Global regulation of X chromosomal genes by the MSL complex in Drosophila melanogaster

Abstract

A long-standing model postulates that X-chromosome dosage compensation in Drosophila occurs by twofold up-regulation of the single male X, but previous data cannot exclude an alternative model, in which male autosomes are down-regulated to balance gene expression. To distinguish between the two models, we used RNA interference to deplete Male-Specific Lethal (MSL) complexes from male-like tissue culture cells. We found that expression of many genes from the X chromosome decreased, while expression from the autosomes was largely unchanged. We conclude that the primary role of the MSL complex is to up-regulate the male X chromosome.

Figure 1.

Delocalization of MSL complexes from the X chromosome following RNAi for msl2 in Drosophila SL2 cells. SL2 cells were treated with GFP-dsRNA or msl2-dsRNA, grown 4 d, fixed, and immunostained with affinity purified rabbit anti-MSL1, anti-MSL2, anti-MSL3, anti-MOF, anti-MLE, or anti-histone H4K16ac followed by anti-rabbit secondary antibodies labeled with Texas Red. DNA was counterstained with DAPI (blue).

Figure 2.

Changes in MSL protein abundance following RNAi for msl2. (A) Real-time RT–PCR quantification of msl2 mRNA. Total cellular RNA was isolated from parallel cultures of cells 4 d after treatment with GFP-dsRNA or msl2-dsRNA. For each sample, transcript levels were normalized to the internal control mRNA pka. The standard deviation was calculated based on three independent experiments. (B) Western analysis of MSL proteins. Crude lysates were prepared from cells 4 d after treatment with GFP-dsRNA or msl2-dsRNA and separated by SDS-PAGE. Western blots were incubated with anti-MSL1, anti-MSL2, anti-MSL3, anti-MOF, anti-MLE, or α-tubulin. Protein extracts were diluted twofold as indicated. Detection was as described in Materials and Methods.

Figure 3.

Microarray gene expression analysis after RNAi for msl2. (A) Distribution of fold change ratios from the X chromosome (red line) and the autosomes (black line). The log fold ratios are centered roughly on zero and are symmetric (black line) for the autosomes, and they are clearly shifted to the left for the X chromosome (red line). In either the t-test or Kolmogorov-Smirnov test, the p-value is <10^-15. (B) Changes in the number of genes reliably detected on microarrays after RNAi removal of MSL2. The black, dark-gray, and light-gray bars indicate the percentage of change in transcripts present for the whole genome, the X chromosome, and the autosomes, respectively. In each case, the decrease is either mostly or entirely from the X chromosome.

Figure 4.

Chromosomal location of genes affected by RNAi for msl2. Genes up-regulated >1.4-fold are indicated in red, and genes down-regulated >1.4-fold are indicated in dark blue. The strongly down-regulated genes are concentrated on the X chromosome.