Genome-wide analysis of FXR target genes in mouse liver

The farnesoid X receptor (FXR, NRIH4) is a member of the nuclear receptor superfamily of transcription factors that regulate expression of their specific target genes after ligand binding. Throughout my doctoral study, I evaluated FXR binding to putative new target genes in the mouse genome using non-biased genome-wide analytical procedures in response to ligand availability to understand the molecular mechanism of FXR-regulated activation.;In this thesis, I utilized the chromatin immunoprecipitation (ChIP)-chip (NimbleGen), combined with a computational tool for analyzing the binding data, to identify and characterize groups of genes that are regulated by the binding of FXR. The analysis of ChIP-chip, which contains tiled arrays of 50-bp probe sets that span the promoter regions of all ∼25,000 annotated genes in the mouse genome, identified 2398 genes that are regulated by FXR upon binding by synthetic ligand GW4064. Interestingly, our data set contained many genes encoding proteins known to be regulated by FXR and many more that were previously unrecognized as FXR target genes.;The ChIP-chip methodology relies on tiled arrays so the analysis is promoter biased and is limited to interrogating binding to the regions covered on the arrays. For the above analysis this was a narrow region around the transcription start sites (TSSs) of known genes. Additionally, the detection methods rely on differential hybridization intensities across the arrays, which are prone to artifacts associated with any hybridization methodology (incomplete hybridization, cross hybridization, incomplete or over-washing etc.). As we were analyzing this data set, new technology became available that allowed us to evaluate the entire genome at once (instead of just promoter regions) and that is based directly on DNA sequencing instead of hybridization. Thus, we re-tooled our efforts to take advantage of this superior technology.;In this analysis, we utilized a completely non-biased genome wide chromatin immunoprecipitation approach with mouse hepatic chromatin enriched with an FXR antibody followed by high throughput DNA sequencing (ChIP-seq, Illumina/Solexa). This identified 1656 FXR binding sites and 10 percent were located within 2 kb of a transcription start site (TSS) which is much higher than predicted by random occurrence. A motif search uncovered a canonical nuclear receptor IR-1 site, consistent with in vitro DNA binding studies reported previously. A gene set enrichment analysis (GSEA) of genes located within 20 kb of a peak showed that genes identified by our ChIP-seq analysis were highly correlated with genes activated by an FXR-VP16 adenovirus in primary mouse hepatocytes providing functional relevance to the genome wide binding study. Gene Ontology analysis showed FXR binding sites close to many genes in lipid, fatty acid and steroid metabolism.;The analysis of ChIP-seq revealed that a separate nuclear receptor half-site for monomeric receptors such as LRH-1 (liver receptor homologue-1) was co-enriched with FXR binding sites and FXR activation of some newly identified promoters was significantly augmented by an LRH-1 expression vector in a co-transfection assay. To demonstrate that LRH-1 acts as a co-regulator of FXR, we evaluated LRH-1 binding to its target genes in the mouse genome by performing non-biased genome-wide SOLiD (ABI) sequencing for liver enriched by LRH-1 antibody. We identified 10,634 genomic sites occupied by LRH-1 protein and 24% were located within 2 kb of a TSS. A motif search for LRH-1 binding sites revealed a LRH-1 motif containing nuclear receptor half site and 33% of total sites contained the motif. Gene Ontology analysis of the genes that contain both LRH-1 and FXR binding sites revealed many genes in lipid, fatty acid and steroid metabolism.;In the last chapter of my thesis, I provide evidence to support a novel role for FXR in protecting mice from acetaminophen (APAP)-induced hepatoxicity. We demonstrated that FXR activation induces the expression of several phase II and phase III genes that are known to be involved in xenobiotic metabolism. We used ChIP-chip, ChIP-seq and gene specific ChIP combined with luciferase reporter genes and animal expression studies, and verified that activation of FXR would provide protection from the hepatoxic effects of APAP.