(564f) Combinatorial Transcriptional Regulatory Network Driving Aberrant Effects of Chronic Alcohol Consumption On Liver Regeneration

Chronic alcohol intake interferes with the onset and progression of liver regeneration after tissue damage; suppression of this repair mechanism is thought to contribute to the development of alcoholic liver disease. Our overall objective is to uncover and characterize the regulatory network affected by chronic alcohol consumption and how alterations in this network lead to an aberrant liver repair and regeneration response. Our strategy is to utilize a combination of experimental analysis of gene expression and transcription factor activity changes combined with computational modeling to evaluate the impact of chronic ethanol consumption on the transcriptional regulatory network. In the present study, we aimed to identify the genes and regulatory patterns associated with the early stage of liver regeneration after partial hepatectomy (PHx) to examine the effects of chronic ethanol intake on this process.

We employed the Lieber-DeCarli alcohol pair-feeding protocol to assess the extent to which the molecular processes underlying liver regeneration in alcohol-fed rats are similar to pair-fed littermate controls in which ethanol calories were replaced by carbohydrates. Our previous studies showed that the alcohol-fed group contained significant changes to transcription factor (TF) localization during the priming phase of liver regeneration (0-6 hours). Genome-wide binding targets of multiple TFs (of NFκB, C/EBPβ, C/EBPα, STAT3) were detected using either Roche NimbleGen ChIP-ChIP microarray platform (ChIP-chip) or ABI SOLiD platform (ChIP-Seq). For each TF, we annotated the localization site data with TF binding site (TFBS) genes and discretized the TFBS data by specifying if a gene was bound by the TF (1) or not bound (0). In order to minimize technical noise, each TFBS was considered to be occupied only if a majority of the biological replicates showed statistically significant binding. We integrated results from multiple TFBS data into dynamic binding activity patterns reflecting the effect of an alcohol diet on combinatorial TF binding during the priming phase of liver regeneration. We examined each pattern for enriched pathways to identify alcohol-affected biological functions.

Our results revealed dynamic changes in binding of several early response TFs at the promoters of key genes differentially co-regulated during alcohol exposure and the onset of liver regeneration. Analysis of binding patterns indicated significant differences in the genome-wide targeting of NFkB activity between chow-fed and isocaloric pair-fed rats. We identified target genes unique to the pair-fed group and unique to the chow-fed group. The former set included genes associated with functions such as positive regulation of caspase activity, protein complex assembly, and apoptosis.  The target gene set unique to chow-fed rats included genes associated with functions such as signaling, positive regulation of organelle organization, and positive regulation of cell growth and size. We also identified genes with a delayed increase in NFkB binding activity in the chow-fed group, including genes related to cell adhesion, and genes with an early increase in NFkB binding activity, including genes related to the cell cycle.  Given the similarity of physiological response in the two dietary groups, it is likely that these differences in NFkB binding are compensated for elsewhere in the global regulatory network driving liver regeneration.

The binding activity dynamics of the TFs of NFκB, C/EBPβ, C/EBPα, and STAT3 at target genes were altered by chronic alcohol intake. Further analysis of the dynamic patterns led to novel insights about the differences in the transcriptional network subtending liver regeneration between alcohol-fed rats and pair-fed controls. The dominant patterns were either novel in the alcohol group or unique to the control group. Functional association of genes in these key dynamic response patterns revealed pathways such as cellular homeostasis, cell cycle regulation, RNA metabolism, and translation, to be affected by novel and missing activity in the alcohol group.  Together, these results indicate that the effects of alcohol on liver regeneration may be mediated by dysregulation of cell cycle related processes and activation of alternative pathways.

Promoter analysis by PAINT revealed that several target genes may be driven by regulatory modules containing co-located binding sites for multiple TFs. We validated a subset of 20 NFκB targets and 5 key target promoters for the other TFs using ChIP-qPCR. Our results reveal that: (1) increased baseline activation of NFκB, C/EBPβ, C/EBPα, STAT3, c-Fos, and c-Jun in regenerating livers is correlated with the promoter binding for a subset of the target genes; (2) similarly, alcohol-fed rats showed a further increase in NFκB binding activity in response to PHx at 6 hours post-PHx occurring at only a subset of target promoters as opposed to pair-fed controls, which showed a consistent increase at all promoters tested during early liver regeneration; and (3) C/EBPβ, C/EBPα, STAT3, c-Fos, and c-Jun binding activity were down regulated at a subset of target promoters in alcohol-fed rats during early liver regeneration.

We conclude that chronic alcohol intake significantly affects the system-wide transcriptional regulatory network, including TF binding to the key genes active during the early stage of liver regeneration. These alterations may be critical in disrupting the normal regulatory network dynamics driving effective liver regeneration.