Short and long-term evolutionary dynamics of subtelomeric piRNA clusters in Drosophila

Abstract

Two Telomeric Associated Sequences, TAS-R and TAS-L, form the principal subtelomeric repeat families identified in Drosophila melanogaster. They are PIWI-interacting RNA (piRNA) clusters involved in repression of Transposable Elements. In this study, we revisited TAS structural and functional dynamics in D. melanogaster and in related species. In silico analysis revealed that TAS-R family members are composed of previously uncharacterized domains. This analysis also showed that TAS-L repeats are composed of arrays of a region we have named "TAS-L like" (TLL) identified specifically in one TAS-R family member, X-TAS. TLL were also present in other species of the melanogaster subgroup. Therefore, it is possible that TLL represents an ancestral subtelomeric piRNA core-cluster. Furthermore, all D. melanogaster genomes tested possessed at least one TAS-R locus, whereas TAS-L can be absent. A screen of 110 D. melanogaster lines showed that X-TAS is always present in flies living in the wild, but often absent in long-term laboratory stocks and that natural populations frequently lost their X-TAS within 2 years upon lab conditioning. Therefore, the unexpected structural and temporal dynamics of subtelomeric piRNA clusters demonstrated here suggests that genome organization is subjected to distinct selective pressures in the wild and upon domestication in the laboratory.

Keywords: Drosophila; TAS; piRNA; subtelomeres; transposable element.

Figure 1

Subtelomeric regions in Drosophila melanogaster. (A) X subtelomeric region from the Dp1187 minichromosome contains four TAS repeats [TAS-A (205 bp), TAS-B (1,872 bp), TAS-C (1,858 bp) and TAS-D (1,575 bp)] located between telomeric elements (HeT-A) and two 0.9 kb repeats. (B–D) Schematic representation of the TAS-R family repeats represented by TAS-B for X-TAS (B), 2R-TAS (C) and 3R-TAS (D) located on the X, second and third chromosomes, respectively. They are composed of several domains [INV-4 solo LTR, T1, T2, T3, T4 and T5 domain]. In order to simplify the comparison between members of TAS-R family, the starting point for each TAS repeat was arbitrary positioned at the beginning of the INV-4 sequence. (E) TAS-L repeats on 2L and 3L chromosomes are composed of six fragments of a sequence found in the T3 domain of X-TAS repeats, that we named TLL (hatched boxes). (F) The TLL domain is also found in the 0.9-kb repeats adjacent to X-TAS. The T3-TLL sequence (B) is taken as the reference here for determining percentage of identity between sequences (E, F). Identity between the TLL of 164 bp in the 0.9 kb repeat and the TLL of 147 bp in TAS-L is 85%. (G) The X chromosome of the Canton-S strain is lacking X-TAS repeats. (H) The X chromosome of the y¹; cn¹bw¹sp¹ line lacks both X-TAS repeats and 0.9 kb repeats. In all schematic representations of telomeric sequences the telomeres are to the left. TAS-R probes used in in situ hybridization were PCR amplified using primers indicated by black arrows (A). Primers used to PCR amplify TAS-L repeats are indicated by arrows (E).

Figure 2

Subtelomeric piRNA clusters in the melanogaster subgroup. (A) D. sechellia Scaffold 31 contains five complete 855 bp repeats. Each repeat is composed of two subrepeats (191 and 138 bp) with sequence identities to D. melanogaster TLLs (hatched boxes), one 129 bp sequence derived from TLL (hatched boxes) (Supplementary Fig. S7) and two unrelated sequences of 274 bp and 123 bp. (B) D. simulans Scaffold 3R contains a 403-bp region composed of one TLL and two sequences of 105 and 129 bp displaying sequence identity with the 129 bp of D. sechellia followed by a T2 and a T5 domains. (C) In situ hybridizations on D. sechellia, D. simulans (Chantemesle) and D. melanogaster (Oregon-R-C) polytene chromosomes were performed using the D. sechellia 855 bp repeats as a probe. The probe was PCR amplified using primers shown by arrowheads in (A). Probe hybridizations at telomeres indicated by arrowheads are consistent with the in silico analysis. Labelling at the D. melanogaster centromeres can also be detected corresponding to smaller domains of identity (see text). (D–E) TLL-containing domains identified in D. sechellia and D. simulans are part of subtelomeric piRNA clusters. Normalized germline small RNAs were mapped to each reference sequences: the 855 bp D. sechellia repeat, the 403 bp D. simulans sequence and the 158 bp-TLL D. melanogaster sequences. (D) Size distribution histograms indicate that the majority of small RNAs produced from telomeric regions in ovaries of the three Drosophila species are between 23 and 29 nt in length. The number in each panel is the proportion of 23–29 nt beginning with a 5′ uridine (1 U bias). A bias exists toward sense (plus strand) vs. antisense (minus strand) strand production. (E) Readmaps show the abundance and the distribution of the piRNAs mapping to each of the reference sequences.

Figure 3

Schematic representation of X subtelomeric regions after X-TAS loss. Four types of X subtelomeres were identified: (A) with one each of the TAS-B, -C, -D and the 0.9 kb repeats like the Dp1187 minichromosome; (B) with only theTAS-D repeat and the 0.9 kb repeats like NA-P(1A); (C) with only the 0.9 kb repeats like Canton-S; (D) with neither TAS repeats nor the 0.9 kb repeats like y¹; cn¹bw¹sp¹. Stocks found to be devoid of X-TAS repeats by in situ hybridization and/or PCR amplification were tested to identify their X subtelomeric structure. This was assayed by PCR amplifications using specific primers for TAS-B (see arrows under), TAS-D (see arrows) and the 0.9 kb repeats (see arrows) (A). Noticeably, the 0.9 kb repeats were systematically amplified in stocks that possess TAS-B and/or TAS-D. The 0.9 kb repeats can also be stably maintained on their own at the tip of the X chromosome as observed for the Canton-S strain. Strains shown to have recently lost X-TAS (see Table 3) are marked by an asterisk (*). Loss of X-TAS might occur by telomeric terminal breakage rather than by internal deletion that might be repaired by transposition of new telomeric HTT (HeT-A, TAHRE).

Figure 4

Distribution of piRNA subtelomeric clusters in recently collected D. melanogaster. Results of analysis of the Lk-P(1A) laboratory line and three recently collected lines [Chantemesle and Reth (Europe) and Cacao H1C (South America)] are shown on first, second, third and fourth columns, respectively. (A) Polytene chromosomes of salivary gland of third instar larvae (DAPI staining) were hybridized with specific probes for TAS-R repeats. TAS-R sequences are present on the X and 2R telomeres in the Lk-P(1A) line and on X, 2R and 3R telomeres in Chantemesle, Reth and Cacao H1C. Other examples of TAS-R localization in various stocks can be found in Supplementary Figs. S8 and S9 and summarized on Tables 1 and 2. (B-G) Graphs show normalized profiles of ovarian small RNAs mapping with 0 mismatches to subtelomeric loci [T3 domain, representative of X-TAS belonging to the TAS-R family (B, C), the 0.9 kb repeats (D, E) and the TAS-L family (F, G)]. Size distribution histograms (B, D and F) and read maps (C, E and G) show the length of small RNA (20–29 nt) and the abundance of normalized piRNAs (23–29 nt) matching to each reference sequence, respectively. Numbers at the bottom right of the size distribution histograms indicate the ratio of 23–29 nt small RNAs starting with an uridine (1 U bias) (ND: Not Determined). Although the piRNA profiles between lines appear similar, differences in the amount of piRNA can be noticed. Absence of TAS-L piRNA in Lk-P(1A) suggests that the 2L and 3L-TAS loci were lost. PCR amplification screen using TAS-L specific primers identified a total of two stocks out of 43 missing the TAS-L family sequences (Tables 1 and 2). This suggested that, contrary to the TAS-R family, complete deletion of TAS-L loci does not affect viability. Other analysis of small RNA profiles can be found in Supplementary Fig. S10.

Figure 5

Complete loss of the X-TAS locus in a two-year period. In situ hybridizations on polytene chromosomes from salivary glands from third instar larvae were performed on stocks at different time points after collection from the wild. Results are presented for two Marsais 53Ja sublines maintained separately (sublines A and B) after 2 years under laboratory conditions. (A) In stock A, the three TAS-R loci are labeled thus presenting the same pattern as when tested a few months after the capture (data not shown). Chromosomal ends of the two 3R arm homologs have been separated during the squash, showing the labeling on each of the homologous chromosomes. (B) In stock B, only two TAS (at 2R and 3R) are labeled, whereas a short time after capture all three TAS-R loci were present (data not shown) indicating the loss of X-TAS in this subline. Therefore, it is possible to witness, in the space of a two-year period, the loss of an X-TAS from a line introduced into the laboratory from the wild. Numbers in parenthesis indicate the number of nuclei studied isolated from ten male larvae. Additional in situ hybridizations are discussed in the Supplementary results.

Figure 6

Model of succession of genomic events during the course of TAS family repeat evolution in the melanogaster subgroup. Shown on the melanogaster subgroup phylogenetic tree, an ancestral TLL gave rise to subtelomeric repeats in D. erecta and D. yakuba on the one hand, while on the other hand duplication of TLL and insertion of sequences leading to the T2 and T5 domains occurred in the common ancestor of D. sechellia, D. simulans and D. melanogaster. During speciation of D. melanogaster, deletion of the T2 and T5 domains followed by amplification events led to the establishment of TAS-L and the 0.9 kb repeats. The TAS-R family arose by insertion of sequences leading to the T1, T4, T3′ and T3″ domains. The T3′, T3″ and TLL correspond to the full-length of the T3 domain. More recently, an INV-4 TE inserted into the locus, recombined so as to leave a solo LTR and then was amplified. A scenario of successive events (insertion, deletion and recombination) giving rise to the three TAS-R family members known today is proposed here. Note that domains and repeat units are not drawn to scale to facilitate the graphic representation. The origin of the ‘T’ domains is unknown and discussed in the text. Telomeres are to the left of the structural scheme for each chromosomal region depicted.