MOLECULAR REGULATION OF LIVER REGENERATION

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The ability of the liver to regenerate provides a unique system to study the in vivo regulation of cell proliferation and gene expression. This remarkable process is critical for its recovery from a number of disease states, including surgical and chemical injuries, and is dependent on the reentry of normally quiescent cells into a state of replication. The ongoing main objective of this research project is to define the role of mRNA stability in the expression of cell cycle- and growth-dependent genes in the regenerating rat liver after partial hepatectomy. The hypothesis is that this posttranscriptional mechanism is primarily responsible for the regulation of mRNA expression beyond the immediate-early (G0-G1) phase of the cell cycle. The specific aims are designed to test the hypothesis and characterize mechanisms involved in the in vivo regulation of mRNA stability. The first specific aim is to evaluate the role of the poly(A) tail in regulating the mRNA decay of genes which exhibit altered mRNA stability in the absence of transcriptional rate changes. The initial degree of polyadenylation and the rate at which the poly(A) tail is shortened will be studied using in vitro polysome decay extracts and RNase H mapping. In addition, the effect of translation in modulating the rate of poly(A) tail removal during regeneration will also be investigated. The second specific aim is to define the role of translation and polysome association in regulating the decay of specific mRNAs during regeneration. Gradient centrifugation of total, free, cytoskeletal- and membrane-bound polysome populations and RNAase protection analysis will be used to determine the relative translational activity of the transcripts. Correlations will be made between poly(A) tail length and association of the mRNA with the different polysome populations. The third specific aim is to characterize the role of the 172 kDa protein factor in modulation of proto-oncogene c-myc mRNA decay. Gel shift assays, UV crosslinking, RNA affinity chromatography and purification techniques used to identify and isolate the 70 kDa c-myc binding protein will be utilized. The role of sulfhydryls and phosphorylation will be studied in vitro to determine their effects on the RNA/protein binding complex. In vitro decay assays will be used to establish the functional significance of the 172 kDa protein in mRNA metabolism. The fourth specific aim is to explore the novel concept that the methylation status of the genomic DNAs regulate their transcript half-lives during liver regeneration. This will be done using Southern blot and PCR analysis, chimeric gene constructs, and in vitro methylation and transfection studies. The long term goal of the project is to formulate a model of posttranscriptional events in the regulation of mRNA stability during hepatic regeneration. The proposed studies will provide important and new information in our understanding of cell growth and the control of gene expression in the ability of the liver to recover from injury.