more of these Y chromosome eQTLs exhibited female bias (166) than male bias (133), indicating a Y chromosome effect on decreasing expression of specific transcripts in males. We mapped the genomic distribution of these sex- biased eQTLs, and they basically shared the hotspot distribution that we observed for the overall female and male eQTLs distribution (Fig 1C, D). DISCUSSION In the current work, we have identified eQTLs separately from male and female bone marrow derived macrophages derived from F2 mice from an AKR x DBA/2 strain intercross. These studies are subject to methodological considerations for both the microarray and QTL analyses. We chose to use Microarray Suite 5.0 (Affymetrix) for array normalization and expression levels, as this method has been shown to yield results similar to those obtained using the more conservative RMA normalization procedure [19– 21]. We also chose not to log transform the gene expression data. Although this will give relatively more weight to the data with large gene expression values, we prefer this over giving equal weight to a doubling of gene expression at the low end of the scale (e.g. 50 vs. 100 arbitrary MAS5 units), where the precision of the measure is expected to be lower and the signal approaches the background level, versus the high end of the scale (e.g. 5,000 vs. 10,000 arbitrary MAS5 units), where the precision of the measure is expected to be higher and the signal is far above the background level. Furthermore, we did not screen the Affymetrix probe sets for SNPs polymorphic between the AKR and DBA/2 strains, but based on a prior screen for polymorphic SNPs between the C57BL/6 and DBA/2 strains [19], only a small fraction of the cis eQTLs identified may be artefactual due SNPs in the probe sequence that could alter hybridization to the array. Nevertheless, some of our cis eQTLs could be due to either a polymorphic SNP overlapping the probe sequence, or a polymorphic copy number variation for the probe target sequence; and either of these would be expected to give rise to strong and highly heritable cis eQTLs. For our QTL analysis, we used the suggestive and significant LOD thresholds of 3.0 and 4.3 [16], as used in prior eQTL studies of mouse strain intercrosses [2,12]; in addition, we performed permutation analysis to directly calculate genome wide p-values of 0.25 and 0.02, respectively, for these LOD thresholds. These genome wide p-values were the same for cis and trans eQTLs at any given LOD threshold. However, it may be argued that we are underestimating the strength of the cis eQTLs, since the genome wide permutations utilized all markers and all probesets, while only one linked marker needs to be used to test the strength of cis eQTLs. We analyzed all eQTLs in single sex cohorts, as sex has been shown be markedly affect gene expression and eQTLs in various mouse tissues [12,13], and this strategy proved particularly important for trans eQTLs which were overwhelmingly sex-specific. At the suggestive LOD threshold of 3.0, there were ,3 to 4 times more trans eQTLs than cis eQTLs in both sexes. However, the average LOD score for the cis eQTLs was much higher than for trans eQTLs, as previously observed [2], presumably due to the direct effect of cis regulation being stronger than the indirect effect of trans regulation. At the significant LOD threshold of 4.3, there were instead more cis eQTLs than trans eQTLs. At this threshold, the vast majority of the cis eQTLs were retained from the suggestive threshold in both sexes, while only ,10% of the trans eQTLs were retained from the suggestive threshold. This shift from predomi- nantly trans eQTLs at the suggestive threshold to predominantly cis eQTLs at the significant threshold was also observed in an eQTL study of adipose tissue from a mouse strain intercross [12]. The occurrence of eQTLs hotspots resembled prior studies in which eQTL hotspots were found [1,2]. Interestingly, the genes Sex Specific Gene Regulation responsible for two trans eQTL hotspots have been identified in yeast, and both are signal transduction proteins rather than transcription factors, one is a G-protein subunit of a pheromone receptor, and the other is a protein that inactivates a transcription factor activator [22]. We found two autosomal eQTL super hotspots in the male F2 cohort on chromosomes 1 and 16. We identified nine candidate genes at the chromosome 1 hotspot, each with a strong cis eQTLs and whose expression is well correlated with the expression of the trans eQTLs mapped to the same locus (Table 2). Five of these nine genes have activities which suggest they could be responsible for the trans regulation of many genes. Nif3l1 encodes a highly conserved protein that has been shown to bind to other nuclear proteins and alter their transcription factor activity [23,24]. Abi2 encodes an SH3 domain containing protein that binds to and modulates c-abl activity with effects on cell morphogenesis and motility [25,26]. Little is known about Raph1, but it encodes a protein that contains both Ras association and plekstrin homology domains, thus it could play a role in signal transduction. Tgfbrap1 encodes a protein that binds to TGFb receptor 1 and plays a role in Smad- mediated signal transduction [27,28]. Unc50, the homologue of the C. elegans unc-50 gene, encodes a nuclear protein with RNA binding activity that has been shown to alter specific gene expression [29]. We identified three candidate genes at the chromosome 16 hotspot, each with a strong cis eQTL and whose expression is well correlated with the expression of the trans eQTLs mapped to the same locus (Table 3). One of these genes, Dgcr8, has an activity which suggests that it could be responsible for the trans regulation of many genes. Dgcr8 encodes an RNA binding protein that associates with Drosha, and which is required for microRNA processing with potentially global effects on gene expression [30–32]. Further work would be required to confirm whether any of these candidates are in fact responsible for these eQTL super hotspots. The third eQTL super hotspot in the males was on the Y chromosome. This is the first report, of which we are aware, of Y chromosome eQTLs. We were able to identify these due to the reciprocal nature of the strain intercross. At the LOD 4.3 threshold there were 334 Y chromosome eQTLs in the male cohort (almost all trans eQTLs), greatly outnumbering the 31 eQTLs on the X chromosome in the males. This indicates that the Y chromosome strain difference had a larger effect on gene expression in male bone marrow derived macrophages than the X chromosome strain difference. There were 171 female eQTLs mapped to the X chromosome at the LOD 4.3 threshold, also outnumbering the 31 male eQTLs mapped to the X chromosome, indicating that strain differences on the X chromosome were more important in regulating gene expression in female than in male macrophages. Although the majority of the cis eQTLs at either LOD threshold were conserved between the male and female cohorts, we were surprised by the low level of sharing for the trans eQTLs, with only ,1 to 2% of female or male trans eQTLs common to both sexes. We considered two possible interpretations of this finding: 1) the sex chromosomes play an enormous role in trans regulation of gene expression; or 2) the majority of trans eQTLs are false positives and therefore not conserved between the sexes. The evidence that supports the first interpretation is: a) the trans eQTLs were identified by the same methods that found the cis eQTLs, which are robustly conserved; and b) our genome wide p- values are based upon permutation analysis which takes into account the data structure, and thus these are likely to be good estimates. However, there is also evidence to support the second interpretation: permuting the sexes did not greatly reduce the number of total trans eQTLs, although it did reduce the trans eQTLs in males by 40%. Another group has also found that trans eQTLs are not reproducible, in this case comparing trans eQTLs PLoS ONE | www.plosone.org 6 January 2008 | Issue 1 | e1435