Jumping genes!

Active transposable elements (TEs) impose a substantial mutational burden on the host genome (1–4). However, there is growing evidence implicating TEs as drivers of key evolutionary innovations by creating or rewiring regulatory networks (5–11). Many TEs harbor a variety of regulatory motifs, and TE amplification may allow for the rapid accumulation of a specific motif throughout the genome, thus recruiting multiple genes into a single regulatory network (12).

In Drosophila miranda, multiple sex chromosome/autosome fusions have created a series of X chromosomes of differing ages (Fig. 1). The ancestral X chromosome, XL, is homologous to the D. melanogaster X and is at least 60 million years old (13). Chromosome XR became a sex chromosome ~15 million years ago and is shared among members of the affinis and pseudoobscura subgroups, whereas the neo-X chromosome is specific to D. miranda and originated only 1 million years ago (14, 15). The male specific lethal (MSL) complex coordinates gene expression on the Drosophila male X to achieve dosage compensation (16). This complex is recruited to the X chromosome in males to high-affinity chromatin entry sites (CES) containing a conserved, roughly 21–base pair (bp)–long GA-rich sequence motif termed the MSL recognition element (MRE) (17). Once bound, the MSL-complex spreads from the CES in cis to actively transcribed genes, where it catalyzes the deposition of the activating histone modification H4K16ac, which ultimately results in a chromosome-wide twofold increase in gene expression levels (16). D. miranda males show MSL binding specific to the X chromosomes, associated with full dosage compensation of chromosomes XL and XR. In contrast, the neo-X shows incomplete dosage compensation (18).

The evolution of dosage compensation on XR and the neo-X involved co-option of the MSL machinery (19) and the creation of CES capable of recruiting this machinery, via MRE sequence motifs at a few hundred locations along the two X chromosomes. We used chromatin immunoprecipitation sequencing (ChIP-seq) profiling of MSL binding to conservatively define 132 CES on chromosome XL, 215 on XR, and 68 on the neo-X (18), and a more realistic estimate identifies 219 CES on XL, 383 on XR, and 175 on the neo-X (fig. S1) (20); we refer to these two groups as our “strict” versus “broad” set of CES. The CES on XR and the neo-X likely arose within the past 15 and 1 million years, respectively, after these chromosomes became X-linked in an ancestor of D. miranda.

Comparison of the genomic regions at strict neo-X CES sequences to their homologous regions in D. pseudoobscura, which are not X-linked and do not recruit the MSL complex, identified the mutational paths responsible for the formation of a MRE at 41 CES on the neo-X (21). In half of these sites, point mutations and short indels at prebinding sites created a stronger MRE. For the remaining half, however, the new MREs appeared to have been gained via a relatively large (~1 kb), D. miranda–specific insertion. Sanger resequencing and manual curation of the genome assembly at these sites allowed us to determine that these insertions are derived from a transposable element [homologous to the ISY element (22)] that is highly abundant in the genome of D. mirandaand its relatives (>1000 copies in D. miranda and D. pseudoobscura) (Fig. 2A). The ISY element (~1150 bp) is a nonautonomous helitron (figs. S2 and S3) (20), a class of DNA-transposable elements that replicate through a rolling-circle mechanism (23, 24). All 21 elements found at strict CES on the neo-X share a 10-bp deletion relative to the consensus ISY element, and we refer to the ISY sequence containing this deletion as ISX (Fig. 3A and fig. S4). ISX is also found at 24 of our broad CES and is present at 43% of strict CES and 30% of broad CES on the neo-X (figs. S5 and S6) (20). This 10-bp deletion creates a sequence motif more similar to the consensus MRE motif inferred from XL relative to the consensus ISY sequence (Fig. 2B) and thus might create a strong recruitment signal for the MSL-complex. The ISX element—but not ISY—is specific to D. miranda and highly enriched on the neo-X relative to other chromosomes (Fig. 3C and fig. S7) and strongly bound by the MSL complex in vivo (Fig. 2C). Additionally, the sequence similarity among ISX elements found at CES on the neo-X (Fig. 3, A and B) is consistent with their recent acquisition on the neo-X, after the formation of the neo-sex chromosomes (20). Together, these results suggest that within the past 1 million years, the D. miranda lineage was invaded by a domesticated helitron that recruits hundreds of genes into the MSL regulatory network on the neo-X. This process involved the formation of a high-affinity MRE sequence motif via a 10-bp deletion, followed by amplification and fixation of this element at dozens of sites along the neo-X chromosome (figs. S8 and S9) (20).

We used a transgenic assay in D. melanogaster to functionally verify that the ISX element attracts the MSL complex and functions as a CES. We targeted our construct to the previously characterized autosomal landing site 37B7 in D. melanogaster (25). Immunostaining of male polytene chromosomes shows that the ISX element can recruit the MSL complex of D. melanogaster, but no staining was detected with the ISY element (Fig. 2, D and E, and figs. S10 and S11). A higher affinity of the MSL complex to ISX versus ISY was also confirmed by means of ChIP–quantitative polymerase chain reaction (fig. S12). We also used mutagenesis assays to convert this ISX element into ISY by inserting the 10-bp sequence (ISX → ISY) and deleted the 10-bp fragment from the ISY element to create ISX (ISY → ISX). Immunostaining confirmed that the ISX → ISY construct could no longer recruit the MSL-complex to an autosomal location, whereas the ISY → ISX transgene was now able to attract MSL to an autosomal landing site in D. melanogaster (fig. S13). Thus, the ISX element alone is able and sufficient to attract the MSL complex, and the 10-bp deletion creates a functional MSL recruitment site. This experimentally confirms that the amplification of this TE along the neo-X chromosome may have resulted in the rapid wiring of neo-X–linked genes into the dosage compensation network. Dosage compensation of neo-X genes is advantageous because ~40% of homologous neo-Y genes are pseudogenized (26); however, because of its ability to recruit the MSL complex and induce dosage compensation, the ISX element should be selected against from autosomal locations. Indeed, out of a total of 82 copies of the ISX element, only two exist on an autosome, within repeat-rich and supposedly silenced regions on the dot chromosome (fig. S14) (20).

In the ancestor of the affinis and pseudoobscura subgroups (~15 million years ago), Muller element D became incorporated into the dosage compensation network after it fused to the ancestral X to form chromosome XR (Fig. 1). We compared all CES sequences on XR to determine whether they were enriched for sequence elements besides the MRE motif that would be indicative of a TE burst. Three repeat elements were present in ~22% of strict (and in 14.4% of broad) XR CES sequences, but not in the homologous regions from D. subobscura, where this chromosome is an autosome (Fig. 1). Furthermore, these elements were all determined to be conserved fragments from a single TE (hereafter referred to as ISXR), which is derived from the same helitron family as the ISY/ISX elements (Fig. 3A and fig. S15). Individual ISXR copies are less similar to each other than the ISX elements, and sequence divergence among the different copies of this TE is consistent with a burst of transposition activity coinciding with the formation of chromosome XR (Fig. 3B). Additionally, ISXR is enriched on chromosome XR (Fig. 3C), and similar to ISX/ISY, its autosomal homologs show less sequence similarity to the MRE consensus motif and cannot recruit the MSL-complex in vivo (fig. S16). ISXR contains a ~350-bp region that is not present in any of the ISY or ISX elements, and this region specific to ISXR contains an additional MRE motif in close proximity to the MRE whose location is conserved between the ISX and ISXR elements (Fig. 3, A and D, and fig. S17). In addition, although the location of the 3′ ISXR MRE is conserved with ISX, there is no evidence of the 10-bp deletion seen in ISX. The presence of this particular sequence region suggests that although ISX and ISXR evolved from a similar helitron progenitor TE, they represent independent TE domestications and chromosomal expansions at different time points (Fig. 3A and fig. S18) (20). Consistent with the more ancient expansion of ISXR, nonfunctional parts of the TE are severely eroded (Fig. 3A and fig. S15).

Similarity-based clustering of the MRE consensus motifs from each helitron subtype reveal that both ISXR MRE motifs are more similar to the canonical XL MRE motif, compared with the ISX MRE motif (Fig. 3D). This suggests that MSL binding motifs supplied by ISX may be suboptimal, whereas ISXR binding affinity is optimized. A large number of substitutions observed at MRE motifs among ISXR copies across the genome (fig. S19) (20) and elevated rate of evolution at homologous ISXR MRE sites relative to XL MREs across species (fig. S20) suggest that the ISXR element initially may have also harbored a suboptimal MRE motif (20). Over time, mutation and selection may have fine-tuned the nucleotide composition at ISXR independently across elements and species, to maximize MSL recruitment by increasing their similarity to the canonical XL MRE motif (Fig. 3D). In agreement with this observation, the TE-derived XR CES show a higher affinity for MSL complex in vivo as compared with that of those on the neo-X (Fig. 3E).

The recently formed sex chromosomes of D. miranda provide insights into the role of TEs in rewiring regulatory networks. The evolutionary pressure driving the acquisition of dosage compensation as well as the molecular mechanism of MSL function and targeting provide clear expectations of which genes should be recruited into the dosage compensation network, as well as when and how. Additionally, the comparison of XR and the neo-X allows us to study the dynamic process of TE-mediated wiring of chromosomal segments into the dosage-compensation network at two different evolutionary stages: both the initial incorporation of the neo-X chromosome by amplification of a domesticated TE and possible subsequent fine-tuning of the regulatory element supplied by the TE on XR, together with the erosion of TE sequence not required for MSL-binding. Our data support a three-step model for TE-mediated rewiring of regulatory networks (domestication, amplification, and potential fine-tuning) followed by erosion of nonfunctional parts of the transposon (Fig. 4). Eventually, the footprints left behind by TE-mediated rewiring will completely vanish, and many ancient bursts of domesticated TEs that rewired regulatory networks are likely to go undetected. Indeed, we do not observe any TE relics within the CES of chromosome XL that acquired MSL-mediated dosage compensation over 60 million years ago, either because they evolved via a different mechanism or deletions and substitutions have degraded the signal of TE involvement to the point at which they are no longer recognizable.

Supplementary Materials

www.sciencemag.org/content/342/6160/846/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S20

Tables S1 to S3

References (28–54)

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Acknowledgments: This work was funded by NIH grants (R01GM076007 and R01GM093182) and a Packard Fellowship to D.B. and a NIH postdoctoral fellowship to C.E.E. All DNA-sequencing reads generated in this study are deposited at the National Center for Biotechnology Information Short Reads Archive (www.ncbi.nlm.nih.gov/sra) under the accession no. SRS402821. The genome assemblies are available at the National Center for Biotechnology Information under BioProject PRJNA77213. We thank Z. Walton and A. Gorchakov for technical assistance.