Chronic Alcohol Effects on Transcriptional Regulation in Liver Regeneration

描述(由申请人提供)：这项探索性申请的主要目的是描述慢性酒精暴露对肝再生启动的影响下转录调控机制的性质和时间进程的变化。酒精性肝病仍然是美国和全世界发病率和死亡率的主要原因。有证据表明，酒精摄入使肝脏更容易受到其他挑战，从而通过损害肝脏的正常修复能力而使肝脏容易受到损害。慢性酒精暴露对转录调控机制的破坏在很大程度上仍未得到解释。本申请的目的是利用最近的两项进展提供的独特的协同机会来解决这个问题：(1)从正常和慢性酒精暴露条件下再生的大鼠肝脏获得高通量基因表达时间序列数据的可用性，以及(2)PAINT的可用性，这是一种用于转录调控网络分析(TRNA)的敏感生物信息学方法工具包，用于推导关键潜在转录因子(TF)的假设。本项目的总体驱动生物学假说是，慢性酒精对肝脏再生的破坏作用涉及作用于网络的一组聚焦的转录因子的改变，以调节系统范围的下游靶基因。我们建议用计算和实验相结合的方法来研究这一假说，分别有两个目的：(1)识别和验证正常肝再生开始背后的‘基线’转录调控；(2)识别和验证慢性酒精暴露对肝再生开始背后转录调控变化的影响。在每个目标中，我们将使用混合效应方差分析模型来分析时间序列基因表达数据，以便识别差异表达基因，并随后进行聚类分析以得出时间表达模式。我们将使用油漆进行tRNA，以假设在慢性酒精暴露下，肝脏再生开始时，候选TF的活性发生了变化。我们将使用基于EL ISA的TFDNA结合活性分析和染色质免疫沉淀以及随后的定量PCR来验证调节网络的优先子集，并评估候选TF在肝脏再生中特定生理意义基因启动子的体内占有率的定量变化。为了得出酒精干扰的组合调控相互作用的假设，我们将遵循对数线性建模方法来比较基线和慢性酒精改变的调控网络。该项目的一个关键成果是定量描述了在肝脏再生开始时，在生理意义基因的特定启动子位置上，慢性酒精依赖的关键转录因子活性的变化。在系统水平上了解酒精干扰的肝脏再生过程中的转录调控机制，将极大地帮助开发治疗靶点，以缓解酒精对肝脏疾病进展的影响。

DESCRIPTION (provided by applicant): The primary objective of this exploratory application is to delineate the changes in the nature and time course of transcriptional regulatory mechanisms underlying the effects of chronic alcohol exposure on the onset of liver regeneration regeneration. Alcoholic liver disease continues to be a major cause of morbidity and mortality in the USA and worldwide. Evidence suggests that ethanol consumption makes the liver more vulnerable to other challenges, thereby predisposing the liver to damage through the impairment of the normal repair capacity of the liver. This disruption of transcriptional regulatory mechanisms by chronic alcohol exposure remains largely unexplained. The present application is aimed at addressing this problem taking advantage of a unique synergistic opportunity presented by two recent advances: (1) availability of high-throughput gene expression time series data being obtained from regenerating rat liver in normal and chronic alcohol exposure conditions, and (2) availability of PAINT, a toolkit of sensitive bioinformatics methods for transcriptional regulatory network analysis (TRNA) for deriving hypotheses on key underlying transcription factors (TFs). The overall driving biological hypothesis of the present project is that the disruptive effects of chronic alcohol on the liver regeneration involve alterations in a focused set of TFs acting in a network to regulate system-wide downstream target genes. We propose to investigate this hypothesis in an integrated computational and experimental approach in two aims, respectively: (1) Identify and validate the 'baseline' transcriptional regulation underlying the onset of normal liver regeneration, and (2) Identify and validate the effects of chronic alcohol exposure on the changes in transcriptional regulation underlying the onset of liver regeneration. In each of the aims, we will employ a mixed-effects ANOVA model to analyze the time series gene expression data in order to identify differentially expressed genes and a subsequent cluster analysis to derive temporal expression patterns. We will perform TRNA using PAINT to hypothesize the candidate TFs with altered activity under chronic alcohol exposure in the onset of liver regeneration. We will validate a prioritized subset of the regulatory network using ELISA-based TF DNA-binding activity assays and chromatin immunoprecipitation followed by quantitative PCR and to assess quantitative changes in the in vivo occupancy of the candidate TFs at promoters of specific physiologically significant genes in the liver regeneration. In order to derive hypotheses on alcohol-perturbed combinatorial regulatory interactions, we will follow a log-linear modeling approach to compare the baseline and chronic alcohol-altered regulatory networks. A key deliverable of this project is a quantitative description of the chronic alcohol-dependent alterations of the activity of key TFs at specific promoter sites of physiologically significant genes in the onset of liver regeneration. Such a systems level understanding of alcohol-perturbed transcriptional regulatory mechanisms in regenerating liver will greatly aid in the development of therapeutic targets for ameliorating alcohol effects on progression to liver disease.