(A) Predicted vs. observed transgene expression levels for dosage compensated transgenes on X relative to transgenes on autosomes for two models of X-chromosome regulation. (Left) Under an X-chromosome-wide model of upregulation, dosage-compensated transgenes on hermaphrodite X chromosomes (2 copies) are predicted to have similar average total expression levels as transgenes on autosomes (2 copies), despite the hermaphrodite-specific repression by the DCC. Moreover, because the DCC reduces gene expression on X by about half, sdc-2(RNAi) animals would be predicted to have two-fold more transgene expression on X relative to autosomes, if X-chromosome upregulation occurred. (Middle) Under a model of no X-chromosome-wide upregulation, dosage compensated transgenes would be expressed at half the level of transgenes on autosomes due to repression by the DCC. In DCC-defective XX animals, transgene expression on X (2 copies) would increase to the level of transgene expression on autosomes (2 copies). (Right) The results of comparing the average expression level of all transgenes on X and autosomes argue against a chromosome-wide model of X-chromosome upregulation. For each reporter, data were normalized to the average autosomal expression level and then combined. Numbers above the graph show the fold change in expression between transgenes on X and on autosomes in control RNAi animals or between transgenes on X in sdc-2(RNAi) animals and transgenes on autosomes in control RNAi animals. The normalized expression level of all transgenes on the X chromosome (light blue) is only 56% of the normalized expression level of all transgenes on autosomes (gray). Expression of transgenes on X is increased to 93% of transgene expression on autosomes in animals treated with RNAi against sdc-2 (dark blue). p≤0.0001, four asterisks. Error bars show the standard error of the mean. (B) Comparison of averaged mRNA expression for all reporter transgenes of each type located at all sites on X or autosomes as quantified in (A), except that expression levels were not normalized to the average autosome expression level. For example, in the first panel, averaged expression from four Pdpy-30:gfp:H2B reporter transgenes inserted on autosomes is compared with averaged expression from four Pdpy-30:gfp:H2B reporter transgenes inserted on X. Expression levels of the Cbr-unc-119 reporter included with each transgene cassette are shown below the expression levels of the fluorescent partner transgenes. The number of asterisks indicates the p-value: p≤0.05, one asterisk; p≤0.01, two asterisks (Student's t-test). Error bars show the standard error of the mean. (C) Comparison between XX and XO L1/L2 larvae of total expression levels for transgenes inserted on X vs. autosomes. For the transgene cassette Pdpy-30:gfp:H2B and Cbr-unc-119, the autosomal cassette was on chromosome IV at site 1, and the X cassette was at site 3. For the transgene cassette Peft-3:gfp and Cbr-unc-119, the autosomal cassette was on chromosome I at site 1 and the X cassette at site 1. Shown below each bar are the sex of the animals in which gene expression was quantified, the copy number of the reporter transgene, and the position of the quantified reporter transgene (either on X or autosomes). For all transgenes, we compared the total level of expression from two reporter copies in XX animals and one reporter copy in XO animals, regardless of whether the reporters were on X or an autosome. The fold change in gene expression between reporters on X and autosomes is given above the graphs. The number of asterisks indicates the p-value: p≤0.05, one asterisk; p≤0.01, two asterisks (Student's t-test). Error bars show the standard error of the mean for at least three biological replicates. Expression of two copies on X was about half the expression of two copies on autosomes. Similarly, expression of the single copy on males was not different from the single copy on autosomes, meaning that expression of one copy on the male X would be half the expression of two copies on male autosomes. Results in (B,C) argue against an Ohno-like mechanism of X-chromosome upregulation in which chromosome-wide transcription of X is increased in expression. The results do not exclude the possibility that diverse gene-specific mechanisms might have arisen to elevate expression of individual X-linked genes with reduced dose.