Sexually Dimorphic Genome-Wide Binding of Retinoid X Receptor alpha (RXRα) Determines Male-Female Differences in the Expression of Hepatic Lipid Processing Genes in Mice

<div><p>Many hepatic functions including lipid metabolism, drug metabolism, and inflammatory responses are regulated in a sex-specific manner due to distinct patterns of hepatic gene expression between males and females. Regulation for the majority of these genes is under control of Nuclear Receptors (NRs). Retinoid X Receptor alpha (RXRα) is an obligate partner for multiple NRs and considered a master regulator of hepatic gene expression, yet the full extent of RXRα chromatin binding in male and female livers is unclear. ChIP-Seq analysis of RXRα and RNA Polymerase2 (Pol2) binding was performed livers of both genders and combined with microarray analysis. Mice were gavage-fed with the RXR ligand LG268 for 5 days (30 mg/kg/day) and RXRα-binding and RNA levels were determined by ChIP-qPCR and qPCR, respectively. ChIP-Seq revealed 47,845 (male) and 46,877 (female) RXRα binding sites (BS), associated with ∼12,700 unique genes in livers of both genders, with 91% shared between sexes. RXRα-binding showed significant enrichment for 2227 and 1498 unique genes in male and female livers, respectively. Correlating RXRα binding strength with Pol2-binding revealed 44 genes being male-dominant and 43 female-dominant, many previously unknown to be sexually-dimorphic. Surprisingly, genes fundamental to lipid metabolism, including Scd1, Fasn, Elovl6, and Pnpla3-implicated in Fatty Liver Disease pathogenesis, were predominant in females. RXRα activation using LG268 confirmed RXRα-binding was 2–3 fold increased in female livers at multiple newly identified RXRα BS including for Pnpla3 and Elovl6, with corresponding ∼10-fold and ∼2-fold increases in Pnpla3 and Elovl6 RNA respectively in LG268-treated female livers, supporting a role for RXRα regulation of sexually-dimorphic responses for these genes. RXRα appears to be one of the most widely distributed transcriptional regulators in mouse liver and is engaged in determining sexually-dimorphic expression of key lipid-processing genes, suggesting novel gender- and gene-specific responses to NR-based treatments for lipid-related liver diseases.</p></div

Sexual dimorphic RXRα-dependent gene regulation in mouse liver.

<p>A) Scatter plot of RXRα scores correlating with Pol2 scores. Genes identified having a RXRα and Pol2 score >6 for male and -<6 for females are represented by the black dots, with the grey dots representing correlation of all genes. Genes listed in order of RXRα score high to low (see also Table S5A and S5B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). B) Ontology analysis and pathway analysis of genes with an RXRα and Pol2 score >6 for male and -<6 for females (see also Table S5C and S5D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>)</p

Stratification of RXRα binding sites in male and female mouse liver according to peak score.

<p>Stratification of RXRα binding sites in male and female mouse liver according to peak score.</p

Gender differential responsiveness to RXRα activation in vivo.

<p>Male and female mice were gavaged once daily for 5 days with the RXRα ligand LG268. A) Relative RNA levels in male and female mouse liver as determined by QPCR in response to RXRα activation by LG268 for selected male and female enriched genes. B) RXRα occupancy in response to RXRα activation on selected male and female enriched genes (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s005" target="_blank">Fig. S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s006" target="_blank">S6</a>). *p<0.05 vs male/veh; ?p<0.05 vs female/veh; #p<0.05 vs female/LG268.</p

Genome wide mapping of RXRα and Pol2 binding sites in male and female mouse liver.

<p>A) Venn diagrams show number of RXRα binding sites in male and female livers with 37602 binding sites shared between genders, and 10243 unique RXRα male and 9275 unique RXRα female binding sites (see also Table S1A and S1B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). B) Venn diagrams show number of Pol2 binding sites in male and female livers, with 19307 binding sites shared between genders, and 6037 unique male and 1332 unique female Pol2 binding sites. C) Venn diagrams representing number of genes associated with RXRα peaks shown in A), with 11660 genes shared between genders, and 1099 unique genes for male and 1021 genes for female. D) Western blot analysis shows increased nuclear RXRα protein levels in female mouse liver compared to male mouse liver. E) Number of RXRα peaks as stratified by peak height (see also Table S2A and S2B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). F) Genomic positions of RXRα binding sites in male and female mouse liver. Data expressed as percentage of total peaks per gender. RXRα binding was significantly increased at TSS (+/−500 bp), TES, within exons and introns with a depletion in binding compared to the expected number of sites based on random distribution (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s001" target="_blank">Fig. S1A and S1B</a>).</p

RXRα and Pol2 binding sites in mouse liver 10 kB surrounding the Transcriptional Start Sites.

<p>A) Venn diagram showing number of genes with shared RXRα regulation between male and female within 10 kB up and downstream of TSS, with 1285 genes found in male only and 1152 regulated by RXRα in females (see also Table S3A–D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). B) Ontology analysis and pathway analysis of RXRα peaks with score >6 or <−6 located within +/−10 kB of TSS (see also Table S3E–F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>).</p

Correlation of gender differential RXRα, Pol2 and RNA expression levels.

<p>A) Correlation of RXRα and Pol2 binding with gene expression levels. B) Scatterplot representation of gender differential RXRα binding with gender differential gene expression on the left-hand side, and scatterplot representation of gender differential Pol2 binding with gender differential gene expression on the right-hand side. C) Genes with a gender specific positive correlation between RXRα binding Pol2 binding and changes in RNA levels (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s004" target="_blank">Fig. S4A–B</a> and Table S6A–H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>).</p

Representatives of gender differential RXRαregulated genes.

<p>A) Predominant RXRα and Pol2 binding for Cyp7b1 in male mouse liver. Left-hand panel: Top 2 tracks show Pol2 binding along the Cyp7b1 gene-span in male and female mouse liver. Bottom 2 tracks show RXRα peaks binding upstream of the TSS of Cyp7b1 TSS. Male enriched RXRα peaks are indicated by arrows P1, P2 and P3. Right-hand panel: Sexual dimorphic RNA levels of Cyp7b1 were confirmed by qPCR (n = 5–6) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s002" target="_blank">Fig. S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s003" target="_blank">S3A–G</a>). B) Predominant RXRα and Pol2 binding for Pnpla3 in female mouse liver. Left-hand panel: Top 2 tracks show Pol2 binding along the Pnpla3 gene-span in male and female mouse liver. Bottom 2 tracks show RXRα peaks binding upstream of the TSS of Pnpla3 TSS. Female enriched RXRα peaks are indicated by arrows P1, P2, P3 and P4. Right-hand panel: Sexual dimorphic RNA levels of Pnpla3 were confirmed by qPCR (n = 5–6) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s002" target="_blank">Fig. S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s003" target="_blank">S3A–G</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s007" target="_blank">S7</a> and S8). *p<0.05</p