Function annotation of hepatic retinoid x receptor α based on genome-wide DNA binding and transcriptome profiling

Abstract

Background: Retinoid x receptor α (RXRα) is abundantly expressed in the liver and is essential for the function of other nuclear receptors. Using chromatin immunoprecipitation sequencing and mRNA profiling data generated from wild type and RXRα-null mouse livers, the current study identifies the bona-fide hepatic RXRα targets and biological pathways. In addition, based on binding and motif analysis, the molecular mechanism by which RXRα regulates hepatic genes is elucidated in a high-throughput manner.

Principal findings: Close to 80% of hepatic expressed genes were bound by RXRα, while 16% were expressed in an RXRα-dependent manner. Motif analysis predicted direct repeat with a spacer of one nucleotide as the most prevalent RXRα binding site. Many of the 500 strongest binding motifs overlapped with the binding motif of specific protein 1. Biological functional analysis of RXRα-dependent genes revealed that hepatic RXRα deficiency mainly resulted in up-regulation of steroid and cholesterol biosynthesis-related genes and down-regulation of translation- as well as anti-apoptosis-related genes. Furthermore, RXRα bound to many genes that encode nuclear receptors and their cofactors suggesting the central role of RXRα in regulating nuclear receptor-mediated pathways.

Conclusions: This study establishes the relationship between RXRα DNA binding and hepatic gene expression. RXRα binds extensively to the mouse genome. However, DNA binding does not necessarily affect the basal mRNA level. In addition to metabolism, RXRα dictates the expression of genes that regulate RNA processing, translation, and protein folding illustrating the novel roles of hepatic RXRα in post-transcriptional regulation.

Figure 1. Global analysis of ChIP-seq and microarray data.

(A) Venn diagrams of RXRα-bound genes (III+IV+V), hepatic genes (I+II+III+IV), and RXRα-dependent genes (II+III). RXRα binding location for (B) RXRα-dependent, (C) RXRα-independent hepatic expressed, and (D) non-hepatic expressed genes. In gene: peak summit located within the coding region; Promoter: peak summit located within −2 kb ∼0 bp to the transcription start site (TSS); Upstream: peak summit located within -100 kb∼−2 kb to the TSS; Downstream: peak summit located within 0 bp ∼100 kb to 3′ end of the gene; Intergenic: peak summit located outside the above mentioned regions. Red circle: RXRα-dependent genes; Blue circle: hepatic genes; Yellow circle: RXRα-bound genes. I: RXRα-independent genes and lack of RXRα binding; II: RXRα-dependent genes but lack of RXRα binding; III: RXRα-dependent genes that have RXRα binding; IV: RXRα-independent genes that have RXRα binding; V: non-hepatic genes that have RXRα binding.

Figure 2. Chromosomal distribution of RXRα peaks in RXRα-dependent genes in mouse liver.

Each bar represents an RXRα binding site on the mouse genome. UIK (green): up-regulated in RXRα KO liver; DIK (red): down-regulated in RXRα KO liver.

Figure 3. Motif Analyses.

(A) Global profiling of RXRα binding motifs in mouse liver genome predicted by Hidden Markov Model. DR: direct repeat; ER: everted repeat; IR: inverted repeat. (B) Out of the top 500 strongest bindings, the most common motif contains three half nuclear receptor binding sites, which may form two overlapped DR1s sharing the middle half site. (C) The other common motif contains a GC box that matches to the Sp1 binding site.

Figure 4. Representative heat maps of functional annotation clustering of RXRα-dependent genes without RXRα binding sites.

Genes up-regulated (A) or down-regulated (B) due to hepatic RXRα deficiency were subjected to DAVID functional annotation. The gene-term association relationship was generated using the functional annotation clustering tool in the DAVID website. Gray areas indicate the gene-term associations have been established by the literatures. Black areas show the gene-term relationships can exist, but requires experimental validation. Explanation for some of the listed terms shown in A: Domain: HIN-200 is a domain of HIN-200 protein, PIRSF018550 is a protein of PIR super family with serial number of 018550, IPR004021 is a domain of protein HIN-200/IF120x. Domain: DAPIN is a domain for apoptosis and interferon response. IPR004020: Pyrin is a subclass of DAPIN domain that interacts with proteins that have pyrin domain.

Figure 5. Expression of genes in wild type and RXRα-null livers.

RNA extracted from wild type and RXRα-null livers (n = 3–4) were subjected to real time PCR to determine the expression level of the studied genes. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level.**: p<0.05 in comparisons between two groups. Microarray experiment showed that fold change in Rarβ, Nr1d1, Nr5a2, Ncor2, and Ppargc1a mRNA levels due to hepatic RXRα deficiency was 1.8, 0.7, 0.5, 1.3, and 0.3, respectively (n = 3, p<0.05).