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. ? ? ?
