Ribosomal DNA deletions modulate genome-wide gene expression: "rDNA-sensitive" genes and natural variation

Abstract

The ribosomal rDNA gene array is an epigenetically-regulated repeated gene locus. While rDNA copy number varies widely between and within species, the functional consequences of subtle copy number polymorphisms have been largely unknown. Deletions in the Drosophila Y-linked rDNA modifies heterochromatin-induced position effect variegation (PEV), but it has been unknown if the euchromatic component of the genome is affected by rDNA copy number. Polymorphisms of naturally occurring Y chromosomes affect both euchromatin and heterochromatin, although the elements responsible for these effects are unknown. Here we show that copy number of the Y-linked rDNA array is a source of genome-wide variation in gene expression. Induced deletions in the rDNA affect the expression of hundreds to thousands of euchromatic genes throughout the genome of males and females. Although the affected genes are not physically clustered, we observed functional enrichments for genes whose protein products are located in the mitochondria and are involved in electron transport. The affected genes significantly overlap with genes affected by natural polymorphisms on Y chromosomes, suggesting that polymorphic rDNA copy number is an important determinant of gene expression diversity in natural populations. Altogether, our results indicate that subtle changes to rDNA copy number between individuals may contribute to biologically relevant phenotypic variation.

Figure 1. Induced deletions in the rDNA locus result in the differential expression of hundreds of genes.

(A) Number of differentially expressed genes for Y chromosomes bearing deletions within the ribosomal DNA (rDNA). Data are given at P<0.05 (first set of data) and other indicated Bayesian Posterior Probabilities. Expected values are calculated from permuted datasets and shown in light gray. rDNA-mild are average numbers for two chromosomes with 87% and 85% wild-type copy number of rDNA, and rDNA-gross is a chromosome with 46% of wild-type rDNA (Figure 6). (B) Venn diagram showing number of differentially expressed genes in each rDNA deletion line relative to the wild-type chromosome (at P<0.001, FDR<0.05). (C) Correlation between the magnitude of change in gene expression (log-fold-changes) for YrDNA-gross (abscissa) and either YrDNA-mild-1 (ordinate – Top panel) or YrDNA-mild-2 (ordinate – Bottom panel). ρ = 0.84 and 0.78, respectively. Fold-changes are for contrasts between each rDNA deletion line and the wild-type chromosome.

Figure 2. Differentially expressed genes are shared in males and females.

(A) Number of differentially expressed genes in XX/YrDNA-gross females (relative to the wild-type Y chromosome in XX/Y females). Data are presented as in Figure 1A. (B) Venn diagram showing number of differentially expressed genes that are unique or common to X/YrDNA-gross males and XX/YrDNA-gross females (at P<0.001). (C) Breakdown of overlapping genes from (B), separately categorizing genes whose expression was increased (up) or decreased (down) relative to the wild-type Y chromosome in the same genetic background.

Figure 3. rDNA–responsive genes are found throughout the genome.

(A) Number of differentially expressed genes either up-regulated or down-regulated as a function of cytological location. Each cytological division shows grouped data for all three Y chromosomes (YrDNA-mild-1, YrDNA-mild-2, YrDNA-gross) relative to the wild-type Y chromosome (at P<0.01). (B) Distribution of microarray spots yielding high quality data (gray bars) with scanning 5-division average (gray line). Overlaid scanning 5-division average (black line) of the number of differentially expressed genes. For each window we show the number of differentially expressed genes grouped for all three chromosomes (YrDNA-mild-1, YrDNA-mild-2, and YrDNA-gross) relative to the wild-type Y chromosome (P<0.01). (C) Scanning 5-division average of number of differentially expressed genes only from males (black) and females (gray) bearing YrDNA-gross (at P<0.01). Cytological divisions are aligned across entire figure (dotted vertical lines). Stylized chromosome map represents euchromatic regions of the genome and location of centromeres and centric heterochromatin (ovals).

Figure 4. Differentially expressed genes are shared between chromosomes with induced rDNA deletions and naturally occurring Y chromosomes.

(A) Venn diagram showing number of differentially expressed genes unique to Y chromosomes with induced rDNA copy number changes (“rDNA Deletions”) or natural Y chromosomes (“wild Y isolates”), and overlap of genes common to both groups (at P<0.005). (B) Event histogram showing that 10,000 randomly-generated datasets produces an average of 38.54 genes shared between rDNA Deletions and wild Y isolates. Arrow shows the observed value of 124 (from (A)).

Figure 5. Correlations between absolute fold changes in rDNA responsive genes identified by induced rDNA deletions and absolute fold changes arising from natural Y chromosome polymorphisms.

(A) Correlation of absolute log-fold-changes comparing differentially expressed genes between YrDNA-gross and wild-type Y (abscissa) to those differentially expressed between YZimbabwe (YZimb.) and YOhio (ordinate); ρ = 0.55, P<10E−16. (B) Correlation of absolute log-fold-changes for YrDNA-gross versus wild-type Y compared to YCongo versus YZimbabwe; ρ = 0.38, P<10E−12. (C) Correlation of absolute log-fold-changes for YrDNA-gross versus wild-type Y compared to YCongo versus YOhio; ρ = 0.25, P<10E−6.

Figure 6. Quantification of rDNA copy number of the chromosomes in this study.

Quantification of rDNA copy number determined by Real Time Polymerase Chain Reaction, presented as percentage of a common wild-type Y chromosome (the progenitor of YrDNA-mild-1, YrDNA-mild-2, and YrDNA-gross). Plots show average ± 1 S.D.