Chronic Alcohol Effects on Transcriptional Regulation in Liver Regeneration

描述（由申请人提供）：本探索性应用的主要目的是描述慢性酒精暴露对肝再生开始的影响的转录调节机制的性质和时间过程的变化。酒精性肝病仍然是美国和世界范围内发病率和死亡率的主要原因。有证据表明，乙醇消耗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使肝脏容易受到损害。长期酒精暴露对转录调控机制的破坏在很大程度上仍然无法解释。本申请旨在利用由两个最新进展呈现的独特协同机会来解决该问题：（1）在正常和慢性酒精暴露条件下从再生大鼠肝脏获得的高通量基因表达时间序列数据的可用性，和（2）PAINT的可用性，一个敏感的生物信息学方法的工具包，用于转录调控网络分析（tRNA），用于推导关键基础转录因子（TF）的假设。本项目的总体驱动生物学假设是，慢性酒精对肝再生的破坏性影响涉及在网络中起作用以调节全系统下游靶基因的一组集中的TF的改变。我们建议在两个目标的综合计算和实验方法中研究这一假设：（1）识别和验证正常肝再生开始的基础转录调控，以及（2）识别和验证慢性酒精暴露对肝再生开始的基础转录调控变化的影响。在每个目标中，我们将采用混合效应ANOVA模型来分析时间序列基因表达数据，以识别差异表达的基因，并随后进行聚类分析，以获得时间表达模式。我们将使用PAINT进行tRNA，以假设在肝再生开始时慢性酒精暴露下活性改变的候选TF。我们将使用基于ELISA的TF DNA结合活性测定和染色质免疫沉淀，然后进行定量PCR，并评估在肝再生中特定生理学显著基因的启动子处候选TF的体内占有率的定量变化，来验证监管网络的优先子集。为了推导出关于酒精干扰的组合调节相互作用的假设，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节网络。该项目的一个关键成果是定量描述肝再生开始时生理学显著基因的特定启动子位点上关键TF活性的慢性酒精依赖性改变。对再生肝脏中酒精干扰的转录调控机制的这种系统水平的理解将极大地有助于开发用于改善酒精对肝脏疾病进展的影响的治疗靶点。

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.