Date of Award

8-10-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Dr. Gregory M. K. Poon

Second Advisor

Dr. W. David Wilson

Third Advisor

Dr. Kathryn B Grant

Abstract

Reporters are genetic constructs that place the biosynthesis of an exogenous probe, such as an indicator enzyme or fluorescent protein, under the control of a cloned promoter. Benefiting from advances in molecular cloning, both the regulatory and indictor elements can be highly customizable to meet specific experimental needs. Reporters are therefore flexible tools for investigating mechanisms of transcriptional regulation in live cells, where detailed characterization of protein/DNA complexes remains challenging. In addition, reporters are attractive cell-based indicators in library screening and downstream pre-clinical characterization of hit compounds. To obtain a better understanding of the mechanism of action of PU.1, a master transcription regulator of hematopoiesis from the ETS family, as well as to establish a cell-based platform for translational efforts, we engineered a series of highly engineered reporters for PU.1. We designed a dual-color fluorescent reporter system for interrogating mammalian cell lines that do not natively express PU.1. The system generates spectrally distinct fluorescent markers that are coupled with the exogenous expression of PU.1 and the transactivation of a PU.1-responsive enhancer/promoter element. We used this reporter system to investigate the evolutionary basis of target selection by the DNA-binding domain of ETS proteins toward PU.1-specific recognition. Further, we designed synthetic enhancer structures varying in number and spacing of PU.1 binding sites to test mechanistic hypotheses of PU.1 auto-regulation via a negatively cooperative DNA-bound dimer. Finally, by (single-cell) flow cytometry and (batch) plate-based fluorimetry, we used the reporter to characterize a panel of novel DNA-targeted heterocyclic diamidines with therapeutic potential as PU.1 inhibitors in acute myeloid leukemia. In summary, fluorescence-based reporters are powerful tools for correlating functional and biophysical properties of PU.1 in living cells. We expect that further development of our reporters to yield useful molecular tools for addressing mechanistic inquiries in transcriptional regulation and quantitative indicators in high-throughput screening applications.

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