DNA methylation in biliary development and disease

描述（由申请人提供）：胆道闭锁（BA）是婴儿阻塞性黄疸的最常见原因，如果不纠正，是致命的。BA影响1/10，000的婴儿，是儿童肝移植的主要适应症。这种纤维炎性疾病的病因仍然难以捉摸，但最近的工作表明细胞因子干扰素-γ（IFN 3）具有关键作用，尽管IFN 3激活的机制尚不清楚。IFN 3水平在患有BA的患者中升高，并且IFNg的靶向缺失抑制BA的恒河猴轮状病毒（RRV）注射小鼠模型的发展。 BA只发生在发育中的肝脏中，表明发育途径在BA的发病机制中起重要作用。表观遗传修饰，如DNA甲基化，作为稳定的基因沉默机制，抑制分化细胞中的基因表达。DNA甲基化的抑制，导致基因表达的去抑制，已被证明对发育有深远的影响，也增加了IFN 3途径基因的表达。我们有证据表明，广泛的化学（氮杂胞苷[azaC]）或遗传（dnmt 1，ahcy）抑制DNA甲基化导致肝胆发育缺陷，与IFN 3途径基因的显着增加，BA患者表现出胆管细胞特异性DNA低甲基化。这些发现使我们推测胆管细胞DNA低甲基化在BA中起致病作用。我们认为这是通过TGF-2介导的，TGF-2在胆道发育中起关键作用，并被IFN-3激活抑制。 本研究的目的是进一步了解DNA甲基化抑制导致发育性胆道缺陷的机制，并确定DNA甲基化在BA发病机制中的重要性。我们的机制研究将利用斑马鱼，其中我们有相当多的经验，检查肝胆的发展。斑马鱼是研究发育的一种简单的模式生物，因为它们离体发育迅速，并且有相当多的工具可用于遗传操作和分析。我们还将利用小鼠模型，并利用费城儿童医院大量的婴儿胆道疾病患者。利用这些资源，我们建议测试的假设，DNA低甲基化导致斑马鱼和小鼠模型和BA患者的胆汁缺陷，IFN 3的激活起着关键作用。

DESCRIPTION (provided by applicant): Biliary atresia (BA) is the most common cause of obstructive jaundice in infants and is lethal if uncorrected. BA affects 1/10,000 infants and is the leading indication for liver transplantation in children. The etiology of this fibroinflammatory disorder remains elusive, but recent work has implicated the cytokine interferon-gamma (IFN3) as having a crucial role, although the mechanism of IFN3 activation is unclear. IFN3 levels are elevated in patients with BA, and targeted deletion of IFNg inhibits development of the rhesus rotavirus (RRV)-injected mouse model of BA. The exclusive occurrence of BA in the developing liver suggests that developmental pathways play an important role in the pathogenesis of BA. Epigenetic modifications, such as DNA methylation, function as stable gene silencing mechanisms that repress gene expression in differentiated cells. Inhibition of DNA methylation, resulting in derepression of gene expression, has been shown to have a profound effect on development, and also increases the expression of IFN3 pathway genes. We have evidence that broad chemical (azacytidine [azaC]) or genetic (dnmt1, ahcy) inhibition of DNA methylation leads to defects in hepatobiliary development associated with a significant increase in IFN3 pathway genes, and that patients with BA demonstrate bile duct cell-specific DNA hypomethylation. These findings led us to hypothesize that bile duct cell DNA hypomethylation plays a pathogenic role in BA. We believe this to be mediated through TGF2, which plays a key role in biliary development and is inhibited by IFN3 activation. The goals of this proposal are to further understand the mechanisms by which inhibition of DNA methylation leads to developmental biliary defects, and to determine the importance of DNA methylation in the pathogenesis of BA. Our mechanistic studies will utilize zebrafish, in which we have considerable experience examining hepatobiliary development. Zebrafish are a facile model organism for studying development, as they develop rapidly ex vivo and there are considerable tools available for genetic manipulation and analysis. We will also utilize mouse models and take advantage of the high volume of patients with infantile biliary disease at The Children's Hospital of Philadelphia. Using these resources, we propose to test the hypothesis that DNA hypomethylation leads to biliary defects in zebrafish and mouse models and in patients with BA, and that activation of IFN3 plays a critical role.