maintained at the LOD 4.3 stringency, a higher retention rate than observed for the autosomal and X chromosome trans eQTLs. Thus in both male and female mice, this more stringent LOD threshold eliminated most of the trans eQTLs but retained most of the cis eQTLs. Significant eQTL Sharing Between the Sexes We examined the LOD 4.3 threshold eQTLs on the autosomes and the X chromosome to determine how many were shared between the female and male cohorts (Table 4). 965 cis eQTLs were shared, representing 73% of the female and 67% of the male cis eQTLs at this stringency. Only nine trans eQTLs at this LOD threshold in both sexes were shared in females and males, representing 1.7% and 0.8% of the female and male trans eQTLs, respectively. These nine shared trans eQTLs had mean LOD scores of 5.97 and 6.82 in the female and male cohorts, respectively. This very low level of sharing could be due to most of the trans eQTLs being sex-specific, or alternatively, most of the trans eQTLs could be false positives. In order to examine this further, we assembled a new set of transcripts that were called present in .1/3 of the pooled male and female expression data, thus enabling new analyses on a single set of transcripts for both sexes. We repeated the eQTL analysis six more times, once using the correct sex assignment, and five times with permuted sex assignments while preserving the number of males and females. At the LOD 4.3 threshold there were 414 and 1132 trans eQTLs in the females and males, respectively, using the correct sex assignments, with a total of 1546 trans eQTLs and 40 shared in both sexes (Table 5). If the trans eQTLs are primarily sex-specific, we would expect a large decrease in their numbers in the permuted datasets. There were on average 642 and 677 trans eQTLs in the permuted female and male datasets, respectively, with an average total of 1319 trans eQTLs and of 40 shared trans eQTLs in both sexes. Since the number of total trans eQTLs was only decreased by an average of 15% in the permuted datasets compared to the correct sex assignments, it is possible that many of the trans eQTLs are false positives. However, for the male cohort, there was an average 40% decrease in the number of trans eQTLs in the permuted datasets, suggesting that many of the male trans eQTLs may be authentic. As in the prior analysis, the majority of the cis eQTLs were shared in two new analyses (Table 5) Sex Effects on Gene Expression We next examined differences in gene expression levels between the male and female F2 macrophages. We pooled the male and female expression data, and set an arbitrary cutoff for expressed genes, in that the transcript must have been called expressed in 1/ 3 of the pooled samples. Altogether, there were 22,056 transcripts that met this criterion. We then performed non-parametric Mann Whitney tests to determine which of these were expressed . .. Table 5. Cis and Trans eQTLs$LOD 4.3 in male and female .... mice with and without 5 gender permutations# .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Female Males Shared Fpeemrmauleted pMearmle uted Spheramreudted . ... Cis eQTLs 1,328 1,442 1,005 1,154618 1,34169 992637 . ... Trans eQTLs 414 1,132 40 6426128 677665 4066 . . .. #based upon a pool of transcripts using the combined male and female . .. expression data. Values for permuted data are mean+S.D. for 5 separate .. gender permutations. . .. doi:10.1371/journal.pone.0001435.t005 Sex Specific Gene Regulation differently in the male and female macrophages. Remarkably 6,719 transcripts (31%) were expressed differently with p- values,2.2761026, meeting the conservative Bonferroni correct- ed p-value of ,0.05. Since the RNA under study was derived from cells cultured two weeks ex vivo, these sex effects on gene expression are likely attributable to X and Y chromosome dosage effects, rather than to endogenous and variable sex steroids in the F2 mice. About half of these (3,304) were expressed higher in female macrophages (female bias), and the other half (3,415) were expressed higher in male macrophages (male bias). These sex biased genes had a large range of fold differences between the sexes, with most having only modest effects of 1.2 to 1.5 fold, but also included 233 transcripts with 2 to 3-fold effects, 40 transcripts with 3 to 10-fold effects, and 10 transcripts with .10-fold effects (Table 6). Supplemental Table S7 gives the details for each of these sex biased transcripts ranked by fold-difference. All three female bias probes with .10-fold effects were not expressed in males and represented the same gene on the X chromosome, Xist. The Xist gene encodes a non coding but functional RNA known to play an important role in X-chromosome inactivation in females [17]; and, it has been previously identified as transcript expressed in female, but not male, mouse blastocyts [18]. Likewise, all seven male bias transcripts with .10-fold effects were not expressed in females and mapped to the Y chromosome. These seven probes represent 4 distinct genes: Ddx3y, encoding a DEAD box RNA helicase; Eif2s3y, encoding a translation initiation factor subunit; Uty, encoding a ubiquitously expressed tetratricopeptide repeat; and Jarid1d, encoding jumonji. In contrast, most of the genes regulated ,10-fold effects mapped the autosomes. Gene ontology analysis for the 3304 female biased transcripts compared to all expressed transcripts revealed many over represented classes (including regulation of metabolism, transcrip- tion, and zinc binding proteins) a few under represented classes (including the extracellular space). Supplemental Table S8 gives a full listing and P-values for the gene ontology findings of the female biased transcripts. Gene ontology analysis for the 3415 transcripts with male biased expression found over representation in the cytoplasm cellular component (p = 0.001), and the biological processes of protein transport, localization, and establishment of protein localization (all p,0.05). 3,974 (59%) of the 6,719 probes that exhibited sexually dimorphic expression were also associated with 2265 female and/or 2852 male eQTLs on the autosomes and X chromosome with LOD scores .3.0, with 74% and 78% of these classified as trans eQTLs in females and males, respectively (Supplemental Table S9). An additional 299 probes exhibited sexually dimorphic expression and had eQTLs on the Y chromosome, with 90% classified as trans eQTLs (Supplemental Table S10). Interestingly, . ... Table 6. Number and fold-effects of transcripts with sex ... biased expression with p,2.2761026. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... Fold effect Female bias Male bias Total . ... 1.0–1.2 1071 770 1841 .... 1.2–1.5 1846 1579 3425 ... 1.5–2.0 229 664 893 . ... 2–3 18 215 233 . ... 3–10 1 39 40 .... .10 3 7 10 ... Total 3304 3415 6719 . . .. . doi:10.1371/journal.pone.0001435.t006 PLoS ONE | www.plosone.org 5 January 2008 | Issue 1 | e1435