Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Xiaolu Yang

Abstract

Daxx, a multifunctional protein with a diverse set of proposed functions, is ubiquitously expressed and highly conserved through evolution. A primarily nuclear protein, Daxx is able to regulate apoptosis, transcription, and cellular proliferation. Despite many studies into the function of Daxx, its precise role in the cell remains enigmatic. Herein, evidence is presented to expand upon the known anti-apoptotic function of Daxx, to establish Daxx as a novel molecular chaperone, and to further its repertoire of transcriptional targets. As an apoptotic inhibitor, Daxx is known to regulate p53 by stabilizing its main negative regulator, Mdm2, via formation of a ternary complex between Daxx, Mdm2, and Hausp. The present study reveals that DNA damage-induced phosphorylation of Daxx is an important step in the disruption of the Daxx-Mdm2-Hausp complex, allowing for p53 activation. A novel activity for Daxx is presented whereby it is able to modulate protein folding in vitro and in vivo. Daxx can refold and reactivate denatured substrates using its paired amphipathic helical and acid-rich domains. This finding was extended to p53, where Daxx was able to solubilize misfolded p53 both in vitro and in vivo. Further, this finding may provide a biochemical rationale as to the varied functionality of Daxx in the cell. Finally, a novel transcriptional target, Cdk6, is described for Daxx. Microarray analysis indicated that Cdk6 is a strongly downregulated gene upon Daxx silencing. Depletion of Daxx in various cancer and primary cell lines led to a decrease in Cdk6 protein levels. Additionally, Daxx can affect transcription of Cdk6, and chromatin immunoprecipitation reveals that Daxx binds to the Cdk6 promoter. Together, these results indicate that Daxx has potential functional plasticity and is involved in an array of cellular functions; in fact, cellular homeostasis relies on the proper execution of multiple biological processes. Elucidation of the biology of Daxx would not only provide insight into the regulation of these processes, but may also establish Daxx as a relevant therapeutic target.

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