Sex Specific Gene Regulation and Expression QTLs in Mouse Macrophages from a Strain Intercross Jeffrey M. Bhasin1, Enakshi Chakrabarti1, Dao-Quan Peng1, Aneesh Kulkarni1, Xi Chen2, Jonathan D. Smith1,3* 1 Department of Cell Biology, Cleveland Clinic, Cleveland, Ohio, United States of America, 2 Department of Quantitative Health Sciences, Cleveland Clinic, Cleveland, Ohio, United States of America, 3 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America Background. A powerful way to identify genes for complex traits it to combine genetic and genomic methods. Many trait quantitative trait loci (QTLs) for complex traits are sex specific, but the reason for this is not well understood. Methodology/ Principal Findings. RNA was prepared from bone marrow derived macrophages of 93 female and 114 male F2 mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 genetic backgrounds, and was subjected to transcriptome profiling using microarrays. A high density genome scan was performed using a mouse SNP chip, and expression QTLs (eQTLs) were located for expressed transcripts. Using suggestive and significant LOD score cutoffs of 3.0 and 4.3, respectively, thousands of eQTLs in the female and male cohorts were identified. At the suggestive LOD threshold the majority of the eQTLs were trans eQTLs, mapping unlinked to the position of the gene. Cis eQTLs, which mapped to the location of the gene, had much higher LOD scores than trans eQTLs, indicating their more direct effect on gene expression. The majority of cis eQTLs were common to both males and females, but only ,1% of the trans eQTLs were shared by both sexes. At the significant LOD threshold, the majority of eQTLs were cis eQTLs, which were mostly sex-shared, while the trans eQTLs were overwhelmingly sex-specific. Pooling the male and female data, 31% of expressed transcripts were expressed at different levels in males vs. females after correction for multiple testing. Conclusions/Significance. These studies demonstrate a large sex effect on gene expression and trans regulation, under conditions where male and female derived cells were cultured ex vivo and thus without the influence of endogenous sex steroids. These data suggest that eQTL data from male and female mice should be analyzed separately, as many effects, such as trans regulation are sex specific. Citation: Bhasin JM, Chakrabarti E, Peng D-Q, Kulkarni A, Chen X, et al (2008) Sex Specific Gene Regulation and Expression QTLs in Mouse Macrophages from a Strain Intercross. PLoS ONE 3(1): e1435. doi:10.1371/journal.pone.0001435 INTRODUCTION The combination of quantitative trait locus (QTL) mapping and gene expression profiling allows for the identification of expression quantitative trait loci (eQTLs), which are loci associated with the expression of each transcript. This method was first applied to a yeast strain intercross, where both cis-acting and trans-acting loci were identified associated with the expression level of hundreds of transcripts [1]. eQTL analysis was applied to mouse tissues from an F2 cohort derived from a strain intercross yielding thousands of eQTLs, which were distributed non-randomly over the genome yielding hotspots that each contained hundreds of eQTLs [2]. eQTLs have also been described using human lymphoblastoid cell lines from defined pedigrees [2–4]. This methodology has been used, in so-called ‘genetical-genomics’ studies [5], as an aid to identify candidate genes for complex phenotypic traits, such as obesity, in mouse strain intercross studies [6–9]; and, it has been a major shortcut in the identification of QTL causative genes, for example the identification of ABCC6 as the gene responsible for dystrophic cardiac calcification in DBA/2 mice [10]. Sex specific effects are quite common in mouse studies, for example PPARc agonist treatment reduces atherosclerosis lesion areas in male, but not female, LDL receptor-deficient mice [11]. Similarly, gene expression studies in male and female F2 mice have shown a large degree of sexually dimorphic gene expression in liver, adipose tissue, muscle, and to a lesser extent in brain [12,13]. Mouse phenotypic QTLs, such as gonadal fat pad mass [12] or atherosclerotic lesion areas [14,15], are also commonly sexually dimorphic, with many specific QTLs found in only male or female cohorts. Likewise, many mouse tissue eQTLs are also sexually dimorphic [12,13]. Prior mouse eQTL studies employed freshly isolated tissues, thus, many sexually dimorphic effects on gene expression could be due to exposure to the different hormonal milieu in male and female mice. In the current study, we employed bone marrow derived macrophages from a mouse strain intercross that was cultured 2 weeks ex vivo. We still found that many eQTLs are sex specific, and remarkably, that 30% of expressed genes were differentially expressed in female vs. male macrophages, suggesting that a large extent of sexually dimorphic gene expression may be directly dependent on X and Y chromosome dosage, rather than on the hormonal environment. RESULTS Suggestive eQTLs Microarray (Affymetrix 430v2) gene expression data were obtained from bone-marrow derived macrophages of 93 female and 114 male F2 mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 backgrounds. Since gene expression in somatic mouse tissues is highly sex specific [12,13], we analyzed eQTLs separately in males and females. We Academic Editor: Justin Borevitz, University of Chicago, United States of America Received July 24, 2007; Accepted December 4, 2007; Published January 16, 2008 Copyright: 2008 Bhasin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by SCCOR grant P50HL077107 from the National Heart Lung and Blood Institute of the National Institutes of Health. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: smithj4@ccf.org PLoS ONE | www.plosone.org 1 January 2008 | Issue 1 | e1435