Transposable element misregulation in Drosophila buzzatii–Drosophila koepferae interspecific hybrids

Els elements transposables (ETs) son unitats genetiques mobils presents en practicament tots els organismes eucariotes sequenciats. La seva capacitat de moure’s, juntament amb el seu caracter repetitiu, els converteix en importants mutagens amb l’habilitat de crear noves variants genetiques susceptibles a la seleccio. Donat que el seu potencial mutagenic pot posar en perill la fitness de l’hoste, els organismes eucariotes han desenvolupat diferents estrategies de regulacio per controlar la mobilitzacio d’ETs. Cal destacar la importancia d’aquestes estrategies en linia germinal, on les mutacions poden ser transmeses d’una generacio a l’altra. En ovaris de Drosophila, el principal mecanisme de regulacio d’ETs es la via dels piRNAs, que contribueix al seu silenciament transcripcional i post-transcripcional. La forta regulacio a la que els ETs estan sotmesos es pot veure relaxada sota diferents condicions d’estres, com es el cas de la hibridacio interespecifica. Diversos estudis han descrit noves insercions d’ETs en hibrids interespecifics, tant d’animals com de plantes. En el cas que ens ocupa, els hibrids de Drosophila buzzatii i Drosophila koepferae, investigacions previes del nostre grup van detectar la mobilitzacio d’almenys 28 ETs. No obstant, els mecanismes responsables d’aquesta activacio son encara desconeguts, tot i que els estudis mes recents del camp semblen apuntar a una desregulacio a nivell d’expressio. Tambe es desconeixen els efectes que la proliferacio d’ETs pot tenir sobre el genoma dels hibrids. En aquest treball, comencem avaluant l’impacte de la hibridacio sobre la mida del genoma dels hibrids de Drosophila buzzatii i Drosophila koepferae al llarg de quatre generacions d’encreuaments hibrids (un primer d’interespecific seguit de quatre retroencreuaments). Demostrem l’existencia d’una expansio genomica sexe-especifica, que afecta nomes les femelles del primer retroencreuament. Aquests resultats representen la primera evidencia d’un augment de la mida del genoma en hibrids interespecifics d’especies animals. La nostra hipotesi es que una desregulacio a nivell transcripcional te lloc a les femelles de la F1, donant lloc a noves insercions que es detecten a la seguent generacio. Per tal de testar aquesta hipotesi, hem realitzat dos estudis d’expressio d’ETs, emprant dues aproximacions diferents. Primer, duem a terme una analisi en profunditat de l’expressio del retrotransposo Helena (un dels ETs que transposen en els nostres hibrids) en ambdos sexes i diferents teixits. Demostrem que l’expressio d’Helena en teixit somatic no es alterada degut a la hibridacio, mentre que en gonades s’observen efectes sexe-especifics. En testicles de la F1, observem una repressio d’Helena, concordant amb l’absencia de canvi en la mida del genoma dels mascles. En ovaris, sembla que Helena es desregula en mosques joves, pero els nivells d’expressio baixen en mosques de major edat. Posteriorment, descrivim una analisi a nivell transcriptomic, on s’avalua si els resultats d’Helena son extrapolables a l’expressio global dels ETs. Per esbrinar quins mecanismes estan involucrats en la desregulacio d’ETs, analitzem tambe les poblacions de piRNAs d’especies parentals i hibrids. Els nostres resultats demostren que els testicles de la F1 tendeixen a presentar nivells d’expressio mes baixos que D. buzzatii, probablement degut a un augment dels nivells de piRNAs. En ovaris, l’efecte mes comu es la sobreexpressio d’ETs, que podria ser explicada per incompatibilitats en la via dels piRNA entre les dues especies parentals. De fet, les proteines d’aquesta via es troben entre les mes divergents entre les dues especies. D’altra banda, alguns casos de desregulacio poden ser explicats per diferencies entre els nivells de piRNAs entre els citoplasmes de D. buzzatii i D. koepferae, com en el cas de la disgenesi hibrida. Finalment, cal destacar que son necessaries altres explicacions per explicar el patro global de desregulacio, com ara un funcionament anormal d’altres vies de regulacio d’ETs o de la modificacio d’histones. Transposable elements (TEs) are mobile genetic units present in almost all the eukaryotic sequenced genomes. Their mobilizing capacity, together with their repetitive nature, makes them powerful endogenous mutators able to create novel genetic variants, which will be then subject to selection. However, their mutagenic potential can also endanger their host’s fitness, which has led to the development of several regulatory strategies against TE mobilization in eukaryotic organisms. These are especially important in the germline, where mutations can be transmitted to the offspring. In Drosophila ovaries, TEs are mainly regulated by a small RNA-mediated silencing mechanism, the piRNA (Piwi-interacting RNA) pathway, which affects transcriptional and post-transcriptional TE silencing. This strong regulation can be relaxed under several stress conditions, including interspecific hybridization, a genomic stressor that promotes TE mobilization. Several cases of transposition events have been described in hybrids of different species, including both animals and plants. In the case that concerns us, D. buzzatii–D. koepferae hybrids, a previous survey in our group detected mobilization of at least 28 TEs. However, the molecular mechanisms underlying this TE release remain elusive, although recent studies on hybrid TE expression seem to point to a transcriptional deregulation. Furthermore, little is known about the effects this phenomenon can have in the genome of the hybrid progeny. In this work, we first assess the impact that hybridization-induced TE proliferation has on the genome size of D.buzzatii–D. koepferae hybrids, throughout four generations of hybridization (an interspecific cross followed by three backcrosses). We demonstrate the existence of a sex-specific genome expansion, that affects only females at the first backcross. These results provide the first evidence of genome size increase in interspecific hybrids of animal species. We hypothesize that a TE deregulation at a transcriptional level occurs in F1 females, leading to new TE insertions that result in a genome size increase in the following generation. In order to test this hypothesis, we address two TE expression studies in the same hybrids, using two different approaches. First, we perform an in-depth analysis of the expression of one of the mobilized transposons, Helena, in both sexes and different tissues. We show that Helena expression in somatic tissues is not altered after hybridization, whereas in gonads sex-biased effects are observed. Indeed, Helena is repressed in F1 testes, in concordance with the unaltered genome size in males. In ovaries, an early Helena overexpression seems to occur in young flies, being then controlled in older ones. We subsequently performed a global analysis using a transcriptomic approach, in order to evaluate if the results for Helena could be extended to other TEs. To disentangle the molecular mechanisms involved in TE deregulatiom, we analysed the piRNA populations of parental species and hybrids. We show that F1 testes indeed tend to present a TE expression lower than D. buzzatii, which is coupled with a global increase of piRNA amounts. In ovaries, TE overexpression is the more common effect, and seems to be mainly due to differences in piRNA production strategies between parental species. Actually, the piRNA pathway proteins are divergent between parental species and could be at the origin of the hybrid instability. Moreover, differences in piRNA amounts between D. buzzatii and D. koepferae cytoplasms could also account for some cases of deregulation, as occurs in hybrid dysgenesis syndrome. Finally, other explanations are needed to account for the whole pattern of deregulation, such as the failure of histone modification’s deposition or of other TE silencing pathways.