Dosage compensation via transposable element mediated rewiring of a regulatory network

Abstract

Transposable elements (TEs) may contribute to evolutionary innovations through the rewiring of networks by supplying ready-to-use cis regulatory elements. Genes on the Drosophila X chromosome are coordinately regulated by the male specific lethal (MSL) complex to achieve dosage compensation in males. We show that the acquisition of dozens of MSL binding sites on evolutionarily new X chromosomes was facilitated by the independent co-option of a mutant helitron TE that attracts the MSL complex (TE domestication). The recently formed neo-X recruits helitrons that provide dozens of functional, but suboptimal, MSL binding sites, whereas the older XR chromosome has ceased acquisition and appears to have fine-tuned the binding affinities of more ancient elements for the MSL complex. Thus, TE-mediated rewiring of regulatory networks through domestication and amplification may be followed by fine-tuning of the cis-regulatory element supplied by the TE and erosion of nonfunctional regions.

Fig. 1. Evolutionary history of Drosophila miranda sex chromosomes

The ancestral X chromosome shared by all members of the Drosophila genus (in red) fused to an autosome roughly 15 MY ago, creating chromosome XR (in orange). Another autosome fused to the Y chromosome about 1 MY ago, creating the neo-X chromosome (in yellow). D. miranda thus harbors three X chromosomes of different ages. Species abbreviations are as follows: D. subobscura (Dsub), D. pseudoobscura (Dpse), and D. miranda (Dmir).

Fig. 2. ISX is a domesticated helitron TE that is associated with chromatin entry sites (CES) on the neo-X chromosome and recruits the MSL complex in transgenic assays

(A) 21 of 68 strict MSL complex CES on the neo-X chromosome overlap D. miranda specific insertions of the ISX element, and 69 (out of 77 total) neo-X linked ISX elements lie within broad CES. (B) A derived 10 bp deletion differentiates ISX from the related ISY element and creates a stronger match to the MRE consensus motif identified from chromosome XL. P-values are from FIMO () and are based on log-likelihood ratio scores between the D. melanogaster canonical MRE consensus motif and the sequence highlighted in grey. (C) MSL3 ChIP-seq data show that MSL complex binds ISX but not ISY elements. (D, E) Ectopic MSL targeting by the ISX element from D. miranda, and lack of activity from the corresponding ISY element. Transgenic polytene chromosomes stained with anti-MSL2 (magenta) to identify regions targeted by the MSL-complex, and DAPI to identify all chromosome arms (cyan). An ISY and ISX element were each targeted to cytosite 37B7 (location denoted by white arrow) on chromosome 2L in D. melanogaster. (D) No staining is detected at 37B7 when the insertion contains ISY (E) but we find robust MSL immunostaining at the location when the insertion contains ISX.

Fig. 3. The ISXR helitron is associated with CES on chromosome XR

(A) Multiple sequence alignment showing helitrons from within neo-X CES (ISX), XR CES (ISXR), and the related ISY element. Grey boxes indicate approximate location of the MRE motifs in the alignment. (B) Divergence of elements from their consensus sequence places the burst of ISXR amplification after the divergence of D. subobscura from the miranda/pseudoobscura ancestor (> 15 MYA) and the ISX burst after the divergence of D. pseudoobscura and D. miranda (~4 MYA). See Supplemental Methods for details. (C) The ISY element is distributed evenly among D. miranda chromosomes (permutation test p > 0.08 in all cases, except for chromosome 4, where it is depleted [permutation test p<0.0001]) while ISXR is enriched on XR and ISX is enriched on the neo-X (permutation test p<0.0001 in both cases)(see Table S2 for additional details). (D) Clustering of the canonical D. miranda and D. melanogaster MRE motifs inferred from the ancient X chromosome along with the consensus from each helitron TE. ISXRcons refers to the ISXR MRE that is conserved with ISX while ISXRuniq refers to the MRE that is unique to ISXR [see () for details]. (E) MSL3 ChIP-seq data show that XR CES overlapping ISXR elements have a higher affinity for the MSL complex in vivo compared to the neo-X CES that overlap ISX elements (comparing the distribution of MSL-enrichment from 21 strict CES created by ISX, vs. 47 strict CES created by ISXR, Wilcoxon test P=0.01).

Fig. 4. TE-mediated evolution of MSL complex chromatin entry sites (CES)

Comparison of the two evolutionary timepoints of acquiring chromatin entry sites on XR and the neo-X suggest a three-step model for the TE-mediated wiring of a newly evolved X chromosome into the dosage compensation network followed by erosion of non-functional elements of the TE. The first step, domestication, involves the acquisition of a MRE sequence motif capable of acting as a CES for the MSL complex. The domesticated TE is amplified across the genome and beneficial on a newly formed X chromosome but selected against on autosomal locations. This results in the accumulation of the domesticated TE, along with the MRE motif that it carries, on the X. The amplified MRE motif may initially be suboptimal, as seen with the younger ISX elements on the neo-X, but over time, secondary fine-tuning mutations within each MRE can refine the ability to recruit optimal levels of MSL complex, as seem to have occurred with the older ISXR elements on XR. This is accompanied by erosion of TE sequences that are not required for MSL-binding, eventually degrading the signature of TE involvement for supplying CES.