A Dissertation entitled HDAC Mediated Integration of NF-κB Transcriptional Regulation by Lindsay Marie Schreiner Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology _________________________________________ Dr. Brian P. Ashburner, Committee Chair _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo August 2014 Copyright 2014, Lindsay Marie Schreiner This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of HDAC Mediated Integration of NF-κB Transcriptional Regulation by Lindsay Marie Schreiner Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo August 2014 Nuclear Factor kappa B (NF-κB) is a family of dimeric transcription factors conserved in structure and function from the fruit fly drosophila melanogaster. NF-κB is involved in survival, inflammatory responses, immune responses, apoptosis, cell cycle regulation, and growth and development. All members of the NF-κB/Rel family contain a Rel-homology domain (RHD) that allows these proteins to dimerize, bind to DNA, and translocate to the nucleus. Certain members of the Rel family contain a transactivation domain (TAD) which activates transcription. Dysregulation of the NF-κB pathway is observed in many disease states including, cancer, heart disease, chronic inflammation, viral infection, cachexia, and neurological disorders. In the classical pathway, NF-κB consists of a heterodimer of p50/p65 that is sequestered in the cytoplasm by IκBα, a member of the IκB family of inhibitory proteins. When cells are induced by a pro-inflammatory cytokine, such as TNFα, intracellular signals that affect NF-κB converge at the IKK proteins. Activated IKK kinases phosphorylate IκBα, which is then ubiquitinated and degraded by the 26s proteasome. After degradation of IκBα, the nuclear localization signal of the p50/p65 heterodimer is iii exposed and NF-κB translocates to the nucleus where it binds to promoters to activate transcription of NF-κB regulated genes. The NF-κB pathway is regulated on several levels. Classical NF-κB regulates transcription of its own inhibitor, IκBα, thus limiting the time NF-κB is transcriptionally active. Coactivators are protiens that enhance the activity of transcription factors. NF- κB activity is enhanced by coactivators, especially the histone acetyltransferases (HATs) CBP/p300 and pCAF. HATs acetylate histones, transcription factors and cofactors to enhance transcription. Corepressors are proteins that repress the activity of transcription factors. NF-κB activity is repressed by various corepressors including histone deacetylases (HDACs) which deacetylate histones to repress transcription. HDACs also deacetylate transcription factors and other non-histone proteins. HAT and HDAC activity help regulate the overall level of transcription taking place by affecting acetylation, which is a post-translational modification. Phosphorylation is another post- translational modification involved in regulating NF-κB activity. Phosphorylation occurs at every step in the NF-κB pathway to signal for kinase activity, degradation, other modifications, or protein-protein interactions. Regulation of the NF-κB pathway is complex and involves the cooperation of many different proteins. HDAC1, HDAC2, and HDAC3 are the focus of the research described in this dissertation because these are the class I HDACs most responsible for regulating classical NF-κB activity. Stable knockdown cell lines were created using a lentiviral RNAi system and shRNAs specific to each gene to study the individual roles of HDAC1, HDAC2, and HDAC3. Individual knockdown of HDAC1, HDAC2, and HDAC3 all caused changes in IκBα gene expression, p65 nuclear localization, and IKKα nuclear localization. iv Knockdown of HDAC3 alone enhanced nuclear NF-κB in HeLa cells treated with TNFα in a similar manner to previous studies that utilized a pan-HDAC inhibitor Trichostatin A (TSA), and individual knockdown of HDAC1 and HDAC2 did not show enhanced nuclear NF-κB. Interestingly, knockdown of HDAC3 caused a decrease in IκBα protein in HeLa cells treated with TNFα, despite causing an increase in IκBα gene expression. It was hypothesized that increased IKK kinase activity in the HDAC3 knockdown cell line was responsible for the decreased IκBα protein upon TNFα treatment; however, the HDAC3 knockdown cell line showed neither increased IKK kinase activity nor increased IκBα phosphorylation compared to control. Surprisingly, increased IKK kinase activity was detected with knockdown of HDAC2 in HeLa cells treated with TNFα, and this observation has yet to be further explored. Gene expression and promoter studies in TNFα treated HeLa cells with HDAC3 knockdown indicate that there is a highly specific and complex system of regulation at each different gene promoter. v I dedicate this dissertation to my mother, Christy Lou Tubbs. She was intelligent, kind, and strong. She was a solver of every problem. She was self-sacrificing and loving, and she had an amazing sense of humor. Although I finished my graduate work after you were gone, I was able to finish because of the lessons you taught me. Thank you. Acknowledgements I would like to thank my mentor, Dr. Brian Ashburner, for his guidance and patience in working on my project, and his advice on life. I would like to thank my committee members for their time, and their guidance on which direction to take my project. Thank you to the University of Toledo for funding and the opportunity to gain experience teaching which is what I would like to continue doing. I would like to thank my family for all their support. My dad, Wesley Tubbs, has been my swim coach since I was a child, and the hard work ethic from athletics translates into all of life’s challenges. My brother, Brian, and sister, Cari, have always been an incredible unconditional loving support network. My mom, Christy (Momma Tubbs) was a constant calming influence, reminding me that if I do my best, that is all anyone can ask. And my husband, Elliott, has taken on many tasks to allow me the opportunity to finish my degree and provided loving support all the way. I would like to thank all of my friends who have been a constant source of joy and support throughout the most challenging parts of my life so far. Katie Halter, you are my soul sister. Natalya Blessing and April Brockman: thank you for making two of the most difficult transitions of my life more bearable and bringing humor and joy to my every-day life. Alexandra Judd (and Judd family): thank you for being my family on the other side of the world. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii 1 Introduction…. .........................................................................................................1 1.1. NF-κB/Rel proteins……..……………………………………………………1 1.2. NF-κB activation……………………………………………………………..4 1.3. Inhibitor of κB (IκB) proteins………………………………………………..4 1.4. IκB kinases…………………………………………………………………..7 1.5. Transcriptional coactivator and corepressor proteins…………………….....11 1.5.1. Coactivators……………………………………………………….11 1.5.2. Corepressors……………………………………………………….11 1.6. HDAC Family of corepressors………………………………………………14 1.7. HDAC1, HDAC2, and HDAC3……………………………………………..16 1.8. Regulation of NF-κB by posttranslational modifications…………………...17 2 Methods…………………………………………………………………………..19 2.1. Cells and Reagents…………………………………………………………..19 vi 2.2. Knockdown of HDACs 1, 2, and 3 using RNAi…………………………….20 2.3. Whole Cell Extracts…………………………………………………………22 2.4 Cytoplasmic and Nuclear Extracts…………………………………………...23 2.5. Western Blot Analysis………………………………………………………24 2.6. Densitometry………………………………………………………………..25 2.7. Electrophoretic Mobility Shift Assay (EMSA)……………………………..25 2.8. RNA Isolation……………………………………………………………….27 2.9. RT PCR……………………………………………………………………...27 2.10 Real Time RT PCR…………………………………………………………29 2.11 Coimmunoprecipitation (CoIP)…………………………………………….29 2.12 IKK Kinase Assay………………………………………………………….30 2.13. Chromatin Immunoprecipitation (ChIP) Assay............................................31 2.14. Luciferase Assay and β-Gal Assay………………………………...………34 2.15. Cell Counting………………………………………………………………35 2.16. Statistical Analysis…………………………………………………………36 3. Results……………………………………………………………………………37 3.1. Individual HDAC1, HDAC2, and HDAC3 shRNA knockdown……………37 3.2. Nuclear p65 is enhanced with individual HDAC1, HDAC2, and HDAC3 knockdown ………………………………………………………………………41 3.3. IκBα gene expression and protein level with individual HDAC1, HDAC2, and HDAC3 knockdown………………………………………………………..45