Insight on the Regulatory Role of the PERIOD 2 Gene in the Cellular Response to DNA-damage Liang Jiang Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Biological Sciences Carla Finkielstein, Chair John Tyson Nikolaos Dervisis Shihoko Kojima May 13, 2019 Blacksburg, VA Keywords: Circadian rhythm, PER2, DNA damage, p53, radiation, MDM2 Copyright 2019, Liang Jiang Insight on the Regulatory Role of the PERIOD 2 Gene in the Cellular Response to DNA-damage Liang Jiang ABSTRACT Circadian rhythm is a ~24-h mechanism that keeps our physiology and behavior in synchrony with environmental changes. Period2 (Per2) is a core component of the circadian clock and a candidate tumor suppressor as its knockout expression results in a cancer-prone animal. p53 is an effector in the DNA damage response and regulates downstream effectors by trans-activation. Recent studies in our lab show that PER2 can bind to p53, and regulates the trans-activation function. This project studied the subcellular distribution of PER2 in response to DNA damage, and explored the role of p53 in the regulation of PER2 subcellular distribution. We found that PER2 accumulates in the nucleus in response to DNA damage, and such accumulation is independent of p53. In addition, we analyzed Single Nucleotide Polymorphisms (SNP) of PER2 in the 1000 Genome project to gain insight onto how missense mutations in PER2 lay at the interface of p53: PER2 binding. In a separate project, we also performed bioinformatics analysis on the iron related genes to discuss the circadian regulation of iron genes in the liver. These findings shed light on the regulation of Per2 under genotoxic stress, genetic variations of Per2 in normal human population, and expression of circadian genes under iron-controlled diets. Insight on the Regulatory Role of the PERIOD 2 Gene in the Cellular Response to DNA-damage Liang Jiang GENERAL AUDIENCE ABSTRACT Circadian rhythm is a ~24-h mechanism that keeps the body in synchrony with the environment. Period2 (Per2) is a gene at the core of circadian rhythm in mammals. In this work, we found that PER2 accumulates in the nucleus of cells in response to DNA damage. In addition, we analyzed the genetic variation of PER2 in general human population to gain insight onto how mutations in PER2 affect the risk of cancer that’s associated to circadian disruption. In a separate project, we performed bioinformatics analysis on the genes related to iron metabolism, and showed pattern of circadian regulation of iron genes in the liver. These findings shed light on how circadian rhythm responds to genotoxic stress, and summarized genetic variations of Per2 in normal human population, and the expression of circadian genes under iron-controlled diets. Contents ABSTRACT .................................................................................................................................................. ii GENERAL AUDIENCE ABSTRACT ........................................................................................................ iii List of figures: .............................................................................................................................................. vi Abbreviations .............................................................................................................................................. vii CHAPTER 1: Introduction ........................................................................................................................... 1 Overview of Circadian Cycle .................................................................................................................... 1 Physiological and Molecular Mechanism of the Circadian Clock ........................................................ 1 The Role of Post-translational Modifications in the Mammalian Circadian Clock .............................. 2 Genetic and Biochemical Properties of PER2 ...................................................................................... 3 Circadian Rhythm Output ..................................................................................................................... 4 Association between DNA Damage and Circadian Clock ........................................................................ 4 Chapter 2: Specific Aims .............................................................................................................................. 6 Aim1: To Determine the Subcellular Distribution of PER2 in Response to Genotoxic Stress ................. 6 Aim2: Determine Molecular Intermediaries Responsible for Circadian Phase Resetting in the Event of Genotoxic Stress. ...................................................................................................................................... 6 Significance of this Study: .................................................................................................................... 6 Chapter 3: Material and methods .................................................................................................................. 7 Cells and antibodies (for all experiments) ................................................................................................ 7 Irradiation (for all experiments) ................................................................................................................ 7 Monitoring Bioluminescence Rhythms in MEF cells (for all experiments associated with Aim2).......... 7 Immunoblotting (for all immunoblot experiments) .................................................................................. 7 Immunofluorescence microscopy (for all IF experiments associated with aim1) .................................... 8 Chapter 4: Results ......................................................................................................................................... 9 DNA damage leads to acute accumulation of PER2 in HCT116 .............................................................. 9 Establishing the experiment context ....................................................................................................... 11 Step1: Establishing the irradiation dosage for HCT116 ..................................................................... 11 Step2: validate the DNA damage response of γH2AX under 10Gy irradiation .................................. 12 Total abundance of PER2 in response to DNA damage is independent of p53 ...................................... 14 The Subcellular Distribution of PER2 in Response to Irradiation is Independent of p53 ...................... 15 Nuclear/cytosolic Distribution of PER2 after DNA Damage ................................................................. 18 Proteasome Inhibition can Rescue the PER2 Nuclear Accumulation from Leptomycin B .................... 20 Perturbation of Circadian Cycle by Small Molecule Inhibitors and Irradiation ..................................... 22 iv Circadian Phase Shift in MEFPer2:luc is Dose Dependent ..................................................................... 22 Circadian Phase Advance in Response to Irradiation can not be Alleviated by Caffeine ................... 23 CK1 Kinase Inhibitor PF670 can Alleviate Circadian Phase Advance in Response to Irradiation .... 24 Chapter 5: Analysis of Immunofluorescence Microscopy Images ............................................................. 25 Chapter 6: Bioinformatics analysis ............................................................................................................. 28 Introduction: ............................................................................................................................................ 28 Single Nucleotide Polymorphism (SNP) Analysis of Human PER2 Protein .......................................... 28 Introduction ......................................................................................................................................... 28 Procedure ............................................................................................................................................ 29 Results ................................................................................................................................................. 30 Effect of Iron Intake on Transcription of Circadian Genes in Mice Liver .............................................. 33 Introduction ......................................................................................................................................... 33 Results ................................................................................................................................................. 34 Materials and Methods ........................................................................................................................ 42 Gene Ontology Analysis of Circadian Iron Genes .................................................................................. 46 Introduction ......................................................................................................................................... 46 Procedure ............................................................................................................................................ 46 Result .................................................................................................................................................