Functional connection between the growth factor independence-1b and post-neonatal regulation of biotransformation genes

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Children signify a population with highly dynamic physiology, particularly during the postnatal stage. We and others have shown that many biotransformation genes (drug metabolizing enzymes and transporters) exhibit a postnatal surge. We have also shown that the surge is accompanied by a rapid downregulation of the growth factor independence-1b gene (GFI-1b), a blood-producing gene essential for the liver to function as a blood- producing organ during the fetal and early stage of life. In addition, we have shown that GFI-1b, a sequence- specific transcription factor, represses the promoter of CES1 (carboxylesterase-1), a most abundant drug- metabolizing enzyme in the liver. The repression requires the DNA binding domain. Very recently, we have shown that interleukin-1β (IL-1β), a proinflammatory cytokine known to downregulate many biotransformation genes, induces GFI1b by 6-fold but decreases CES1 mRNA by 80%. The central hypothesis of the proposed project is that GFI-1b is intimately involved in the postnatal surge and inflammatory downregulation of many biotransformation genes. The specific aims are: (1) to ascertain the global engagement of GFI-1b in regulating biotransformation genes in the postnatal surge, and (2) to functionally characterize the response element(s) for GFI-1b to downregulate CES1. To link GFI-1b directly to the repressed expression of a large number of biotransformation genes, overexpression and knockdown of GFI1b as well as chromatin-immunoprecipitation- seq will be performed. To locate the element(s) in the CES1 gene that supports GFI1b repression, a set of molecular assays will be performed including reporter dissection, electrophoretic mobility assay and site- directed mutagenesis. The functionality of these reporters will be tested in GFI-1b overexpression and knock- down cells. To establish the role of induced GFI-1b in suppressed expression of CES1, GFI-1b knockdown cells will be treated with this cytokine and monitored for the reversal of suppressed CES1 expression. The reversal will be confirmed with CES1 reporter assays to gain sequence specificity. The scientific premise of this project is strong and original. The originality stems from the novelty of GFI-1b as a critical regulator in the expression of biotransformation genes, particularly during the early stage of life. It is the de-repression of GFI- 1b that supports the postnatal surge of many biotransformation genes. The project will also investigate a role of GFI-1b in inflammatory regulation of biotransformation genes, pointing to a novel mechanistic connection between developmental and inflammatory regulation. Overall, completion of this project will have filled knowledge gaps, delivered sustained impact in this research area with strong pathophysiological significance, and laid a foundation for a large project on GFI-1b based enhancement of hepatic differentiation and normalization of the expression of biotransformation genes under inflammatory conditions.