Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2011

Thesis Director

Gary Bulla

Thesis Committee Member

Gary Fritz

Thesis Committee Member

Zhiwei Liu

Abstract

Extinction of tissue-specific genes in mammalian somatic cell hybrids is a well known epigenetic phenomenon and knowledge of this process is useful in understanding molecular mechanisms underlying tissue specific gene expression. Signaling proteins critical for the activation of transcription including transcription factors, co-activators, and repressors, play a role in the establishment and maintenance of tissue-specific phenotypes. Tissue-specific transcription factors are the key driving force of tissuespecific gene expression. However, mechanisms responsible for gene silencing are largely unknown.

Extinction of liver specific traits in somatic hybrids generated by the fusion of hepatoma and fibroblast cells has been well documented and is correlated with the loss of liver-enriched transcription factors HNFla and HNF4 in the hybrids. However, loss of these liver transcription factors does not solely define the extinction phenomenon.

Although extinction ofliver-specific gene expression has been well-studied, the silencing of fibroblast-specific gene expression has not been well defined. Here, we asked about the validity of the reported bidirectionality of the extinction phenomenon, focusing on the extent of silencing of fibroblast-specific genes that occurs in somatic cell hybrids. The aim of this work was to specifically analyze the extent of extinction of fibroblastspecific gene expression in somatic hybrids and also to identify candidate genes that are responsible for gene extinction. In this study, whole genome microarrays were utilized in order to compare the expression profiles of three rat cell lines: hepatoma cells (FT02B), fibroblasts (RAT-I) and the resulting somatic hybrid cell lines (FR). Using the ratio of gene expression levels in RA T-1 compared to FT02B cells, a total of 944 genes were determined to be >5-fold higher expressed in fibroblasts (RA T-1 ). To identify genes that were more likely to be involved in gene silencing in the cell hybrids, we focused on genes involved in transcriptional regulation and signaling pathways that were also diminished in hybrid cell line by at least 5 fold. We report 18 candidate genes potentially responsible for the extinction of fibroblast specific genes. Candidate genes include the fibroblast specific genes Short stature homeobox 2 (Shox-2) and paired related homeobox 1 (PMXl). Shox-2 plays a role in controlling the expression of Osteopontin, a highly fibroblast specific gene which is known to be the crucial component of the osteodifferentiation process during bone formation. PMXl plays a role in the c-Fos gene activation that regulates the cellular proliferation and differentiation leading to the fibroblast cell type. Also identified were several candidate genes associated with signaling pathways that have yet to be evaluated for their role in gene regulation and tissue-specific gene expression.

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