Genetic analysis of transcriptional regulation is a rapidly emerging field of investigation that promises to shed light on the regulatory networks that control gene expression. Although a number of such studies have been carried out, the nature and extent of the heritability of gene expression traits have not been well established. We describe the inheritance of transcript levels in liver tissue in the first filial (F1) generation of mice obtained from reciprocal crosses between the common inbred strains A/J and C57BL/6J. We obtain estimates of genetic and technical variance components from these data and demonstrate that shrinkage estimators can increase detectable heritability. Estimates of heritability vary widely from transcript to transcript, with one-third of transcripts showing essentially no heritability (<0.01) and one-quarter showing very high heritability (>0.50). Roughly half of all transcripts are differentially expressed between the two parental strains. Most transcripts show an additive pattern of inheritance. Dominance effects were observed for 20% of transcripts and a small number of transcripts were identified as showing an overdominance mode of inheritance. In addition, we identified 314 transcripts with expression levels that differ between the reciprocal F1 animals. These genes may be related to maternal effect.
MICROARRAY technology has enabled the measurement of transcript levels for thousands of genes simultaneously. It has been used to identify gene expression changes in response to treatments or over time (Brown and Botstein 1999; Parmigiani et al. 2003; Choudhuri 2004). However, there is growing interest in the application of microarray technology to investigate the genetic control of transcript levels. The expression level of each gene in a genetically segregating population can be treated as a quantitative trait for genetic analysis (Darvasi 2003). For example, Schadt et al. (2003) conducted microarray experiments on 111 mice from an intercross population and mapped 4339 quantitative trait loci (QTL) that are associated with variation in gene expression. Monks et al. (2004) profiled 167 individuals in 15 human families using microarray and estimated the heritability of genes that are differentially expressed in the children. They found that 31% of these genes are heritable and median heritability is 0.34. Additional studies have been carried out in yeast (Brem et al. 2002), mouse (Hitzemann et al. 2003; Bystrykh et al. 2005; Chesler et al. 2005), rat (Hubner et al. 2005), and human (Morley et al. 2004) mapping populations.
Another aspect of the genetic analysis of gene expression as quantitative traits is the characterization of the pattern of genetic inheritance in F1 hybrids (Gibson and Weir 2005). Gibson et al. (2004) compared the gene expression between inbred parents and hybrid F1 Drosophila using microarray and found that 33% of the genes were different between F1's and parents. Interestingly, only 25% of transcripts were found different between the two inbred parents and only 2% of these transcripts show additive patterns of inheritance. Auger et al. (2005) analyzed the expression of 30 maize genes using Northern blotting and found that 20 genes exhibited significant dominance effects. These studies indicate that inheritance patterns of gene expression show extensive nonadditivity. Vuylsteke et al. (2005) studied three Arabidopsis accessions and two pairs of reciprocal F1 hybrids using cDNA arrays. They found that ∼30% of genes analyzed are differentially expressed between each pair of the three accessions and only ∼10% of the genes show significant dominance effects (Vuylsteke et al. 2005). A shortcoming of these studies is that pooled biological samples were often used without biological replication and statistical inferences are based on technical variance only. In this study, we compare transcript levels in liver samples from inbred mouse strains A/J (A) and C57BL/6J (B) and their F1 hybrid progenies from reciprocal crosses (A × B and B × A) using three independent biological samples from each group and two technical replicates of each biological sample. This design allows us to partition the total variation in each transcript into genetic, biological (but nongenetic), and measurement components. These inferences apply to the populations of inbred mice. We applied a shrinkage estimator for each variance component and demonstrate that this improves the estimated heritability of gene expression.
MATERIALS AND METHODS
Animals and tissue collection:
Mice were obtained from The Jackson Laboratory and maintained on standard (6% fat) chow diet in a clean mouse room facility on a 14:10 light:dark cycle. F1 mice were obtained from reciprocal crosses between parental strains A and B. Three female mice were sampled from each strain (A, B, A × B, and B × A) for microarray analysis. Mice were fasted starting at 7:00 am on the day of collection and collections were completed between 10:00 and 11:30 am. All mice were 8 weeks (± 4 days) at the time of tissue collection. Two tissue samples were collected from each mouse and stored directly in RNAlater (Ambion, Austin, TX) following dissection and later homogenized in TRIzol (Invitrogen, Carlsbad, CA).
Total RNA was isolated by standard TRIzol methods according to the manufacturer's protocols, and the quality was assessed using a 2100 Bioanalyzer instrument and a RNA 6000 Nano LabChip assay (Agilent Technologies, Palo Alto, CA). Following reverse transcription with an oligo(dT)-T7 primer, double-stranded cDNA was synthesized with the Superscript double-stranded cDNA synthesis custom kit (Invitrogen). In an in vitro transcription (IVT) reaction with T7 RNA polymerase, the cDNA was linearly amplified and labeled with biotinylated nucleotides (Enzo Diagnostics, Farmingdale, NY). Fifteen micrograms of biotin-labeled and fragmented cRNA was then hybridized onto MOE430v2.0 GeneChip arrays (Affymetrix, Santa Clara, CA) for 16 hr at 45°. Posthybridization staining and washing were performed according to manufacturer's protocols using the Fluidics Station 450 instrument (Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 laser confocal slide scanner. The images were quantified using GeneChip Operating Software (GCOS) v1.2 (Affymetrix).
Statistical analysis of microarray data:
Probe intensity data from all 24 arrays were read into the R software environment (http://www.R-project.org) directly from .CEL files using the R/affy package (Gautier et al. 2004), which was also used to extract and manipulate probe level data to assess data quality and to create expression summary measures. Normalization was carried out using the robust multiarray average (RMA) method (Irizarry et al. 2003) to form one expression measure for each probe set on each array. RMA processing was performed on all probe intensity data sets together. Briefly, the RMA method adjusted the background of perfect match (PM) probes, applied a quantile normalization of the corrected PM values, and calculated final expression measures using the median polish algorithm.
Statistical analyses of the expression summaries were conducted using the R/maanova package version 2.3.0 (http://www.jax.org/staff/churchill/labsite/software/Rmaanova) in the R environment with models and tests specified as below.
Probe set-specific ANOVA models:
To identify transcripts that are differentially expressed across the 12 mice in the experiment, we fitted a fixed-effect ANOVA model to the expression data from each probe set using the model(1)where μ is the overall probe set mean, Sj (j = 1, … , 12) is the deviation from the overall mean in the jth mouse, and εjk is the deviation of the kth (k = 1, 2) measurement from the mean of mouse j. This model ignores the strain information and treats the two samples from each mouse as independent technical replicates.
To estimate effects of each strain and test for overall genetic effects, a mixed-ANOVA model was fitted to each gene:(2)Here, the Sj effect in Equation 1 is decomposed into a fixed genetics (strain) effect, Gi (i = 1, 2, 3, 4) and a random mouse effect Mj(i) (j = 1, 2, 3) nested within strain Gi. This model was also used to identify transcripts that show significant variation among mice within strain by testing the mouse effect Mj(i) in model (2).
Estimating variance components:
We estimated the variance components for strain (), mouse (), and technical replicates () by treating all effects except μ as random in model (2). These variance components were estimated using the restricted maximum-likelihood (REML) method (Searl et al. 1992; Witkovsky 2002). The estimated variance estimates were then stabilized using a shrinkage estimator based on Stein's method (Cui et al. 2005). For estimating the mouse variance in each strain, the data were split into four subsets corresponding to the four strains. The mouse variance for each strain was estimated using the same model as model (1) except that the sample effect Sj (j = 1, 2, 3 here) was treated as random.
Heritability was computed using the estimated strain, mouse, and technical variance components for each transcript as(3)
Testing for strain effect, mouse effect, and strain contrasts:
To test the overall difference among the 12 mice, the variation explained by the Sj term in model (1) was compared to the technical variance using the shrinkage-based Fs-test (Cui et al. 2005). The empirical distribution of the Fs-statistic was established through permutation analysis, where the rows of the design matrix corresponding to the Sj term were shuffled 1000 times randomly while the data were kept unchanged (Kennedy and Cade 1996; Cui et al. 2005). The Fs-statistics calculated from the permutations were pooled across genes that are not significant (Xie et al. 2005) at the nominal 0.1 level according to a conservative gene-specific F-test to form one overall empirical distribution. The percentile of the Fs from observed data in the empirical distribution provides an estimate of the P-value for each gene. To test the natural biological variation after accounting for strain effect, the Mj(i) term in model (2) was tested against the technical variance using the Fs-test. To test the strain effect (Gi) in model (2), the mouse effect Mj(i) was treated as a random effect and the variance explained by the strain effect was tested against the combination of mouse variance and technical variance using the Fs-test. Differences between parental strains, between reciprocal F1 animals, and between the F1 and parental animals were tested by contrasting the appropriate components of the strain effect (Gi) in model (2), using a shrinkage-based t-test (Cui et al. 2005). The permutation for these later tests is carried out with the constraint that data from technical replicates must remain paired in the permutated data.
Gene lists were generated using a false discovery rate (FDR) of 0.05 (Benjamini and Hochberg 2000) unless otherwise specified. The fraction of differentially expressed transcripts was estimated using the method mix-o-matic (Allison et al. 2002). Briefly, the distribution of P-values is modeled as a mixture of a uniformly distributed component representing the nondifferential transcripts and a beta-distributed component representing the differentially expressed transcripts. The proportion of nondifferential genes () is estimated using maximum likelihood. A web implementation of this method at http://services.ssg.uab.edu/mixomatic/ was used to analyze the P-values obtained from each test. The is very similar to the obtained from the qvalue package (Storey 2002, 2003) at http://www.bioconductor.org/packages/bioc/stable/src/contrib/html/.
Estimation of the additive and dominance effects:
The expression level of each gene within each strain was estimated using generalized least squares for the Gi term in model (2) after estimating the variance components for the residual (εjk) and mouse [Mj(i)] terms (Searl et al. 1992). The additive effect, a, is estimated as half of the observed difference between the parental strains. The dominance effect, d, was estimated as the difference between the F1 and the midparent values.
Statistical tests for additive, dominance, and overdominance effects:
The additive effect was tested (H0: ) by contrasting the two parental strains. The dominance effect was tested (H0: ) by contrasting the F1 and the mean of the two parental strains. Overdominance effect was identified when the F1 expression value fell outside the range of the two parental strains and was significantly different from both parental strains. All tests were based on Fs with permutation-based critical values and significance level of FDR of 0.05.
The hierarchical clustering analysis was based on the correlation distance () of the gene expression among samples (Wu et al. 2003). Bootstrapping (100 times) was used to evaluate the cluster stability (Kerr and Churchill 2001) and a consensus tree was used to summarize the common features of the bootstrapped trees (Margush and McMorris 1981). Clades that appeared in >80% of the bootstrapped trees were shown in the consensus tree.
SNP effect assessment:
The allele-specific probe list for the Affymetrix MOE430v2.0 chip was downloaded from http://arrayanalysis.mbni.med.umich.edu/MBNIUM.html. This list contains SNPs for many mouse strains on the basis of information in the dbSNP database (build 126). SNPs between strains C57BL/6J and A/J were used to identify the specific SNP-containing probes and these were removed from the Affymetrix chip description file (CDF) using the R package ProbeFilter 1.4.0. Probe sets with fewer than four probes remaining were discarded from further analysis. The .CEL files were preprocessed again using the customized CDF file.
Determination of overrepresented biological categories:
Overrepresented categories within lists of differential expressed genes were identified by testing for association with gene ontology “biological process” terms in Expression Analysis Systematic Explorer (EASE) (Hosack et al. 2003) or gene product relationships available in a curated database of biological networks (Ingenuity Pathways Analysis) (http://www.ingenuity.com). Enrichment of pathway members among differentially expressed transcripts was assessed with the one-tailed Fisher's exact test for 2 × 2 contingency tables (Ingenuity) or a conservative adjustment to the Fisher's exact test result (EASE score) that weights significance according to the number of transcripts identified in a list. In the EASE analysis, only transcripts mapping to a unique EntrezGene identification were included for analysis. Because biological themes supported by a single gene in Fisher's exact test are not stable (Hosack et al. 2003), only results containing multiple genes were explored further.
Total RNA (1 μg) was reverse transcribed, employing standard random hexamer priming methods and Superscript III enzyme (Invitrogen) according to the manufacturer's protocols. Diluted reaction products were then used in a subsequent PCR reaction containing Taqman Univeral PCR master mix (Applied Biosystems). Gene-specific primers and probe sets were obtained from the Applied Biosystems Assay on Demand service and used according to manufacturer's protocols. Real-time PCR reactions were performed using the ABI PRISM 7900HT sequence detection system (Applied Biosystems) with recommended thermal cycling protocols and 40 cycles of amplification. Threshold cycle (Ct) values were determined using the supplied sequence detection system (SDS v2.2) software package. Three replicated reactions were conducted for each of the 12 samples used in the microarray study. The GUS gene was used as the internal control for all samples.
Overall sample difference and sample clustering:
To provide a picture of the overall relationship of gene expression among the 12 mice (3 from each of the four strains: A, B, A × B, and B × A) profiled in this experiment, we first fit an ANOVA model (Equation 1) to each transcript, treating the 12 mice as independent samples without considering their strain identities. A shrinkage-based Fs-test (Cui et al. 2005) was applied to each of the 45,000 transcripts present on the array. A total of 6143 transcripts were identified as significantly different among these 12 mice at the high-stringency significance level of FDR 0.001 (Benjamini and Hochberg 2000). On the basis of the estimated expression level of these transcripts we clustered the 12 mice using a hierarchical clustering method (Wu et al. 2003). As expected, the genetically defined groups cluster together (Figure 1). The two reciprocal F1 hybrids cluster together and the two inbred parental strains cluster together, which is similar to what was described in the Drosophila study (Gibson et al. 2004).
Natural variation among mice within strain:
Some of the differentially expressed transcripts among the 12 samples can be attributed to nonheritable natural variation among individual mice. To identify these transcripts we tested the mouse effect after accounting for strain effect in model (2) and found 1754 transcripts (FDR < 0.05) with significant biological variation among mice within a strain. Gene ontology analysis of these genes shows that they are enriched for fatty acids metabolism, amino acid metabolism, and hormone related processes, which indicates that some genes in these biological processes have high natural variation among individual mice with same genotype.
F1 hybrids are often more uniform in phenotype than inbred individuals (Crow 1998), possibly due to the buffering effects of heterozygosity (Hartman et al. 2001; Hartwell 2004). To test whether the gene expression of hybrids has this characteristic, we estimated and compared the variation of gene expression in F1 mice to variation within the parental strains. Cumulative plots of within-strain variance confirm that the B × A hybrid mice show less individual variation in gene expression than either of the two parental strains (Figure 2). However, the F1 hybrid mice from the A × B cross show more variation than the parental strain B. Thus gene expression in F1 hybrids does not necessarily resemble the heterosis phenomena associated with whole-organism phenotypes (Birchler et al. 2003). It is curious that the two reciprocal hybrids differ in this regard. Further investigation may be required to confirm that this is a general phenomenon or that it may be particular to these individuals.
Heritability of gene expression:
The variation of gene expression can be decomposed into heritable and nonheritable variation. The latter can be decomposed into the within-strain mouse variation and measurement error variation. The fraction of the total variation explained by genotype is known as the broad sense heritability (Falconer 1986). We calculated the heritability for each transcript on the basis of the estimated variance for genotype, mouse, and measurement. Due to the small number of biologically independent samples variance estimates are not very accurate. We applied a shrinkage adjustment to improve the estimation (Cui et al. 2005). The shrinkage adjustment is adaptive to the distribution of each variance component. After shrinkage the spread of the measurement variance was reduced substantially, reflecting a level of variation that is consistent with statistical estimation error. The strain and the mouse variances had minimal shrinkage due to their markedly heterogeneous variance distributions (Figure 3, A and B). Both of these variance components have bimodal distributions with about one-third of genes having estimates close to 0 (<10−6). However, only 8% of genes have both strain and mouse variance components close to 0. About 60% of the genes show both strain and mouse variance components >10−6 and the correlation (on the standard deviation scale) of these two variance components is 0.47. The high correlation between strain and mouse variance components indicates that genes that have high within-strain variation also tend to have high variation across strains.
We computed heritability as the ratio of between-strain variance to the total variance for both the raw and the shrinkage estimates of variance. The application of shrinkage increased the heritability in many genes. For example, 6503 (14% of the total) transcripts showed heritability >50% without shrinkage. With shrinkage, the number increased to 8131 (18% of the total transcripts). The median heritability of all the transcripts increased to 22% from 16%. Figure 3C shows the histograms of the heritability from transcripts with heritability >1% (∼75% of the transcripts present on the chip). The overall median heritability (22%) obtained here is larger than the 11% estimated from the mouse brain expression profile mapping experiment using recombinant inbred (RI) lines (Chesler et al. 2005).
To identify the heritable transcripts, we tested for the strain effect on the basis of the combination of biological variation and technical variation in a mixed-effect ANOVA model (2). We identified 8979 transcripts as significantly heritable (FDR < 0.05) among the four strains of mice. The median heritability of these genes is ∼70%. This gene list has 312 overlaps with the gene list for natural biological variation, which indicates that although these genes are naturally variable, the genetic effect is still significant after the nongenetic variation is accounted for. The heritable genetic effect was decomposed into three contrasts among the strains: the contrast between the two parental strains, the contrast between the two F1 strains, and the contrast between the mean of the two parental strains and the mean of the two F1 strains. These contrasts yield 8950, 357, and 67 significant transcripts, respectively, at FDR of 0.05. Treating the two reciprocal F1 hybrids as the same in the third contrast may result in reduced power due to the large difference between the two hybrids. To avoid this problem, we also compared the two hybrids with the midparent value separately. The A × B hybrid gave 317 significant genes while the B × A hybrid gave only 24 significant genes.
Additive and dominance effects:
The inheritance pattern, the difference of gene expression between parental strains and F1 hybrids, can be decomposed into an additive effect (a) and a dominance effect (d) as in conventional quantitative genetics (Falconer 1986). For purely additive effects (), the phenotype of the F1 will be equal to the midparent values. When a dominance effect is present (), the F1 gene expression differs from the midparent value by an amount d. Due to the presence of technical and biological variation, the estimated a and d effects are never exactly 0. We rely on statistical tests that contrast the parental and F1 trait values as described earlier to test for the a and d effects. Figure 4 shows the inheritance of the significant genes from some of the tests for the A × B hybrid. A large number of transcripts are significant for the additive effect and some of them are also significant for the dominance effect. Transcripts that are significant for the overdominance effects tend to have small additive effects and therefore are centered around the axis.
The numbers of significant genes from testing for the additive and dominance effects are also represented by the excess of small P-values in the P-value histograms (Figure 4). Under the assumption that the P-values would be uniformly distributed from 0 to 1 if there were no true difference among transcripts, the presence of excessively small P-values would indicate the presence of true differences. The histogram of P-values from testing the dominance effect shows an excess of small P-values but much less than those from the tests for additive effects.
Degree of dominance:
To describe the relative size of the dominance effect compared to that of the additive effect in gene expression, we computed the ratio of d/a as a measure of nonadditivity (Gibson et al. 2004). A purely additive inheritance model will have and a fully dominant trait will have . If the absolute value is >1, the effect is considered to be overdominance. To illustrate the degree of dominance, we plotted the histograms of estimated d/a for each F1 hybrid strain (Figure 5). It appears that many transcripts have a d/a ratio >1, indicating overdominance. However, this pattern may also be explained by variation in the estimates of the dominance ratios that have associated errors. The excess of transcripts with large dominance ratios may be due to the small additive effect estimates that occur by chance. This is evident when we plotted the additive effect against the degree of dominance. Most transcripts with large d/a ratios have small a estimates (Figure 5) and these genes are less significant for the additive effect. Therefore, formal statistical tests are needed to determine whether the dominance or overdominance effects are significant by considering both the size of the effect and the variation from both biological and technical sources.
Overdominance is commonly associated with hybrid vigor but may also have a negative effect on fitness (Johnson 1980; Pan et al. 2005). To declare overdominance, we required that a transcript mean expression in at least one hybrid F1 lies outside of the range of the parental strains and it should be significantly different from both parents at a significance level of FDR 0.05. We found 158 significantly overdominant transcripts for A × B hybrids (Figure 4) and most of these have a relatively small additive effect. The test for overdominance among the B × A hybrids identified only 18 transcripts with significant overdominance effect. The small number of significant genes with overdominance effect further indicates that using just the d/a ratio is not a reliable method to identify overdominant genes.
Estimating the proportion of differentially expressed transcripts:
The length of a significant gene list depends on the choice of the significance levels, multiple testing adjustment, the power of the study design, and the size of the actual effects. To obtain an estimate for the percentage of differentially expressed genes independent of the significance level, we applied the mix-o-matic analysis to all P-values obtained from testing individual transcripts. As shown in Figure 4, the number of differentially expressed transcripts from each test is represented by the excess of small P-values as compared to the uniform distribution expected under the complete null. The proportion of differentially expressed genes is estimated as the proportion of the beta component in the mixture of a beta and a uniform distribution using maximum likelihood in the mix-o-matic analysis (Allison et al. 2002). On the basis of this analysis, 73% of transcripts are differentially expressed among the 12 mice and 68% of transcripts are differentially expressed among the four strains after accounting for both biological and technical variation. About 23% of transcripts are differentially expressed among individual mice within strain and about half (56%) of the transcripts are differentially expressed between the two parental strains. Dominance effects were estimated to be present in ∼20% of transcripts. Compared with the relative small proportion of transcript with dominance effects, the large proportion of transcripts with additive effects suggests that most transcripts show an additive pattern of inheritance, with F1 levels similar to the midparent value. The tests for overdominance were not subjected to the estimation for the proportion of differentially expressed transcripts because they were selected with a combination of tests and the assumption of uniform P-value distribution under the complete null may not hold. For testing the two F1 hybrids, 31% of transcripts were estimated to be different between the two reciprocal F1's, indicating that the expression of a large number of genes may be affected by the maternal environment.
Probe designs for the Affymetrix array are based on the reference C57BL/6J sequence and it is plausible that strain polymorphisms could alter their hybridization properties. Thus there is a potential to observe differences in hybridization intensity even if the mRNA levels are the same in the two strains. To evaluate the effect of SNPs in this study, we identified individual probes containing known SNP(s) between A/J and C57BL/6J in the dbSNP database. A total of 3453 probe sets (∼10% of the total probe sets on the chip) were found to contain such SNPs. We reconstructed the Affymetrix CDF file to exclude the individual probes that contain any SNP from these probe sets. Eight probe sets were completely removed from further analysis because they had ≤3 probes remaining. We reanalyzed the data and compared the results with those from our original analysis (Table 1). The comparison showed only very minor or no changes in the clustering of samples, the heritability, and the variance components. These global characteristics may not be sensitive to changing a small subset (10%) of probe sets. We also compared the expression fold change between A/J and C57BL/6J for the SNP-containing probe sets and found that ∼50% of them increased and 50% of them decreased. This result suggests that SNPs have not significantly reduced the expression measurements for the A/J strain samples.
The number and identity of significant genes from each test after removing the SNP-containing probes were largely unchanged, with allowance for some numerical differences in borderline calls that result from the permutation analysis (Table 1). For almost all tests, ∼10% of significant probe sets contain SNPs, which is consistent with the overall rate of SNPs in probe sets on the chip. Therefore, there is no enrichment of SNP-containing probe sets, which might be the case if SNPs contribute heavily to the expression difference between the two strains. However, some differences are observed between the identities of the significant SNP-containing probe sets. For example, the contrast of the two parental strains identified 801 and 792 of the 3453 SNP-containing probe sets as significant before and after removing the SNP-containing probes. The 648 probes sets identified in common between these two analyses showed similar estimated fold change values. The 129 probe sets identified only in the first analysis show reduced fold change after removing SNP-containing probes, while the 120 genes significant only in the second analyses show increased fold change (Figure 6). Dramatic fold change differences were observed for only a few probe sets (the points far away from the identity line). These results indicate that SNPs in individual probes have a relatively small impact on the overall comparison of gene expression changes between strains.
Overrepresented biological categories:
To identify biological processes that may be overrepresented in each comparison, we performed statistical tests for overrepresentation of functional categories determined using the EASE analysis platform and the known functional relationships available in the Ingenuity Pathways Knowledge Base (see materials and methods). Multiple processes, including components of metabolism (including metabolism of proteins, DNA, RNA, phosphates, cofactors, and pyruvate), transcription and RNA processing, cell division, and biosynthesis pathways, were induced in B relative to A. Literature-supported pathways involving the expression of genes related to the metabolism of arachidonic acid, fatty acids, tryptophan, xenobiotic signaling, and amino groups were statistically overrepresented in gene lists that had lower expression in B compared to A. Other processes were expressed at higher levels in A relative to B, including probe sets mapping to genes associated with ion transport, protein localization, metabolism of alcohols and monosaccharides, biogenesis, and signaling. Transcripts associated with the literature-supported processes of N-glycan biosynthesis, the phototransduction pathway, and linoleic acid metabolism were overrepresented in gene lists, showing greater expression on A compared to B. These differences are summarized in supplemental Tables S1 and S2 at http://www.genetics.org/supplemental/.
Tests for the presence of significant dominance effects contrasting the parental strains and F1 hybrids were conducted separately for each hybrid. For the A × B hybrid, genes that showed lower expression in the hybrid than the midparent value were associated with macromolecule (protein) biosynthesis. For genes that showed higher expression in the A × B hybrid, categories related to metabolism (fatty acids, amino acids, and amino sugars), signaling (IL-10, IL-6, Integrin, and B cell receptor), and localization were overrepresented. In the test of dominance for the B × A hybrid, probe sets involved in protein localization were more highly expressed in the parental strains and probes sets associated with antigen presentation and the metabolism of fatty acids were more highly expressed in the B × A hybrids.
Differentially expressed transcripts expressed at a higher level when the expression of A × B was higher than B × A included bile acid biosynthesis, arginine and proline metabolism, and death receptor signaling. Ubiquinone biosynthesis and oxidative phosphorylation were overrepresented when the expression of A × B was lower than B × A.
Real-time PCR validation:
To validate the results from the microarray measurements, we chose six transcripts that show differential expression between the two parental strains and the expression in F1's is more similar to that in parental strain A. The same RNA samples used in the microarray experiment were assayed via real-time PCR with three technical replicates per RNA sample. The results showed that the relative expression among the strains for each transcript was similar to microarray results in both the direction of the difference and the relationship between F1 hybrids and the parental inbred line (Figure 7). Only the Cap1 transcript did not confirm the microarray results. Its real-time PCR did not show any difference among the strains while the microarray results showed large differences. This inconsistency could be explained by the fact that the primers used in the real-time PCR experiments are located at the upstream, exon 2/3, of the transcript while the Affymetrix probe set hybridizes to the 3′-untranslated region.
Comparing with similar studies in other organisms:
In this study we compared the gene expression of two parental strains and their reciprocal F1 hybrids in mouse liver. We tested the additive, dominance, and overdominance effects and identified lists of genes that are significant for each effect. At an FDR of 0.05, 19.9, 0.8, and 0.4% of all transcripts showed significant additive, dominance, and overdominance effects, respectively. Compared with other studies on gene expression inheritance (Gibson et al. 2004; Auger et al. 2005; Vuylsteke et al. 2005), our significant gene lists appeared to be substantially shorter in general (Table 2). One obvious reason for the discrepancy is that these studies examine different organisms and different tissues, which are likely to have a large impact on expression patterns (Wang et al. 2006). One potential explanation for the differences between the various studies is the effect of the design of these experiments. Our mouse study used three biological replicates per strain and two technical replicates per sample, while most others used one pool of many samples with more technical replicates. Because of the large number of technical replications, the measurements in some of these studies appear to be very precise and the tests appear to be more powerful, especially when replicated spots were treated as independent replicates in the Arabidopsis study (Vuylsteke et al. 2005). In studies without true biological replication the tests for differential expression depend on the technical variation in the microarray assay and the scope of the inference is limited. With biological replicates it is possible to construct tests that account for individual (nongenetic) variation in gene expression; the resulting tests have lower power but the conclusions apply to the population of animals from which the sample was obtained (Churchill 2002). For many genes, there is a substantial biological variance component and thus it can be expected that fewer genes will be detected.
One striking feature of Table 2 we would like to draw attention to is the dramatically different criteria used to select differentially expressed genes, which makes any direct result comparison across experiments impossible. Some studies even employed different significance criteria for different gene lists. One way to avoid inconsistency of significance criteria is to estimate the proportion of differentially expressed genes independent of significance levels using the P-value distributions. For example, the mix-o-matic algorithm (Allison et al. 2002) estimates the proportion of genes that are differentially expressed on the basis of the distribution of the P-values from individual gene tests. It provides an overall estimate of the proportion of genes that are differentially expressed. In our study, although the significance gene list for additive effect at an FDR of 0.05 is only 19.8%, the mix-o-matic estimation showed that >50% of transcripts are differentially expressed between the two parental strains. However, the identities of these differentially expressed genes are not known. It is not possible to conduct a functional analysis of these 50% transcripts. In addition, this approach requires the assumption of uniformity under the null, which may not hold in some tests. For example, the null hypothesis for testing the overdominance effect is that the expression of F1 hybrids is within the range of the two parental strains. Under this null hypothesis, the P-values will probably not follow a uniform distribution. Therefore, we cannot estimate the proportion of genes that show overdominance effect using the mix-o-matic method.
Another striking difference across these studies is in the degree of nonadditivity, which can be assessed by comparing the lengths of significant gene lists obtained from testing the additive and dominance effects within each study. The fly study (Gibson et al. 2004) showed a very high degree of nonadditivity, with ∼40% of genes showing dominance or overdominance effects vs. only 25% of genes showing additive effects. In contrast, our mouse study identified 19.9% of genes that were significant for additive effects but only <1% of genes that were significant for dominance or overdominance. The findings from the Arabidopsis study (Vuylsteke et al. 2005) fall between these extremes. Liver tissue is composed largely of tetraploid cells, which may explain the low levels of nonadditivity observed in this study. Another potential cause could be the difference in genetic divergence between the parental inbred strains used in different studies. It has been suggested that in crosses between more distantly related strains there should be greater differences in expression and that the expression of the F1 hybrid will diverge more from the midparent values (Enard et al. 2002; Landry et al. 2005; Lemos et al. 2005). The two inbred mouse strains are relatively close (Beck et al. 2000) while the two Drosophila inbred strains may be more distant. The larger number of genes with additive effects compared with those with dominance effects in our study suggests that most of the difference of gene expression between the two parental strains may derive from cis-regulatory variation. This is consistent with the finding that most of the expression QTL (eQTL) obtained in expression mapping studies in mouse are cis-eQTL (Schadt et al. 2003; Bystrykh et al. 2005; Chesler et al. 2005).
After significant gene lists are obtained, it is common to run some classification or gene category analysis to examine which biological processes or pathways are overrepresented in the significant gene lists. If we compare the results from these similar studies, it is easy to identify some common terms among these studies. For example, “metabolism,” “signaling,” and “transportation” appear to be overrepresented in the significant gene lists for nonadditive inheritance pattern in multiple studies even though the studies differ in many aspects as discussed above, which indicates that there are some common processes or pathways showing dominance in gene expression inheritance. These results are consistent with the finding that genes involved in the metabolism and stress response are more likely to show nonadditive inheritance due to their role in responding to environmental stress (Kristensen et al. 2005). However, it should be pointed out that these terms are abundant in the array annotations. The overrepresentation of these terms in different studies may not be the direct result of underline biology.
Heritability of gene expression:
In classical quantitative genetics, phenotypic variation is decomposed into genetic and environmental variances (Falconer 1986). For inbred lines, the environmental variance can be estimated using the within-strain variation among genetically identical individuals. In this study we used biological replicates of inbred lines and repeated (technical) measurements to further decompose the environmental variance of gene expression into a biological component and a measurement component. Genetic and environmental variation was estimated from the variation between and within strains, respectively. The broad-sense heritability was estimated for the expression of each transcript. About one-quarter of the transcripts have heritability <1% but 18% of transcripts show heritability >50% and the median heritability of all genes is 20%. This heritability is substantially higher than the 11% found in the mouse brain gene expression from mouse RI mapping lines (Chesler et al. 2005). The application of shrinkage to improve variance component estimation in our study improved the overall heritability and resulted in more transcripts with high heritability. The median heritability increased from 16 to 20% with shrinkage. If we examine only the genes that have heritability >1%, the median heritability after shrinkage is 31%, which is similar to the 0.34 estimated from human family cell lines (Monks et al. 2004).
The authors are especially indebted to Sandy Daigle for help with RNA isolation and to the Gene Expression Service in the Microchemistry Department at The Jackson Laboratory for help with RNA quality and microarray analyses. We thank Manhong Dai at University of Michigan for help with custom CDF files. The Jackson Laboratory is American Association for Laboratory Animal Science accredited and all animal procedures were approved by The Jackson Laboratory Animal Care and Use Committee. This study was supported by National Institutes of Health grants HL55001 (G.A.C.) and HL072241 (Jurgen Naggert) and by the National Cancer Institute core grant CA34196 (The Jackson Laboratory).
Communicating editor: J. A. Birchler
- Received May 2, 2006.
- Accepted July 31, 2006.
- Copyright © 2006 by the Genetics Society of America