| The purpose of my dissertation is to use multidisciplinary approaches to characterize the expression and regulatory mechanisms of the drug-processing genes during postnatal liver maturation. The ontogenic expression and the regulatory mechanisms have been examined for various drug metabolizing enzymes and transporters in the liver. By characterizing the ontogeny of the drug-processing genome in the liver, the present study has provided novel insights into identifying and further understanding the molecular targets for effective and safe drug treatments for children.;First, cluster analysis demonstrated that the ontogenic expression of drug-processing genes separate into 4 distinct patterns: perinatal enriched, early adolescent enriched, late-adolescent enriched, and adult enriched. In addition, the mRNA ontogeny of over 70 transcription factors was examined. These mRNAs also separate into 4 patterns. Critical nuclear receptors, including the xenobiotic sensor pregnane X receptor (PXR, N1I2), as well as the bile-acid sensor farnesoid X receptor (FXR, NR1H4), are crucial in regulating the expression of drug-processing genes during liver development. Initiation of bile acid signaling, mediated largely via FXR, is a hallmark of the neonatal induction of major liver transporters involved in the enterohepatic circulation (EHC), whereas PXR is more important for the induction of xenobiotic-processing genes in adolescent and adult period, when the organism is exposed to more xenobiotics.;The accessibility of transcription factors to the target genes was determined by the methylation status of histones and gene promoters. The present studies have illustrated that various drug-processing genes and transcription factors are expressed in distinct dynamic patterns in developing mouse livers, and their expressions correlated with the chromatin architecture. Among various types of epigenetic signatures, histone H3 lysine 4 di-methylation (H3K4Me2) appeared to be the choice of nature to induce numerous drug-processing genes during postnatal liver development. These drug-processing genes include some cytochrome P450s (Cyps), some UDP glucuronosyl transferases (Ugts), some glutathione S-transferases (Gsts), and some transporters, such as the sodium-taurocholate cotransporting polypeptide (Ntcp, Slc10a1). In addition, the ontogeny of several transcription factors also appeared to be associated with altered occupancy of H3K4Me2, including the xenobiotic sensor aryl hydrocarbon receptor (AhR), the lipid sensor peroxisome proliferator-activated receptor alphaPGC-1alpha (PPARalpha, NR1C1), as well as the master endobiotic metabolism regulator and transcription co-activator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC1alpha, PPargc1).;Under a permissive chromatin environment, FXR triggered the "day-1" surge pattern of liver transporters involved in the EHC. Both the mRNA and protein expression of these transporters were decreased in livers of the FXR-null mice at 1 day of age. The bile-acid bio-synthetic enzymes from the classic pathway, including cytochrome P450 7a1 and 8b1, were more activated than enzymes from the alternative pathway during the neonatal period. Using LC-MS/MS analysis, I have detected 11 bile acids in serum and 17 in the liver. Cholic acid, which can only be synthesized from the classic pathway, was the predominant bile acid in newborns, and appeared to be responsible for the FXR-mediated neonatal upregulation of transporters.;Very little is known about the PXR DNA-binding signatures in vivo, or how PXR regulates novel direct targets on a genome-wide scale. Therefore, to further consolidate critical roles of PXR in regulating drug-processing genes during the early adult period, a roadmap of hepatic PXR bindings in the entire mouse genome was generated by ChIP-Seq. The most frequent PXR DNA-binding motif was two AGTTCA-like direct repeats with a 4bp spacer (DR-4). Surprisingly, there were also high frequencies of motifs with spacers of a periodicity of 5bp, forming a novel DR-(5n+4) pattern for PXR binding. For the PXR-target gene Oatp1a4, DR-9 (n=1) was the only motif within the ChIP DNA sequences bound by the PXR protein. ELISA-based transcription factor binding assay validated that PXR binds to DR-(5n+4) like sequences, including DR-4, DR-9, DR-14, and DR-19. This novel finding challenges the existing paradigm for the current understanding of nuclear receptor consensus sequences. (Abstract shortened by UMI.)... |
