In partial fulfillment of the requirements for the degree of

Master of Science in Biology

in the

School of Biological Sciences

Jessica Pfliger

Will defend her thesis

“CHARACTERIZATION OF A CHROMATINIZED HYDROGEN PEROXIDE BIOSENSOR IN CANCER CELLS”

14, February 2022

 1:00pm

https://bluejeans.com/126058378

Thesis Advisor:

Dr. Yuhong Fan

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Dr. Alfred Merrill

School of Biological Sciences

Georgia Institute of Technology

 Dr. Hengbin Wang

Department of Biochemistry and Molecular Genetics

School of Medicine

University of Alabama at Birmingham

Abstract:

Hydrogen Peroxide (H2O2) is an important eukaryotic signaling molecule regulating cellular processes. As one of the most abundant reactive oxidative species (ROS), H2O2 can cause oxidative stress and cytotoxicity at elevated concentrations leading to DNA damage and programmed cell death. Despite the recent advancements in modulating ROS responses for potential cancer therapies, little is known about nuclear ROS temporal emergence and dynamics. To investigate the role of nuclear ROS and H2O2 in chromatin, we established a genetically engineered chromatin-targeted biosensor for H2O2, H2B-HyPer, by fusion of HyPer, a specific H2O2 biosensor, with core histone H2B. In this thesis, I utilized fluorescent microscopy and fluorescent recovery after photobleaching (FRAP) to study the H2O2 dynamics within the chromatin of HCT116 colon cancer cells containing genetically integrated H2B-HyPer. I demonstrate that H2B-HyPer is localized in the nucleus of HCT116/H2B-HyPer cells with an average residence time and mobile fraction of 9.14 minutes and 20.59%, respectively, comparable to the core histones in chromatin. I then show that H2B-HyPer is sensitive to changes in H2O2 levels post addition of H2O2 or DTT in culture medium. Further analysis of H2B-HyPer kinetics revealed a rapid increase and recovery of H2B-HyPer signal intensity in HCT116/H2B-HyPer cells upon treatment with and the removal of H2O2. Altogether, these studies establish H2B-HyPer as an effective biosensor for real-time spatio-temporal tracking of chromatin-proximal H2O2 dynamics.