Date

July 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

The mechanisms that regulate gene expression are complex, however many features have been well-conserved through evolution. Repulsive guidance molecule c (RGMc), or hemojuvelin (HJV), is a member of a three gene family that in most vertebrates plays a critical role in iron metabolism, yet virtually nothing is known about the regulation of RGMc gene expression. To better understand the mechanisms that regulate RGMc expression, this dissertation investigates the molecular biology and biochemistry of the RGM family, presents the first detailed analysis of the RGMc promoter and data to support a post-transcriptional mechanism for RGMc gene regulation, and integrates the findings into an understanding of the molecular evolution of the RGM family of genes. This dissertation discusses three main topics: (1) the genomic structure of the RGM family of genes, (2) the mechanisms of transcriptional and post-transcriptional regulation focusing on RGMc, and (3) the molecular evolution of the RGM family. The long-range and overarching goal of this dissertation is to enhance understanding of the molecular mechanisms by which evolution has shaped the regulation of gene expression in a family of genes, like the RGM family. Following a brief introductory chapter on gene regulation, this work addresses the molecular biology and biochemistry of the RGM family, beginning with the structures of the genomic loci and organization of the genes across multiple organisms. In addition, chapter 2 attempts to define and critically evaluate what is known about the RGM family, and identify critical gaps in our understanding of the gene family. The molecular evolution of the gene family is presented along with the first ab intio structural model to permit future work for investigators in the field. Chapter 3 of this thesis focuses on defining the detailed gene struture of RGMc, its transcripts, and mechanisms of transcriptional regulation of the gene. Using reporter gene experiments, three critical regions of the proximal promoter are identified that are responsible for RGMc transcriptional activation in skeletal muscle, comprising paired E-boxes, a putative Stat and/or Ets element, and a MEF2 site. In non-muscle cells, expression of the muscle transcription factors myogenin and MEF2C can stimulate RGMc promoter function suggesting that these factors are important components for the expression of the gene in skeletal muscle. As these elements are highly conserved in RGMc from multiple mammalian species, the results presented in chapter 3, coupled with the evolutionary analysis in chapter 2, support the hypothesis that RGMc has been a muscle-enriched gene throughout its evolutionary history. Finally, the 4th chapter reveals a novel region, called the e-element, in the untranslated region of the RGMc transcript that operates via a post-transcriptional mechanism. RT-PCR results demonstrate a constant steady-state level of mRNA whether this element is present or absent, but an order of magnitude increase in reporter expression only when the element is present. Additional data reveals that RGMc is not regulated by iron levels prior to the formation of nascent protein. These data suggest that the e-element controlling RGMc expression may be an example of a small, but growing number of regulatory mechanisms that utilize the 5‘-untranslated region (UTR) to enhance translation of specific mRNA transcripts into a nascent protein. In summary, this dissertation reveals the promoter of RGMc (the first example in the entire three gene RGM family), a possible positive translational control element in the 5‘UTR of RGMc, the first ab initio structure of the RGM protein family, and provides a foundation to understand the molecular evolution of RGMc, and how this knowledge may be applicable to gaining insight into the structure, function, and development of gene families and their tissue-specific patterns of expression.

Identifier

doi:10.6083/M40K26J3

School

School of Medicine

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