siRNA that participates in Drosophila dosage compensation is produced by many 1.688X and 359 bp repeats

Abstract

Organisms with differentiated sex chromosomes must accommodate unequal gene dosage in males and females. Male fruit flies increase X-linked gene expression to compensate for hemizygosity of their single X chromosome. Full compensation requires localization of the Male-Specific Lethal (MSL) complex to active genes on the male X, where it modulates chromatin to elevate expression. The mechanisms that identify X chromatin are poorly understood. The euchromatic X is enriched for AT-rich, ∼359 bp satellites termed the 1.688X repeats. Autosomal insertions of 1.688X DNA enable MSL recruitment to nearby genes. Ectopic expression of dsRNA from one of these repeats produces siRNA and partially restores X-localization of MSLs in males with defective X recognition. Surprisingly, expression of double-stranded RNA from three other 1.688X repeats failed to rescue males. We reconstructed dsRNA-expressing transgenes with sequence from two of these repeats and identified phasing of repeat DNA, rather than sequence or orientation, as the factor that determines rescue of males with defective X recognition. Small RNA sequencing revealed that siRNA was produced in flies with a transgene that rescues, but not in those carrying a transgene with the same repeat but different phasing. We demonstrate that pericentromeric X heterochromatin promotes X recognition through a maternal effect, potentially mediated by small RNA from closely related heterochromatic repeats. This suggests that the sources of siRNAs promoting X recognition are highly redundant. We propose that enrichment of satellite repeats on Drosophilid X chromosomes facilitates the rapid evolution of differentiated sex chromosomes by marking the X for compensation.

Keywords: Drosophila melanogaster; siRNA; roX; 1.688X satellite repeats; 359 bp repeats; X recognition; dosage compensation; epigenetics; small RNA.

Fig. 1.

Original and reconstructed 1.688^X pWIZ transgenes. a) Left—1.688^3F, 1.688^1A, and 1.688^3C amplicons used in original pWIZ-ds1.688^X transgenes (Menon et al. 2014). Arrows represent ∼359 bp repeat units. EcoR1 sites present in many 1.688^X repeats are arbitrarily designated as the endpoint of each repeat unit. The dotted line in 1.688^3C represents a region with limited repeat homology. Center—orientation of inserts in original constructs. Right—dsRNAs produced by each construct. b) Inserts in reconstructed pWIZ-ds1.688^1AR and pWIZ-ds1.688^3CR are the same size, orientation, and phasing as the original pWIZ-ds1.688^3F. The orientation of inserts in pWIZ-ds1.688^1AR-inv and pWIZ-ds1.688^3CR-inv is inverted.

Fig. 2.

Expression of reconstructed ds1.688^X transgenes rescues roX1^SMC17A roX2Δ males. a) roX1^SMC17A roX2Δ females heterozygous for the p[w⁺Sqh-GAL4]2 driver were mated to males carrying pWIZ transgenes. The recovery of male offspring was normalized by multiplying males of each category by two and dividing by the total number of daughters. b) The original pWIZ-ds1.688^3F and pWIZ-ds1.688^1A (hatched bars) and three independent insertions of each reconstructed transgenic (dark bars) were tested. The survival of males with each pWIZ transgene but without driver is shown by light gray bars. Error bars represent the SEM of four biological replicates. *P < 0.05; **P < 0.01, ***P < 0.001, as determined by Student’s t test.

Fig. 3.

Biologically active and inactive dsRNAs differ in small RNA production. a) Alignment of small RNA from larvae carrying pWIZ-ds1.688^1AR-inv (top; insertion 35) pWIZ-ds1.688^1A (middle; insertion 2A) or no transgene (control, bottom). Two independent RNA preparations are shown for each genotype. The orientation of 1.688^1A sequence within pWIZ is illustrated to the left. Tandem 1.688^1A repeats proximal to tyn (left) are depicted by arrows. Related 1.688^4A repeats are 4.3 kb distal to CG43689 (middle). All preparations contained abundant small RNA aligning to the autosomal, hairpin RNA-producing esi-1 locus (right). Alignments are depicted on a linear scale. b) Small RNAs were aligned to the transcript produced by pWIZ-ds1.688^1AR-inv, schematically represented with 1.688^1A sequence highlighted. The SV40 terminator is upstream of two polyadenylation sites marked with red arrow heads. Alignments to the pWIZ-ds1.688^1AR-inv transcript are presented on a logarithmic scale. All alignments are viewed on igv genome browser (Robinson et al. 2011). Expression of all transgenes is driven by p[w⁺Sqh-Gal4]2.

Fig. 4.

Expression of reconstructed transgenes modulates expression near an autosomal 1.688^3F integration. a) roX1 and 1.688^3F recruiting elements are integrated in a 40 kb intron of the haf gene (arrow head). Primers to measure transcript accumulation amplify a haf exon and lie within a Rab3GP1 intron (verticle bar). b) Accumulation of transcripts in male larvae with integrated recruiting elements roX1, 1.688^3F, or both is shown by gray bars (insertions [1.688^3F]^22A3, [roX1]^22A3, and [roX1+1.688^3F]^22A3; Joshi and Meller 2017). Additional expression of ds1.688^1AR or ds1.688^1AR-inv (blue), and ds1.688^3CR or ds1.688^3CR-inv (green) increased haf expression only when 1.688^3F was present in haf. Expression is normalized to dmn and that in lab reference yw males is set to 1 (line). The significance of expression with recruiting elements alone is with respect to yw males as indicated by asterisks. All other statistical comparisons are between males with recruiting elements alone and those with each recruiting element and dsRNA expression. Error bars represent SEM from three biological replicates. *P < 0.05; **P < 0.01, ***P < 0.001, as determined by Student’s t test.

Fig. 5.

Pericentromeric 359 bp satellites influence dosage compensation. a) Eclosion of roX1^ex33 roX2Δ males that are wild-type for Zhr (Zhr⁺) and two independent roX1^ex33 roX2Δ Zhr¹ recombinants. Error bars represent SEM of four biological replicates. b) roX1^ex33 roX2Δ Zhr^1/+ mothers are mated to reference yw males (+++/Y) and male offspring pooled for DNA extraction and qPCR of 359 bp repeats. c) Abundance of 359 bp repeats in roX1^ex33 roX2Δ Zhr⁺ (left) is set to 1. Zhr¹ (center) lacks the 359 bp repeat array (see Supplementary Table 2 for primers). Pooled sons from roX1^ex33 roX2Δ Zhr^1/+ mothers (right) reveal approximately equal numbers of Zhr¹ and Zhr⁺ sons. Error bars represent SEM from four biological replicates. **P < 0.01, ***P < 0.001, as determined by Students t test.