferase) promote trans-reactivation (green arrows). (C) High throughput RNA-Seq reads mapped on the Drosophila genome at the white gene locus for long and short RNA species purified from Malpighian tubules of Δw, w sey , w m4h and w sey /w m4h lines. (D) FPKM values plot obtained for the overall amount of long RNA species mapped at the w gene in the Δw, w sey , w m4h and w sey /w m4h lines. (E) Eye pigment quantification of w m4h females resulting from the injection of w m4h eggs with buffer (-) or total RNA extracted from testis of either Δw or w sey Drosophila males. The asterisks indicate the statistical significance (< 0.05) of differences in eye pigment using Student's t-test. doi:10.1371/journal.pgen.1005444.g006

Role for ncRNA in transcriptional regulation
Over the last decade we have come to appreciate that the eukaryotic chromatin is full of coding and non-coding overlapping transcripts, and that a lot of the genetic information is transacted by coding and non-coding RNAs. Indeed, the vast majority of genomes of all metazoans are transcribed into complex patterns of ncRNAs. Recent observations strongly suggest that ncRNAs contribute to the complex transcriptional networks needed to regulate cell function [36,37]. However, despite a rich variety of mechanisms by which RNA acts by repressing transcription have been identified, fewer studies have explored the role of RNA in the positively regulation of transcription. RNA is an integral component of chromatin [38] and many transcription factors and chromatin modifying enzymes have the capacity to bind RNA or complexes containing RNA or RNA-binding proteins [8]. Moreover, RNA-directed processes help to establish chromatin architecture and epigenetic memory [10]. Indeed, ncRNAs can act locally to regulate the epigenetic state of the nearby chromatin, often recruiting either repressing or activating chromatin remodeling complexes [8]. Given the high degree of sequence and locus specificity information, RNA could recruit chromatin remodelers and modifiers in a sequence-specific manner. Indeed, recent observations are beginning to reveal that RNA molecules can stimulate gene transcription. These RNA activators employ a wide array of mechanisms to up-regulate transcription of target genes, often functioning as DNA-tethered activation domains, as coactivators or modulators of general transcriptional machinery [39].

trans-reactivation: A new level of gene expression control of silenced genes?
In search for a possible role played by cellular RNA pools produced by homologous genomic loci in changing the transcriptional state of a silenced gene, we found that non-functional alleles of the classic fly marker white could trans-reactivate the expression of wild type copy of white silenced by heterochromatin. We called this new epigenetic phenomenon trans-reactivation. This process is heritable over many generations, it relies on the presence of diffusible RNAs, and it is affected by mutations in genes involved in post-transcriptional gene silencing. Our data strongly suggest that the presence of a gene that does not produce a full coding transcript but only spurious transcription could trans-reactivate the expression of a functional copy of the same gene silenced by heterochromatin, defining a new unpredicted level of gene expression control in the context of heterochromatic genes.

The mechanism underlying trans-reactivation
The mechanisms of action and many features of trans-reactivation appear to be different from the previously characterized epigenetic reprogramming events occurring in paramutation, transinduction and transvection. In trans-reactivation genomic regions, transcribing RNAs unable to produce a full coding transcript, can positively influence the transcriptional state of an homologous locus silenced by heterochromatin. However, how can the information present in the non-functional RNA molecules be 'read' and telecasted to specific silenced homologous genomic regions? RNA molecules have the potential to pair with other RNA molecule or with DNA. Possibly through their ability to form paired structures, RNA may function as a sensor and/or a messenger of homology. RNA can form duplexes with sequence homology with genomic promoter sequences thus promoting transcription [40]. Indeed, there is a great deal of evidence that enhancers and other promoter regulatory sequences are transcribed in the cell in which they are active, and that enhancer transcription is often needed to activate the transcription on their target gene [41]. Moreover, given that mutations in genes encoding for factors involved in posttranscriptional gene silencing modulate trans-reactivation, it remains to be determined whether some sequence specific small ncRNAs, that we failed to identify, may explain the mechanism of trans-reactivation. Furthermore, we do not know why some non-functional w Ã null or hypomorphic alleles failed to trans-reactivate their homologous silenced locus. Indeed, differences in trans-reactivation ability between different w Ã alleles will need future investigation, and may provide key information to understand the mechanism of action of trans-reactivation. However, independently from the mechanistic details of transreactivation, we are convinced that we identified a new epigenetic phenomenon of allelic communication that acts through diffusible RNA molecules. Nevertheless, much work need to be done to study the evolutionary conservation and the general ability of trans-reactivating RNAs in resetting the epigenetic and transcriptional state of homologous genetic loci.

Drosophila stocks and genetic crosses
Flies were raised and crossed at 20°on K12 Medium (US Biological). Unless otherwise stated, strains were obtained from Bloomington, Szeged, and DGRC Stock Centers. Recombination mapping of the trans-reactivating effect of w Ã alleles is described in S1 Fig. The white alleles used for the trans-reactivation assays are listed in S1 Table. The w 1 ; gyn-2 1 ;gyn-3 1

Pigment and variegation assays
The eye pigment assay was performed in triplicate for each of the three biological replicate we conducted, as previously described [22]. Eyes from representative individuals of the genotype tested were photographed with an Olympus SZ61 stereomicroscope. For the dominant Stubble variegation (Sb V ) assay ten pairs of major dorsal bristles were analysed in 20 flies. The extent of Sb variegation was represented as the mean of Sb and WT bristles per genotype tested in three biological replicate.

Fluorescence in situ hybridization (FISH)
Cytological preparations and FISH analysis were carried out as previously described [42] The P(CaSpeRA) plasmid containing the genomic sequences of the mini white as well as the hsp70 genes was used as a template for generating the FISH probe. FISH signals were acquired with a Leica DM 4000B microscope (Leica).

Semi-quantitative RT-PCR
Total RNA was purified from 20 heads of adult flies of the desired genotype, using TRIzol reagent following the manufacturer's (Invitrogen) recommended protocol. First strand cDNA was synthesized using MuLV reverse transcriptase (Applied Biosystems). Semi-Quantitative PCR was conducted using specific primer pairs that amplify the fourth, fifth and sixth exons of the white gene (forward, 5'-CTATAGGTCATATCTTGTTTTTATTGGCAC-3') (Reverse 3'-CGTGGGTGCCCAGTGTCC-5') and the following parameters: denaturation 2 min at 94°C, followed by 20 cycles of 30" at 94°C, 30" at 53°C, 2' at 72°C and final extension at 72°C for 3'. The PCR products were analyzed by agarose electrophoresis and the images were acquired and quantified with the ChemiDoc XRS imager (BioRad). The Act5C RNA was amplified as loading control with the primer pairs (forward, 5'-CCTCGTTCTTGGGAATGG-3', Act5C reverse, 3'-CGGTGTTGGCATACAGATCCT-5') using the same PCR cycle parameters.

RNA-sequencing
Total long RNAs were extracted from malpighian tubuli tissues using the miRNeasy Mini Kit, following an RNeasy MinElute Cleanup Kit (Qiagen) step for the enrichment of small RNAs. The 100bp paired-end sequencing was performed at IGA using a HiSeq2000 TM machine. Long RNAs were mapped against the Drosophila genome (assembly BDGP Release 5) using TopHat (PUBMED 19289445) with default parameters. Mapped reads were associated with gene annotations (FlyBase 5.12), and transcript levels of annotated genes, expressed as Fragments per Kilobase of exon Per Million fragments mapped (FPKM), were estimated by CuffCompare (PUBMED 20436464). For small RNAs, sequencing adapters were trimmed from sequence reads with custom scripts. Resulting sequences were mapped against the Drosophila genome using Bowtie (PUBMED 19261174) allowing at most two substitutions within the first 20 base pairs.

RNA-injection
w m4h ; CyORoi/Sco 3h starved flies were allowed to lay eggs for 50 minutes on apple juice agar plates with yeast. Approximately 100-150 decorionated eggs were injected with 1-2 pl of an injection solution containing 5 mM NaCl, 5% Texas Red conjugated dextran (Molecular Probes), and 1 ng/μl of total RNA extracted and purified from testis of either Δw or w sey Drosophila males.
Supporting Information S1 Fig. Screen scheme and genetic mapping. Detailed description of the cross schemes and fly genotypes used for the identification of w Ã alleles modifying w m4h variegation. Each of the w Ã allele tested (S1 Table) was screened for its ability to modify w m4h variegation when inherited from the father (A) or from the mother (B) using multiple isogenic modifier-free w m4hcarrying stocks (C, D). The advantage of testing w Ã alleles from the father is that in the F1 progeny it is possible to score not only the experimental trans-heterozygous w m4h /w Ã female but also the w m4h /Y internal control males, to exclude autosomal background effects. (E, F) Eye pigment quantification of parental stocks (P) and the resulting trans-heterozygous females (w m4h /w Ã ) derived from reciprocal crosses employing w sey /w sey and w 1118 /w 1118 females, are shown together with representative eye pictures for each genotype tested. P and F1 eye pigment quantification graphs have different scale. To map the genetic loci responsible for the increase in eye pigmentation in w m4h /w Ã trans-heterozygous we used classic recombination mapping on the X chromosome using recessive markers (G). Single recombinant F2 males carrying the w Ã interacting alleles flanked by the recessive y 1 and cv 1 markers were retested with In(1)w m4h stocks confirming that the genetic interaction scored mapped the tip of the X chromosome between cytological map 1A5 and 5A13, where the w gene maps in 3B6 (H). To help follow the transmission of individual chromosomes in the above described crosses, female chromosomes were represented in pink while male chromosomes are shown in blue. (JPG) S2 Fig. Hypomorphic white alleles increasing w m4h eye color pigmentation. Eye pigment quantification of parental stocks (P) and of the resulting trans-heterozygous female (w m4h /w Ã ) and control male (w m4h /Y) progeny (F1) derived from the hypomorphic w is (A), w bf (B), w ch (C), w a4 (D), w a (E) and w Bwx (F) alleles (see S1 Table and Experimental Procedures for the exact genotypes and nature of the lesion of the alleles tested), are shown together with representative eye pictures for each genotype tested. P and F1 eye pigment quantification graphs have different scale. Note that, the parental hypomorphic w Ã alleles tested (P) look with darker eyes when compared to the parental w m4h line (P). However, the eye color pigment present in the population of the parental w m4h is greater than the one read from the hypomorphic w Ã alleles. This apparent contradiction can be explained by the fact that while the hypomorphic w Ã flies have all an homogenous eye color, the w m4h line eye shown in the picture is a representative example of a much more heterogeneous population containing few very dark pigmented eyes that contribute to an overall higher pigment reading. Loss of function mutations in smaug (smg 1 ) and pumilio (pum 13 ), two RNA binding protein involved in mRNA destabilization in flies, are embryonic lethal. Thus, the ability of smg 1 and pum 13 loss of function alleles to modify the levels of eye pigmentation (trans-reactivation) we scored in w m4h /w Ã females (+) was tested in heterozygosis (pum and smg). Polytene chromosome FISH using genomic probes covering the entire w gene (with the exception of the first intron) and coding sequences for the hsp70 gene (mapping chromosome 3R) on homozygous w wt /w wt (B), and transheterozygous w m4h /Δw (C) combinations. FISH signals for white (red) and hsp70 (green) genomic sequences are indicated by arrowheads. The asterisk indicate the region of pericentric heterochromatin. The X indicates the chromosome where the w gene maps. (JPG) S6 Fig. Cross schemes used to test trans-generational inheritance of w m4h trans-reactivation. Detailed description of cross schemes and fly genotypes tested as experimental and control classes to measure trans-generational inheritance of w m4h trans-reactivation from (F1) trans-heterozygous w m4h /w Ã females when crossed with parental w Ã /Y (A) or Δw /Y (B) males. To help follow the transmission of individual chromosomes, female chromosomes are represented in pink while male chromosomes are shown in blue. (C) Trans-generational inability of a wild type w + allele (coming from an OreR stock) to trans-reactivate in F2 the w m4h locus. Eye pigment quantification and representative eye pictures of the parental (P), F1 and F2 progenies for each genotype tested are shown. (JPG) S7 Fig. Cross schemes used to test that trans-reactivation is mediated by a diffusible factor or influenced by mutations in PTGS genes. Detailed description of cross schemes and fly genotypes tested as experimental (A) and control (B) classes to measure trans-reactivation of w m4h during the process of gynogenesis. (C) Cross scheme used to test in heterozygosis the ability of loss of function alleles (Mut) to modify trans-reactivation. (D) Eye pigment quantification was conducted on adult flies of the indicated genotype and the Log 2 ratio of w m4h /w Ã trans-reactivation in the mutant background over the normal levels of w m4h /w Ã trans-reactivation (normalized for the respective readings on the w m4h /w m4h lines alone) was used as a measure of the influence of the mutation tested in the onset of trans-reactivation. To help follow the transmission of individual chromosomes, female chromosomes are represented in pink while male chromosomes are shown in blue. (JPG) S1 Table. Genotypes and nature of the lesion of the w Ã alleles tested. Genotypes of stocks carrying the w Ã alleles tested for their ability to modify in trans-heterozygosity the w m4h eye color variegation. Stocks that increased eye color variegation are highlighted in orange (Suppressors), the ones that did not have any effect are highlighted in light red (-). Tested white genomic deletions are highlighted in light grey. Finally, the mutation class, the mutagen employed and the Nature of the lesion, for each w Ã allele tested is reported when known. (DOCX)