Transcription Factor Engineering: Tools and Applications

Transcription factors play a vital role in the biology of every organism. By controlling gene expression they regulate growth, development, metabolism, reproduction, signaling, and response to the environment. They have also provided the basis for many useful tools in molecular biology. The estrogen receptor alpha is one of the most studied human transcription factors and acts as a ligand controlled regulator of transcription. The modular design of this and other transcription factors allows for the rational design of artificial gene switches to control expression of desired genes. In this thesis I explore some tools for, and applications of, engineering the estrogen receptor. Beginning with two ligand binding domain mutants previously engineered to recognize the small molecules 4,4’-dihydroxybenzil (DHB) or 2,4-di(4-hydroxyphenyl)-5-ethylthiazole (L9), I showed that they could be used as a gene switch to independently control reporter genes in yeast and mammalian cells. By using different DNA binding domains, activation and repression domains, promoter elements, and a luciferase reporter I implemented the logic functions AND, OR, NAND, and NOR in HeLa cell culture. My research revealed some of the limitations of both the modular engineering approach, and the yeast two-hybrid screening assay used to engineer the ligand binding domain. I explored the feasibility of performing directed evolution of gene switches in mammalian cells through a protoplast fusion method, which combines the benefits of simple library creation with screening in a functionally relevant system. Although individual steps of the process were successful, the method proved unsuitable for large scale screening of libraries. The endogenous gene vascular endothelial growth factor-A (VEGF-A) was targeted for control by a gene switch. The effect of construct design was evaluated using a VEGF-A promoter controlled luciferase gene and performance was impacted by the choice of DNA binding domain, activation domain, and the order of domain use. Endogenous VEGF-A protein secretion in HeLa cells was successfully upregulated twofold by a DHB ligand controlled gene switch. Finally, I developed a useful biosensor for estrogenic compound detection that has the advantage of requiring no added substrates for signal generation. Through fusing the N and C terminal halves of the fluorescent protein Venus to the receptor ligand binding domain, fluorescence complementation generated a robust signal upon addition of an estrogenic ligand. The biosensor was capable of responding to a range of endogenous, pharmaceutical, environmental, and industrial compounds with sensitivities that correlated with their relative binding affinity. The signal characteristics were seen to depend on the length of the LBD region used, with some constructs distinguishing between agonists and antagonistic ligands.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Computnl Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• McLachlan, Michael J.

Contributors dc:contributor

• Zhao, Huimin
• Katzenellenbogen, John A.
• Zhong, Sheng
• Gerlt, John A.

• Transcription factor
• estrogen receptor alpha
• protein engineering
• biosensor
• vascular endothelial growth factor-A (VEGF-A)

Statement dc:rights

• Copyright 2010 Michael McLachlan

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/18624

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/18624