Year of Publication

2012

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Microbiology, Immunology, and Molecular Genetics

First Advisor

Dr. Brett T Spear

Second Advisor

Dr. Thomas Roszman

Abstract

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression.

My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

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