Student Theses and Dissertations

Date of Award

1990

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell James Laboratory

Abstract

An analysis is presented of the molecular mechanisms involved in maintaining the liver specific transcriptional profile of the rat and mouse serum albumin genes. Using DNA transfection and recombinant viral infection, the minimal rat promoter and mouse enhancer elements were identified, functionally dissected and shown independently to be hepatocyte specific elements. Based on these studies, multiple cis-acting DNA regulatory regions were elucidated and the cognate proteins which interact with them characterized and where possible their identity determined. It was found that the promoter proximal -156 nucleotides of the rat albumin gene were necessary and sufficient to insure albumin transcriptional specificity. Furthermore, the transcription factors involved in insuring this effect included several liver enriched proteins and others of a more ubiquitous cellular distribution. It is therefore, the interaction of several factors (only some of which are liver specific or enriched) which dictates the liver specificity of this promoter. The albumin enhancer element was localized much farther upstream (-10.5 to -8.43kbp) and shown to consist of both positive and negative regulatory domains. Two of the proteins which interact with the albumin enhancer are newly described, NLS1 and ANF, a positive and negative acting protein respectively, which interact in a modular fashion to stimulate the rate of albumin transcription. NLS1 (non-liver specific 1) DNA binding activity is present in all cell types, yet it stimulates albumin transcription only in the liver. ANF (albumin negative factor) negates the action of NLS1 and can act upon heterologous promoter and enhancer elements in this same fashion. This negative factor is present ubiquitously, but its activity is overridden in the liver by a third protein factor. This protein was determined to be C/EBP, a liver enriched transcription factor, which for the enhancer element performs this novel modulatory role. The biological action of these different factors was subsequently evaluated by correlating their relative levels with both the specificity and rate of albumin gene transcription in a series of rat hepatoma cell lines, which each transcribe the albumin gene at a different rate. An analysis of the expression of the rat transthyretin gene was also included in this series of experiments, and the question of coordinate hepatocyte gene control was addressed. Lastly, the control of some of these transcription factors themselves were analyzed by measuring both their transcription rate and protein binding activity in the rat liver and several hepatoma cell types. These factors are regulated in a complex fashion, involving both transcriptional and post transcriptional mechanisms.

Comments

A thesis presented to the faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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