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. 2010 Feb 23;8(2):e1000320.
doi: 10.1371/journal.pbio.1000320.

Expression in aneuploid Drosophila S2 cells

Yu Zhang  1 , John H Malone, Sara K Powell, Vipul Periwal, Eric Spana, David M Macalpine, Brian Oliver
Affiliations

Expression in aneuploid Drosophila S2 cells

Yu Zhang et al. PLoS Biol. .

Abstract

Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression) genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets of autosomes, or 2X;4A). Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system "anticipates" the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal-independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. S2 cell DNA copy number.
(A–D) DNA density and copy number profiles of the X chromosome (A, B) and chromosome 2L (C, D), showing copy number of aneuploidy segments along chromosome length. The RPKM DNA-Seq density in nonoverlapping 1 kb windows was plotted against the chromosome coordinates and the final deduced copy number is indicated (color key). The copy number was determined by Bayesian change point analysis (CPA) (A, C) and CGH (B, D). The CGH results are projected onto the DNA-Seq data. The average DNA densities of each aneuploid segment between predicted breakpoints (black lines) are shown.
Figure 2
Figure 2. Expression at varying copy numbers.
(A, B) Boxplots showing the distribution of DNA-Seq read densities (in non-overlapping 1 kb windows) mapped to chromosome arms in S2 cells (A) and the distribution of RNA-Seq expression values at the gene-level (B). Pie charts (A, B) show the distributions of copy numbers on each chromosome arm (for expressed genes only). See Figure 1 for copy number color key. The X chromosome is in red. (C, D) Boxplots showing the distribution of RNA-Seq expression values by copy number (C) and expression per copy (D). Equivalent expression medians for two copies on the X and four copies on the autosomes are indicated (dashed line). For all boxplots, the 25th to 75th percentiles (boxes), medians (lines in boxes), and ranges (whiskers, 1.5 times the interquartile range extended from both ends of the box) are shown. Asterisks indicate significant differences from all other chromosome arms (A, B) or from the 2X or 4A baseline (C).
Figure 3
Figure 3. msl2 and mof RNAi.
(A) Western analysis showing changes in MSL protein abundance following RNAi for msl2 and mof in S2 cells. (B) K-means clustering (k = 3) of H4K16ac ChIP/input ratio for expressed genes on the X chromosome and chromosome 3R in RNAi and mock treated S2 cells. Genes enriched (yellow) and depleted (blue) for H4K16ac are indicated. (C) Boxplots showing the distribution of H4K16ac ChIP/input ratios in mock treated cells for expressed genes on different chromosome arms. (D–E) Boxplots showing the distribution of H4K16ac ChIP ratios between msl2 RNAi cells (D) or mof RNAi cells (E) and mock treated cells for expressed genes on different chromosome arms. Significant differences (p<10−2) among chromosome arms (C) and between RNAi and mock treated cells (D, E) are indicated by asterisks.
Figure 4
Figure 4. Expression following msl2 or mof RNAi.
Boxplots showing the distribution of expression RPKM values at indicated copy number on the X chromosome (left) and autosomes (right) in RNAi and mock treated S2 cells. Equivalent expression of two copy X chromosome genes and four copy autosomal genes in mock treated cells is shown (dashed line). See Figure 2 for boxplot format. Asterisks indicate significant expression decrease in RNAi cells compared to mock treated cells.
Figure 5
Figure 5. Mof and Msl2 effects on expression.
(A, B) Boxplots showing the distribution of expression ratios between msl2 RNAi cells (A) or mof RNAi cells (B) and mock treated cells by chromosome arms. The expected fold decrease in X chromosome expression after RNAi treatment is indicated (red dashed line). (C, D) Boxplots showing the expression ratios for msl2 (C) and mof (D) RNAi treated cells at indicated gene copy numbers. The X chromosome (left) and autosomes (right) are shown separately. (E, F) The relation between gene expression and fold expression change in msl2 (E) and mof (F) RNAi treated cells plotted as a moving average (20 gene/window).
Figure 6
Figure 6. Characterization of dose-response curves.
(A, C) Median expression RPKM values plotted against the DNA copy for X chromosome and autosome genes in RNAi and mock treated S2 cells based on absolute (A) or scaled (C) data. Fitted trend lines for the X chromosome (red) and autosomes (black) following mock (solid), msl2 (dashed), and mof (dotted) RNAi treatment are indicated. (B) Boxplots and table showing the distribution of expression ratios among different copy numbers. Expression fold change values were calculated based on real median RPKM values (bold) or projected expression values. Asterisks indicate significant variation for the expression fold change between X chromosome and autosome genes at an equivalent dose in RNAi cells (p<10−2).
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