Degree Name

Master of Science (MS)

Semester of Degree Completion

2019

Thesis Director

Gary A. Bulla

Thesis Committee Member

Antony O. Oluoch

Thesis Committee Member

Britto P. Nathan

Abstract

Mammalian development involves a complex system of regulatory signals and reactions resulting in highly differentiated cell types with specific structure and function. The liver is a major organ that has been studied extensively to understand underlying genetic processes responsible for specification, establishment and maintenance of tissue identity. Hepatoma and hepatoma variant cell lines have been used as models to understand genetic networks responsible for liver function. Whole genome microarray analysis of hepatocyte cell lines has revealed candidate genes that may serve as regulators or master regulators of liver specificity. In two previous studies in our lab, the role of candidate gene Cellular Repressor of EIA Stimulated Gene (CREG1) in regulation of liver-specific gene expression was determined using transfection studies combined with utilization of quantitative real-time polymerase chain reaction (qRTPCR). Both studies found strong activation (10-100-fold) of transcription factor Hnf6 and the downstream gene Serpinal (a marker gene used to identify liver function). These findings suggested that CREG1 might act through Hnf6 to regulate Serpinal via a Locus Control Region (LCR). However, while one study showed that CREG1 overexpression in a hepatoma variant cell line resulted in modest activation of liver-specific transcription factors Hnf1, Hnf4 and Hnf3, the second study suggested robust activation of these genes as measured by qRT-PCR. In the current study, a CREG1 expression vector was reintroduced into one of the variant cell lines (H11) and gene activation profiles monitored to establish validity of the previous studies. However, we postulated that CREG-1 overexpression, validated by qRT-PCR, can activate expression of hepatocyte transcription factor Hnf6 and the downstream gene Serpinal along with other liver-specific downstream genes. Results showed that CREG1 can fully rescue expression of liver-specific genes including transactivation genes, suggesting its role as a master regulator of liver function. CREG1 action appears to act at least partially through HNF6 gene activation, as well as through activation of HNF4/HNF1 pathway, both of which act to increase expression of the serpin locus through LCR activation through HNF6.

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