Multi-species microarrays reveal the effect of sequence divergence on gene expression profiles

Abstract

Interspecies comparisons of gene expression levels will increase our understanding of the evolution of transcriptional mechanisms and help to identify targets of natural selection. This approach holds particular promise for apes, as many human-specific adaptations are thought to result from differences in gene expression rather than in coding sequence. To date, however, all studies directly comparing interspecies gene expression have been performed on single-species arrays, so that it has been impossible to distinguish differential hybridization due to sequence mismatches from underlying expression differences. To evaluate the severity of this potential problem, we constructed a new multiprimate cDNA array using probes from human, chimpanzee, orangutan, and rhesus. We find a large effect of sequence divergence on hybridization signal, even in the closest pair of species, human and chimpanzee. By comparing single-species array analyses with results from multispecies arrays, we examine how estimates of differential gene expression are affected by sequence divergence. Our results indicate that naive use of single-species arrays in direct interspecies comparisons can yield spurious results.

Figure 1.

Experimental error was estimated using four replicates of a human self-hybridization. The number of probes (y-axis) with a given Cy5/Cy3 log₂ ratio (x-axis) is shown with black bars for the human probes and with clear bars for the (A) chimpanzee probes (mean difference 0.03), (B) orangutan probes (mean difference 0.05), and (C) rhesus probes (mean difference 0.04). The difference between the distributions of log₂ ratios from different probe sets reflects the experimental error due to the hybridization to different probes on the array.

Figure 2.

Results for interspecies competitive hybridization. The number of probes (y-axis) with a given nonhuman/human log₂ ratio (x-axis) is shown with black bars for the human probes and with clear bars for (A) chimpanzee probes (mean difference 0.50), (B) orangutan probes (mean difference 0.78), and (C) rhesus probes (mean difference 1.15). All three values are significantly higher than the experimental error estimated from the self-hybridization (P <10^–4), indicating that sequence differences affect hybridization intensity. Note that the normalization based on both species probe sets leads to a symmetric distribution (see Methods).

Figure 3.

The difference between gene-expression estimates from multi- and single-species array analyses is plotted on the y-axis. Thus, positive values indicate that a greater difference was found in the multispecies analysis than in the single species one, while negative values point to the reverse. Probes are ordered by their log₂ expression difference, as estimated from the multispecies analysis (x-axis). (A) Human–human (for which the human and chimpanzee probes were used—mean absolute log₂ difference 0.07 ± 0.06) (B) Human–chimp (mean absolute log₂ difference 0.18 ± 0.16) (C) Human–orangutan (mean absolute log₂ difference 0.24 ± 0.21) (D) Human–rhesus (mean absolute log₂ difference 0.25 ± 0.20)