Study On The Developmental Expression And Epigenetic Regulation Mechanism Of Human Hepatic UGT1A1

UDP-glucuronosyltransferases(UGTs) are key phase II enzymes that catalyze the glucuronidation of various compounds, including drugs and environmental pollutants. For instance, 35% of prescription drugs are metabolized by UGTs. UGTs are classified into two main families: UGT1 and UGT2. UGT1A1 is the unique enzyme responsible for bilirubin glucuronidation. Therefore, the complete or partial absence of its activity results in unconjugated hyperbilirubinemia, such as occurs in Crigler–Najjar and Gilbert’s syndromes.Neonatal hyperbilirubinemia is a very common phenomenon affecting60%–80% of newborn infants worldwide. Numerous factors have been implicated in neonatal hyperbilirubinemia, such as breast feeding, weight loss, premature birth, and UGT1A1 genetic polymorphism, among others. Recent studies suggest that, owing to delayed and inadequate UGT1A1 expression, the activity of this enzyme is diminished in fetuses and neonates, which is the principal cause of this condition,suggesting that the research on the development of UGT1A1 is the key point to control the blood bilirubin with normal level in neonates.Studies have shown that during an individual’s development and maturation,UGT1A1 exhibits marked changes. Its expression and activity do not follow a linear pattern from the fetal period to adulthood, which has a direct impact on neonatal and pediatric drug administration. Newborn infants and children are susceptible to adverse drug reactions, as metabolic functions have not yet matured. For example, the gray baby syndrome reported in the late 1950 s is caused by a neonatal UGT deficiency, resulting in the delayed clearance of chloramphenicol. Therefore, studying the developmental pattern of UGT1A1 will help to put in place age-dependent drug adjustment strategies for neonatal and pediatric patients.Transcription factor, also called trans-acting factor, can identify and combine with the core sequence of cis-acting elements involved in the transcriptional regulation of target genes. Some hepatic transcription factors can regulate the expression of genes involved in the metabolism of endogenous and exogenouscompounds, such as CYPs, UGTs and so on. While phase I enzymes have been the focus of many investigations, the mechanisms regulating ontogenetic changes in UGT1A1 by transcription factors remain elusive.More and more experimental evidences show that epigenetic modification plays an important role in the regulation of gene transcription. Epigenetic modification mainly includes DNA methylation, histone modification, chromatin remodeling and non-encoding RNA etc. Histone methylation is an important histone modification,which refers to the methylation in the N-terminal lysine or arginine residues of histone H3 and H4 with the catalysis of histone methyltransferase. Many studies confirm that H3K4me2, as an active marker of histone modification, promotes gene expression, while H3K27me3, as an inhibitory marker, silences gene expression. Li et al has reported that H3K4me2 and H3K27me3 are involved in the developmental regulation of Cyp3 a in fetal and adult rat. However, as for the ontogenetic regulation of hepatic UGT1A1, the study concerning epigenetic modification mechanism has rarely been reported.In conclusion, this study focuses on the molecular mechanisms of histone modification involved in the development of human hepatic UGT1A1, which will aid the development of individualized therapy strategies for pediatric patients..Part one Hepatic expression of transcription factors affecting developmental regulation of human hepatic UGT1A1ObjectiveComplete or partial inactivity of UGT1A1, the unique enzyme responsible for bilirubin glucuronidation, is commonly associated with hyperbilirubinemia. We investigated the dynamic expression of UGT1A1, and that of the TFs involved in its developmental regulation during human hepatic growth.Methods1. Ninety prenatal, pediatric and adult liver samples were obtained from Han Chinese individuals.2. Quantitative real-time polymerase chain reaction was used to evaluate m RNA expression of UGT1A1 and transcription factors(TFs) including PXR, CAR, HNF1 A,HNF4A, PPARA etc.3. UGT1A1 protein levels and metabolic activity were determined by Western blot and high-performance liquid chromatography.4. Direct sequencing was employed to genotype UGT1A1*6(211G? A) and UGT1A1*28(TA6? TA7) polymorphisms.Results1. Dynamics of UGT1A1 expression during human hepatic development.UGT1A1 expression was minimal in prenatal samples, but significantly elevated during pediatric and adult stages. m RNA and protein levels and metabolic activity were prominently increased(120-, 20-, and 10-fold, respectively) in pediatric and adult livers compared to prenatal samples.2. Correlation between UGT1A1 and TF expression in human livers. Dynamic expression of TFs, including PXR, CAR, HNF1 A, HNF4 A and PPARA, was consistent with UGT1A1 levels at each developmental stage. A pronounced correlation between expression of these TFs and that of UGT1A1(P<0.001) was observed.3. Polymorphisms affect UGT1A1 m RNA and protein expression and metabolic activity. UGT1A1*6 and UGT1A1*28 polymorphisms reduced levels of UGT1A1 by up to 40–60%.Part two Histone modification involved in the regulation of dynamic expression of UGT1A1 during human hepatic developmentObjectiveHistone methylation is one of the important epigenetic modification. As an activated marker of histone modification, H3K4me2 promotes the activation of gene expression, while as an inhibitory marker, H3K27me3 inhibits gene expression. In this study the differences of enrichment peaks of histone modification between fetal and adult liver during human hepatic ontogeny were detected to investigate the influence of histone modifications on the UGT1A1 dynamic expression.Methods1. 4 cases of liver samples were selected randomly(including 2 cases of fetal and2 cases of adult) for chromatin immunoprecipitation experiment by specific antibody of H3K4me2 and H3K27me3.2. Samples of enrichment DNA were sent to the Huada Company, library was built and high-throughput sequencing and bioinformatics analysis were applied.3. Difference of H3K4me2 and H3K27m3 of whole genome between fetal liver and adult liver was analyzed.4. Ch IP-q PCR was used to verify the results of Ch IP-seq with 6 cases(3 fetal liver and 3 adult liver selected randomly).Results1. Difference distribution of H3K4me2 enrichment peak between fetal liver and adult liver. There was an enrichment peak of H3K4me2 with a length of 5873 bp located near the promoter region of UGT1A1 in adult liver. Moreover, there were one or more enrichment peaks of H3K4me2 located in the genes of transcription factors,including HNF1 A, HNF4 A, GR, PXR, PPARA, CAR and RXRA in adult liver.2. Difference distribution of H3K27me3 enrichment peak between fetal liver and adult liver. There was an enrichment peak of H3K27me3 with a length of 226 bp located near the 12 kb upstream of UGT1A1 promoter region in fetal liver. Moreover,there were one or more enrichment peaks of H3K27me3 located in the genes of transcription factors, including GR, CAR, RXRA and HNF4 A in fetal liver.3. Validation of ChIP-seq results by ChIP-qPCR. Compared with fetal liver,average enrichment fold of H3K4me2 was 3.2 in adult liver. Moreover, compared with adult liver, average enrichment fold of H3K27me3 was 3.7 in fetal liver. So the result of Ch IP-q PCR was consistent with Ch IP-seq, which confirmed the reliability of the Ch IP-seq.Part three Molecular mechanism of histone modification involved in the regulation of dynamic expression of UGT1A1 during human hepatic developmentObjectivesWe have demonstrate that histone modification H3K4me2 and H3K27me3 affecting the regulation of the dynamic development of hepatic UGT1A1 in part two,but the molecular mechanisms involved in the developmental regulation of the UGT1A1 by histone modifications is not clear. This part intends to investigate the interaction between transcription factor and histone modifying enzyme involved in the regulation of UGT1A1 by methods of si RNA, Ch IP-seq and Co-IP.Methods1. The expression of HNF1 A was knocked down by si HNF1 A transfection. After transfection, the cells were cultured for 24 h-48 h, and then collected for q RT-PCR,Co-IP or Ch IP analysis.2. In order to investigate the changes of histone modification in the promoter of UGT1A1, the cells were collected for Ch IP analysis by specific antibody of H3K4me2, H3 Acetylation, NCOA6 and HNF1 A. Then enriched DNA fragments were collected for q RT-PCR analysis after si HNF1 A transfection.3. After si HNF1 A transfection for 48 h, the cells were collected for Co-IP analysis in order to investigate the interaction between transcription factor HNF1 A and histone methylation enzyme MLL1, as well as the interaction between HNF1 A and NCOA6(nuclear receptor cofactor 6).4. The expression levels of EZH2 and UGT1A1 were detected and the correlation between them was analyzed.Results1. Detection of HNF1 A and UGT1A1 gene expression after si HNF1 A transfection. In both Hep G2 and LS174 T cell lines, compared with the negative control, the HNF1 A and UGT1A1 protein expression level in si HNF1 A group decreased by about 70-80%.2. Changes of histone modification located in the promoter region of UGT1A1 after si HNF1 A transfection. In both cell lines, compared with the negative control group, the enrichment of H3K4me2 and H3 Acetylation located in the promoter region of UGT1A1 in si HNF1 A group were significantly decreased, as well as the enrichment of transcription factors HNF1 A and NCOA6.3. In both cell lines, there was interaction between MLL1 and HNF1 A as well as NCOA6 and HNF1 A.4. EZH2 protein level was high in the fetal livers and was low in the adult livers.In contrast, UGT1A1 protein level was low in the fetal livers and was high in the adult livers. EZH2 protein level was negatively correlated with both UGT1A1 m RNA level and UGT1A1 protein level.Conclusions1. Hepatic expression of transcription factors was associated with developmental regulation of UGT1A1 in the Han Chinese population.2. Histone modified of H3K4me2 and H3K27me3 were involved in the developmental regulation of human hepatic UGT1A1.3. Transcription factor HNF1 A may activate transcription expression of UGT1A1 by recruitment of histone modifying enzymes and nuclear receptor cofactor. Histone methylase EZH2 may be involved in the regulation of UGT1A1 expression.