Nucleolar dominance of the Y chromosome in Drosophila melanogaster

Abstract

The rDNA genes are transcribed by RNA polymerase I to make structural RNAs for ribosomes. Hundreds of rDNA genes are typically arranged in an array that spans megabase pairs of DNA. These arrays are the major sites of transcription in growing cells, accounting for as much as 50% of RNA synthesis. The repetitive rDNA arrays are thought to use heterochromatic gene silencing as a mechanism for metabolic regulation, since repeated sequences nucleate heterochromatin formation in eukaryotes. Drosophila melanogaster carries an rDNA array on the X chromosome and on the Y chromosome, and genetic analysis has suggested that both are transcribed. However, using a chromatin-marking assay, we find that the entire X chromosome rDNA array is normally silenced in D. melanogaster males, while the Y chromosome rDNA array is dominant and expressed. This resembles "nucleolar dominance," a phenomenon that occurs in interspecific hybrids where an rDNA array from one parental species is silenced, and that from the other parent is preferentially transcribed. Interspecies nucleolar dominance is thought to result from incompatibilities between species-specific transcription factors and the rDNA promoters in the hybrid, but our results show that nucleolar dominance is a normal feature of rDNA regulation. Nucleolar dominance within D. melanogaster is only partially dependent on known components of heterochromatic gene silencing, implying that a distinctive chromatin regulatory system may act at rDNA genes. Finally, we isolate variant Y chromosomes that allow X chromosome array expression and suggest that the large-scale organization of rDNA arrays contribute to nucleolar dominance. This is the first example of allelic inactivation in D. melanogaster.

Figure 1

H3.3 marks the active rDNA genes on the sex chromosomes of Drosophila. Mitotic chromosome spreads from larval neuroblasts were fixed and stained with DAPI (red). Signals from hybridization probes to 18S rDNA (blue) and H3.3-GFP protein (green) are shown. (A) A spread from a male larva hybridized with an 18S rDNA probe shows the location of the rDNA arrays on the X and the Y chromosome. (B–F) Larvae carrying an H3.3–GFP construct were induced to produce a pulse of tagged histone, and the localization of the protein was assessed on mitotic spreads. The active rDNA genes are the major sites of H3.3–GFP deposition after the pulse. (B) Spreads from a female larva show H3.3–GFP deposition on the rDNA arrays of both X chromosomes. The X chromosomes always show equivalent signals. (C) Spreads from a male sibling show H3.3–GFP only on the Y^OR chromosome rDNA array, indicative of Y nucleolar dominance in this strain. (D and E) Males carrying variant Y chromosomes show H3.3–GFP on both sex chromosomes, indicative of nucleolar codominance of the X and these Y chromosomes. (F) Males carrying the variant Y^sv392 chromosome show H3.3–GFP only on the X chromosome. (G) Hybridization with an 18S rDNA probe (blue) to spreads from a male carrying the Y^sv392 chromosome demonstrates that this variant Y is deleted for rDNA genes.

Figure 2

Transcription of an X-specific rDNA retrotransposon fragment is repressed by nucleolar dominance. (A) Schematic of two rDNA units showing the transcribed region (arrow) and the position of the 167-bp-long 5′-truncated R2 element (solid triangle, called R2¹⁶⁷). A single copy of R2¹⁶⁷ is present on the X chromosome used in this study (Eickbush and Eickbush 2003). The indicated primers (red) were used to measure abundance of the R2¹⁶⁷ transcript. (B) R2¹⁶⁷ transcript abundance measured by qPCR on cDNA pools generated from 10–20 adult animals. Ct values for the R2¹⁶⁷ transcript were normalized to Ct values for the Adh mRNA in the same samples and to the value for X/Y^OR males. The y-axis shows the fold change in expression. The standard error determined by RMS of each measurement is shown as black bars. The genotypes are (1) w¹¹¹⁸ females, (2) w¹¹¹⁸/Y^OR males, (3) C(1)DX/Y^OR females, (4) w¹¹¹⁸/Y^B males, (5) w¹¹¹⁸/Y^C males, (6) w¹¹¹⁸/Y^H males, (7) w¹¹¹⁸/Y^K males, (8) w¹¹¹⁸/Y^S males, (9) w¹¹¹⁸/Y^sv254 males, (10) w¹¹¹⁸/Y^sv391 males, and (11) w¹¹¹⁸/Y^sv392 males. The parent stock for each Y chromosome is listed in the Materials and Methods. (C) R2¹⁶⁷ transcript abundance in males carrying Su(var) mutations. ΔCt is displayed as in B. The genotypes are (1) w¹¹¹⁸ females, (2) w¹¹¹⁸/Y^OR males, (3) w¹¹¹⁸/Y^OR; Su(var)2–5⁰⁵/+ males, (4) w¹¹¹⁸/Y^OR; Su(var)3–9¹/+ males, (5) w¹¹¹⁸/Y^OR; Su(var)3–9¹/Su(var)3–9² males.

Figure 3

rDNA copy number polymorphism does not account for nucleolar dominance. The copy number of intact rDNA units and those carrying R1 or R2 retrotransposon insertions were assessed by qPCR. Copy numbers for a single w¹¹¹⁸ X chromosome were measured in females and that of the variant Y chromosomes in C(1)DX/Y females.

Figure 4

Codominant Y chromosomes derepress heterochromatic silencing near the X chromosome rDNA array. Effects of three Y chromosomes on silencing with In(1)w^m4 (left) and with bw^D/+ (right). Silencing of w^m4 chromosome is severe in males carrying the nucleolar dominant Y^OR chromosome, but is derepressed in males carrying either of the codominant Y^sv254 or Y^sv391 chromosomes. In contrast, silencing associated with bw^D is similar with all three Y chromosomes.

Figure 5

Codominant X and Y chromosomes are persistently paired. (A) Schematic of the X and Y chromosomes showing the rDNA arrays (blue) and the adjacent 359-bp satellite block (yellow) on the X. (B) Hybridization with probes to the rDNA (blue) and to the 359-bp satellite (yellow) of a mitotic chromosome spread from a male larva shows the position of these sequences on the X and Y chromosomes. DAPI is in red. (C and D) Examples of interphase nuclei from larval brains hybridized with rDNA (blue) and 359-bp satellite (yellow) probes, combined with antibody detection of Fibrillarin (red, a component of the nucleolus). In C there is only one block of rDNA signal, indicating that the two rDNA arrays are paired. In D two blocks of rDNA signal are apparent, and the Y chromosome array coincides with the nucleolus. (E) Quantitation of rDNA pairing in females and males with different Y chromosomes. At least 500 nuclei were scored in each genotype. * indicates comparisons that are statistically significant (<0.0001, χ² test).