Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

James A. Goodrich

Second Advisor

Jennifer F. Kugel

Third Advisor

Dylan J. Taatjes

Fourth Advisor

Hubert Yin

Fifth Advisor

Robin D. Dowell

Abstract

Transcription of mRNA by RNA Polymerase II (Pol II) is a fundamental step in gene expression. In mammalians, this is a highly regulated process that requires the assembly and coordination of a multitude of nuclear factors that determine the level of transcription of any particular gene. This thesis investigates the mechanisms of transcriptional regulation by NFAT and AP-1 proteins, two families of transcription factors that play pivotal roles in tumorigenesis, proliferation, and the immune response. Using the human interleukin-2 (IL-2) gene as a model system, we characterized the mechanism of transcriptional synergy by NFATc2 and cJun in activated T cells. We propose a model by which cJun homodimers are recruited to the IL-2 promoter as a coactivator of NFATc2, resulting in high levels of transcription. This recruitment is mediated by a unique interaction between the C-terminal activation domain of NFATc2 and the DNA binding domain of a cJun homodimer.

Given the distinct role of cJun homodimers in regulating transcription, we aimed to develop DNA aptamers that are capable of distinguishing between cJun homodimers and cJun heterodimers, such as cJun/cFos. SELEX targeting DNA-bound cJun resulted in DNA aptamers that bind cJun homodimers with high affinity and specificity. For the highest affinity aptamer, we characterized the mechanism of binding, its secondary structure, and identified the minimal binding region. We determined that this aptamer effectively inhibits cJun from binding its corresponding DNA recognition element, and in cells we show that the aptamer can inhibit cJun homodimer activated transcription.

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