Chronic Alcohol Effects On the System-Wide Transcriptional Regulatory Network Modules and Dynamics During Rat Liver Regeneration

Liver regeneration is a complex, yet remarkably coordinated, process characterized by system-wide gene expression changes underlying the response of the liver tissue to acute damage through mechanical, chemical or viral mechanisms. This normal regenerative response is impaired by acute or chronic alcohol exposure. Such an alcohol-mediated disruption of repair mechanisms presumably contributes to the onset of liver damage in alcoholic liver disease. While progress has been made in understanding several aspects of liver regeneration, the system-wide mechanisms by which these are impaired by chronic alcohol exposure remain largely unexplained. This study aims to characterize how chronic ethanol treatment affects the transcriptomic regulatory dynamics in regenerating liver. 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 take an integrated experimental and computational approach to investigate the dynamics of the system-wide transcriptomic changes, use sensitive bioinformatics methods to infer regulatory modules comprising of transcription factors (TFs) and target genes, and validate these for changes in the activity of TFs and their binding at target gene promoters. In our experimental approach, rats were fed a liquid diet containing 36% of total calories derived from ethanol; corresponding pair-fed calorie-matched controls received liquid diets in which ethanol calories were replaced either by carbohydrate or by fat. After five weeks, rats were subjected to 2/3d PHx by surgical excision of left-lateral and medial (LLM) lobes. Remnant liver tissues were harvested after 1, 6 and 24h to represent the priming phase, early G1, and G1/S transition, respectively. LLM tissues obtained at t=0 served as within-animal controls. We used Affymetrix Rat Gene 1.0 ST arrays with ~25000 probe sets to obtain global gene expression data from each liver sample (n=4 replicate animals, 72 arrays total). Analysis of the gene expression data obtained from the LLM tissue samples (12 replicates per diet group) revealed a total of approximately 400 differentially expressed genes (>1.5-fold change) in ethanol-adapted animals compared to either control diet. No significant differences between the high-fat and high-carbohydrate control groups were obtained. Functional annotation analysis using DAVID Bioinformatics resource (v. 6.7) indicated significant alterations in gene clusters related to circadian rhythm, lipid and steroid metabolism, and ER membrane-related functions. The effect of PHx, analyzed using a 3-way ANOVA, revealed a total of approximately 5000 transcript clusters as responsive to PHx in at least one of the diet groups at either 1, 6 or 24h post PHx (false discovery rate < 15%). Hierarchical clustering yielded >35 distinct dynamic gene expression response profiles, several with altered response pattern in ethanol fed rats compared to those fed carbohydrate or fat control diets. Within the control and post PHx clusters, alcoholic livers showed distinct expression profiles and clustered separately from those of the carbohydrate and high fat fed rats. In each of the gene expression clusters, over-represented transcriptional regulatory elements were identified in corresponding promoters using our PAINT bioinformatics software, indicating that chronic ethanol intake significantly affects the transcriptional regulatory network dynamics in response to PHx. We experimentally validated the effects of chronic alcohol exposure on 16 TFs actively involved in liver regeneration. Our results reveal that ethanol exposed rats showed: (1) suppression of FosB, JunD and JunB, GATA-1, C/EBP-a, PPAR-a, PPAR-?, NRF2 and ATF2, (2) delayed activation of NF-kB, HNF1, STAT3, p-CREB and C/EBP-ß, (3) transient activation of c-Fos and c-Jun. We applied a novel regulatory module identification approach that was previously employed on genome-wide location analysis data to our PAINT bioinformatics predicted TF binding sites. Our analysis revealed that these TFs are likely to operate in combinatorial modules with co-localized regulatory elements. We predicted NF-kB binding at 21 target promoters using our PAINT bioinformatics approach and validated these predictions using Chromatin Immunoprecipitation assay. Our results reveal a baseline activation of NF-kB in alcoholic livers, further increase in binding activity at 1h post PHx for all promoters but increase at 6h post PHx occurring at only a subset of the target genes. We propose that the baseline increase in NF-kB activity due to chronic alcohol intake limits further changes in promoter binding at key genes, and this may underlie the adverse affects of chronic alcohol treatment on liver regeneration. Taken together, our results provide a first system-wide view of the regulatory network dynamics in liver regeneration as it is affected by alcohol, and implicate combinatorial regulatory modules as participating in the adverse liver regeneration response. Supported by: AA008714, AA014986, AA017261, AA016919, and AA018873.