Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

<p>Abstract</p> <p>Background</p> <p>Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the <it>HeT-A </it>retrotransposon. The sequence of the <it>HeT-A </it>retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the <it>HeT-A </it>is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different <it>HeT-A </it>scenarios.</p> <p>Results</p> <p>A detailed study on the variability of <it>HeT-A </it>reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of <it>HeT-A </it>subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the <it>HeT-A </it>RNA. Interestingly, our results also suggest that most <it>HeT-A </it>copies are actively expressed regardless of which telomere and where in the telomere they are located.</p> <p>Conclusions</p> <p>Our study demonstrates how the <it>HeT-A </it>sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.</p

Caracterització del cicle de vida dels retrotransposons telomèrics HeT-A i TART de Drosophila melanogaster

[cat] Els telòmers són estructures dels extrems dels cromosomes linials d’organismes eucariotes. La longitud telomèrica es manté mitjançant l’enzim telomerasa en la majoria d’eucariotes, on s’hi troba altament conservat. Drosophila, en canvi, empra un mecanisme de replicació telomèrica alternatiu: els retrotransposons de tipus no-LTR anomenats HeT-A, TART i TAHRE. Estudis anteriors han hipotetitzat el cicle de vida que duen a terme aquests transposons per elongar els telòmers, no obstant, aquest encara no ha estat demostrat ni es coneixen els factors cel•lulars que hi participen. A més, no s’ha estudiat mai anteriorment la localització de les proteïnes endògenes de HeT-A i TART, així com només s’ha pogut observar la localització dels seus trànscrits en organismes mutants que sobreexpressen aquests retrotransposons. Aquesta tesi doctoral presenta una caracterització detallada de les proteïnes i els RNAs de HeT-A i TART a diferents teixits salvatges i mutants de Drosophila melanogaster. S’hi identifiquen també diverses proteïnes cel•lulars que interaccionen amb les proteïnes telomèriques de Drosophila, i que podrien estar participant en el seu cicle de vida. D’aquestes, les més rellevants són Nap-1, Z4, Lost i Tral, les quals s’han analitzat en major detall. Algunes de les funcions que aquestes proteïnes estarien duent a terme en el cicle de vida telomèric de Drosophila són: regular l’expressió de HeT-A, localitzar i ajudar al transport correcte de les proteïnes i els RNAs telomèrics dins la cèl•lula, processar els trànscrits telomèrics o regular-ne la traducció, i participar en l’estabilitat telomèrica. En global, aquest estudi contribueix a incrementar els coneixements que es tenien fins ara sobre el cicle de vida telomèric de Drosophila, així com obre tot un ventall de noves proteïnes i factors a investigar en futurs estudis sobre els telòmers d’organismes eucariotes.[eng] Telomeres are structures at the end of the chromosomes of eukaryote organisms. Telomere length is maintained by telomerase enzyme in most eukaryotes, however, Drosophila uses an alternative mechanism to replicate telomeres: the non-LTR retrotransposons HeT-A, TART and TAHRE. Previous reports have hypothesized the life cycle that these transposons perform in order to increase telomere length but it has never been confirmed, and little is known about which cellular factors may participate in it. Even more, the localization of the endogenous proteins of HeT-A and TART has never been studied, and their transcripts have only been observed in mutant flies that overexpress these retrotransposons. This PhD thesis analyzes the localization of HeT-A and TART proteins and RNAs in wild-type and mutant tissues of Drosophila melanogaster. Different cellular proteins that interact with the telomere proteins, and might be involved in the life cycle of HeT-A and TART, are identified. Among them, Nap-1, Z4, Lost and Tral are the most relevant, so we have studied them in detail. Some of the roles that these proteins may be doing in the life cycle of Drosophila telomeres are: regulation of HeT-A expression, cellular localization and transport of the telomere proteins and RNAs, processing and translation regulation of HeT-A and TART transcripts, and telomere stability. Overall, this study contributes to increase the knowledge about the life cycle of Drosophila telomeres, and opens up a range of new proteins and factors to be investigated in future studies about eukaryote telomeres

Localization of the <i>HeT-A</i> Gag and <i>TART</i> Pol proteins in <i>wt</i> and mutant neuroblasts.

<p>Immunofluorescence detection of <i>HeT-A</i> Gag (green) and <i>TART</i> Pol (red). DNA stained in blue. First column (at left): merge of the three channels. Second column (middle): <i>HeT-A</i> Gag (green). Third column (at right): <i>TART</i> Pol (red). First and second row: <i>wt</i> neuroblasts. Third and fourth row: <i>GIII</i> neuroblasts. Arrowheads indicate co-localization of <i>HeT-A</i> Gag and <i>TART</i> Pol (visible in yellow).</p

Distribution of the <i>TART</i> sense RNA in <i>wt</i> and mutant ovaries.

<p>Fluoresence in situ hybridization to <i>HeT-A</i> sense RNA (red). DNA is shown in blue. Images were obtained using 40x magnification, except for those specified otherwise. (A-D) <i>wt</i> ovaries. (A) Follicle cells (external view) of a stage 5–6 egg chamber. (B) Stage 9 egg chamber. (C) Stage 10. (D) Magnification of a nurse cell nucleus from (C). Arrow indicates localization inside the nucleus of a nurse cell. This image was obtained using 63x magnification. (E,F) <i>GIII</i> ovaries. (E) Follicle cells (external view) of stage 5–6 egg chamber. Arrow shows localization inside the nucleus of follicle cells. (F) Stage 9. (G-I) <i>aub</i> mutant ovaries. Arrows indicate detection in the nucleus of nurse (left) and follicle cells (right). (G) Early stages (germarium and stages 3–4 and 5, from left to right). Arrow shows localization inside the nucleus of a nurse cell. (H) Stage 8. Arrow indicates detection in the nucleus of a nurse cell. (I) Stage 9. Arrow shows localization inside the nucleus of a nurse cell.</p

Distribution of <i>HeT-A</i> sense RNA in <i>wt</i> and mutant ovaries.

<p>Fluoresence in situ hybridization to <i>HeT-A</i> sense RNA (red). DNA is shown in blue. Images were obtained using 40x magnification, except those specified otherwise. Images for the three genotypes were captured with the same microscope settings. Images in (E-N) have been adjusted to a lower intensity in order to visualize the mRNA distribution. (A-D) <i>wt</i> ovaries. (A) Stage 5–6 egg chamber. Arrows indicate localization close to the nucleus of nurse cells. (B) Stage 9. Arrow shows localization inside the nucleus of nurse cells. (C) Stage 10. (D) Magnified view of the border cells boxed in (C). Arrow indicates localization in the nucleus of border cells. (E-H) <i>GIII</i> ovaries. (E) Stage 8. Arrow indicates intense localization in the nucleus of nurse cells. (F) Stage 10. (G) Magnification of the border cells boxed in (F). Arrow shows intense localization in the posterior side of border cells cytoplasm. (H) Stages 10a and 10b. This image was obtained with 20x magnification. (I-N) <i>aub</i> mutant ovaries. (I) Stage 4. Arrow indicates localization inside the oocyte. (J) Stages 6–7 (left) and 8 (right). Arrows show localization in the oocyte. (K) Stage 8. Arrows indicate localization in the oocyte and in the cytoplasm of nurse cells. (L) Stage 9. Arrows show localization in the oocyte and in the cytoplasm of nurse cells. (M) Stage 10. Arrow indicates localization at the anterior side of the oocyte. (N) Stage 10 nurse cells. Arrow shows detection in the cytoplasm of a nurse cell.</p

Localization of <i>HeT-A</i> Gag protein in <i>wt</i> and mutant ovaries.

<p>Immunofluorescence detection of <i>HeT-A</i> Gag (red). DNA is stained in blue. Images were obtained using 40x magnification, except those specified otherwise. Arrowheads correspond to differences in localization between mutant and <i>wt</i> ovaries. (A-C) <i>wt</i> ovaries. (A) Germarium and stage 1 egg chamber. (B) Stage 3–4. (C) Stage 5–6. (D) Magnified image of follicle cells. (E-G) <i>GIII</i> ovaries. (E) Germarium and stage 1. Arrow indicates high levels of detection in follicle cells. (F) Stage 2–3. Arrow shows high levels of detection in follicle cells. (G) Stage 5–6. Arrow indicates intense signal in follicle cells. (H-M) <i>aub</i> mutant ovaries. (H) Germarium (I) Stage 2–3. (J) Stage 4–5. Arrow shows localization in the oocyte. (K) Stage 8. Image obtained using 20x magnification. Arrow indicates detection in the nucleus of nurse cells. (L) Magnification of boxed nurse cell nucleus in (J). Image obtained using 63x magnification. Arrow shows localization in the nucleus of a nurse cell. (M) Stage 8. Image obtained using 20x magnification. Arrow indicates detection in the oocyte.</p

Localization of the <i>TART</i> Pol protein in <i>Drosophila melanogaster wt</i> and mutant ovaries.

<p>Immunofluorescence detection of <i>TART</i> Pol (red). DNA stained in blue except for (B). Images were obtained using 40x magnification, except those specified otherwise. <i>(</i>A-E) <i>wt</i> ovaries (A) Germarium. (B) Stage 2–3. Arrow shows perinuclear detection in nurse cells. (C) Stage 4. Arrow indicates localization inside the nucleus of nurse cells. (D) Stage 10. Image obtained with 20x magnification. (E) Magnified view of the border cells in (D) (posterior up). (F-H) <i>GIII</i> ovaries. (F) Germarium and stage 1. (G) Stage 2–3. (H) Stage 4–5. (I-K) <i>aub</i> mutant ovaries. (I) Germarium and stage 1–2. (J) Stages 2 and 4. (K) Stage 5–6. Arrow indicates localization in the nucleus of nurse cells.</p

Detection of Gag and Pol proteins in S2 cells and larval brain extracts.

<p>(A) Western blot developed with α-<i>HeT-A</i> Gag antibody. Lane 1: S2 cells; Lane 2: S2 cells transfected with <i>HeT-A</i> Gag-GFP; Lane 3: S2 cells transfected with <i>HeT-A</i> Gag-Flag; Lane 4: brains of 3^rd instar <i>GIII</i> larvae. Both the recombinant and the endogenous HeT-A Gag proteins are detected. (B) Western blot developed with α-<i>TART</i> Pol antibody. Lane 1: extract from stable transfected S2 cells <i>TART</i>-RT-CTAP; Lane 2: extract from brains of 3^rd instar <i>GIII</i> larvae. Both the recombinant and the endogenous RT proteins are detected.</p

The Chromosomal Proteins JIL-1 and Z4/Putzig Regulate the Telomeric Chromatin in <em>Drosophila melanogaster</em>

<div><p><em>Drosophila</em> telomere maintenance depends on the transposition of the specialized retrotransposons <em>HeT-A</em>, <em>TART</em>, and <em>TAHRE</em>. Controlling the activation and silencing of these elements is crucial for a precise telomere function without compromising genomic integrity. Here we describe two chromosomal proteins, JIL-1 and Z4 (also known as Putzig), which are necessary for establishing a fine-tuned regulation of the transcription of the major component of <em>Drosophila</em> telomeres, the <em>HeT-A</em> retrotransposon, thus guaranteeing genome stability. We found that mutant alleles of <em>JIL-1</em> have decreased <em>HeT-A</em> transcription, putting forward this kinase as the first positive regulator of telomere transcription in <em>Drosophila</em> described to date. We describe how the decrease in <em>HeT-A</em> transcription in <em>JIL-1</em> alleles correlates with an increase in silencing chromatin marks such as H3K9me3 and HP1a at the <em>HeT-A</em> promoter. Moreover, we have detected that <em>Z4</em> mutant alleles show moderate telomere instability, suggesting an important role of the JIL-1-Z4 complex in establishing and maintaining an appropriate chromatin environment at <em>Drosophila</em> telomeres. Interestingly, we have detected a biochemical interaction between Z4 and the HeT-A Gag protein, which could explain how the Z4-JIL-1 complex is targeted to the telomeres. Accordingly, we demonstrate that a phenotype of telomere instability similar to that observed for <em>Z4</em> mutant alleles is found when the gene that encodes the HeT-A Gag protein is knocked down. We propose a model to explain the observed transcriptional and stability changes in relation to other heterochromatin components characteristic of <em>Drosophila</em> telomeres, such as HP1a.</p> </div

<i>HeT-A</i> expression normalized to copy number in different mutant alleles.

<p><i>HeT-A Gag</i> (A) and <i>HeT-A</i> 3′UTR (B) transcripts decrease in <i>JIL-1</i> mutants (<i>JIL-1^z60</i>/+, <i>JIL-1^z2</i>/+ and <i>JIL-1^z60</i>/<i>JIL-1^z2</i>) and in <i>Z4</i> mutants when combined with <i>JIL-1^z2</i> null allele (<i>JIL-1^z2</i>/<i>z4^7.1</i>, <i>JIL-1^z2</i>/<i>Z4^2.1</i> and <i>JIL-1^z2</i>/<i>pzg⁶⁶</i>). <i>Z4^7.1</i> allele also affects <i>HeT-A Gag</i> transcripts but in this case an increase in the expression is observed. The <i>Su(var)2-5⁰⁵</i> mutant allele, was used in this experiments as a positive control. <i>HeT-A</i> transcript levels were normalized to actin transcripts and corrected for the respective <i>HeT-A</i> copy number in each analyzed stock. Error bars represent standard deviations of three independent experiments. Asterisks indicate statistically significant differences using the t-test (one asterisk, <i>P</i><0.05 to 0.01; two asterisks, <i>P</i><0.01 to 0.001; three asterisks, <i>P</i><0.001) in <i>HeT-A</i> expression of each mutant compared to <i>w¹¹¹⁸</i>.</p