Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2018 Kdm6b is Required for Self-Renewal of Normal and Leukemic Mouse Stem Cells Under Proliferative Stress Cates Mallaney Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Genetics Commons, and the Oncology Commons Recommended Citation Mallaney, Cates, "Kdm6b is Required for Self-Renewal of Normal and Leukemic Mouse Stem Cells Under Proliferative Stress" (2018). Arts & Sciences Electronic Theses and Dissertations. 1637. https://openscholarship.wustl.edu/art_sci_etds/1637 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Human and Statistical Genetics Dissertation Examination Committee: Grant A. Challen, Chair Todd E. Druley Daniel C. Link Jeffrey A. Magee Laura G. Schuettpelz Matthew J. Walter Kdm6b is Required for Self-Renewal of Normal and Leukemic Mouse Stem Cells Under Proliferative Stress by Cates N. Mallaney A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2018 St. Louis, Missouri © 2018, Cates N. Mallaney Table of Contents List of Figures .................................................................................................................. v List of Tables .................................................................................................................. vii Acknowledgments ......................................................................................................... viii Abstract .......................................................................................................................... xii Chapter 1: Introduction .................................................................................................... 1 1.1 Epigenetic Modifications are Dynamic and Reversible .......................................... 1 1.2 KDM6B as a Demethylase ..................................................................................... 2 1.3 KDM6B Plays an Important Role in Development .................................................. 5 1.4 KDM6B Regulates Transcription in a Demethylase-Independent Manner ............. 9 1.5 KDM6B as a Regulator of Inflammatory Response .............................................. 11 1.6 KDM6B as a Tumor Suppressor .......................................................................... 15 1.7 KDM6B as an Oncogene ..................................................................................... 17 1.8 KDM6B in Hematopoietic Malignancies ............................................................... 18 Chapter 2: Kdm6b is Required for Self-Renewal of Normal and Leukemic Stem Cells Under Proliferative Stress ............................................................................................. 20 2.1 Introduction .......................................................................................................... 20 2.2 Results ................................................................................................................. 21 2.2.1 Loss of Kdm6b results in depletion of phenotypic hematopoietic stem cells ..............21 2.2.2 Loss of Kdm6b results in depletion of functional long-term repopulating HSCs .........23 ii 2.2.3 Kdm6b is required for HSC self-renewal ...................................................................24 2.2.4 Kdm6b is required for self-renewal of leukemia-initiating cells ...................................25 2.2.5 Interferon response and NF-κB signaling are increased in Kdm6b-deficient HSCs ....26 2.2.6 Inflammatory stress forces differentiation of Kdm6b-deficient HSCs..........................28 2.2.7 Kdm6b is necessary for HSC maintenance in response to proliferative stress ..........31 2.3 Summary .............................................................................................................. 33 2.4 Methods ............................................................................................................... 36 2.4.1 Mice ..........................................................................................................................36 2.4.2 Bone Marrow Transplantation ...................................................................................36 2.4.3 Flow Cytometry .........................................................................................................37 2.4.4 Western Blotting ....................................................................................................... .39 2.4.5 H3K27me3 quantification ..........................................................................................39 2.4.6 LPS, pIpC, and 5-FU injections .................................................................................39 2.4.7 Methocult Plating.......................................................................................................40 2.4.8 Plasmids and Viral Transduction ...............................................................................40 2.4.9 Quantitative Real-Time PCR .....................................................................................41 2.4.10 RNA-SEQ data, quality control and analysis ...........................................................42 2.4.11 ChIPmentation ........................................................................................................43 2.4.12 Statistics..................................................................................................................44 2.5 Figures ................................................................................................................. 45 2.6 Tables .................................................................................................................. 74 iii Chapter 3: Discussion ................................................................................................... 96 3.1 Conclusions ......................................................................................................... 96 3.1.1 Introduction ...............................................................................................................96 3.1.2 Kdm6b in Stem Cell Fate Decisions ..........................................................................97 3.1.3 Regulation of Stress Response in Hematopoiesis Requires Kdm6b ..........................................................................................................................................98 3.1.4 Kdm6b as a Therapeutic Target for AML ................................................................. 100 3.2 Future Directions ................................................................................................ 101 3.2.1 Combinatorial Treatment of IFN and KDM6B inhibition ........................................... 101 3.2.2 Fos/Jun AP-1 Transcription Factor as a Possible Regulator of Kdm6b Demethylase- Independent Gene Expression ......................................................................................... 101 3.2.3 Does Genetic Inhibition of AP-1 Transcription Factor Genes Rescue Kdm6b Phenotype? ...................................................................................................................... 103 3.3 Figures ............................................................................................................... 105 3.4 Tables ................................................................................................................ 106 References .................................................................................................................. 107 iv List of Figures Figure 2.1: Loss of Kdm6b results in depletion of phenotypic hematopoietic stem cells45 Supplementary Figure 2.1.1: Loss of Kdm6b does not generate hematopoietic malignancies ................................................................................................................. 47 Supplementary Figure 2.1.2: Progenitor analysis in young and aged Kdm6b-deficient mice ............................................................................................................................... 49 Figure 2.2: Loss of Kdm6b results in depletion of functional long-term repopulating HSCs ............................................................................................................................. 51 Supplementary Figure 2.2.1: Progenitor analysis in young and aged Kdm6b-deficient mice ............................................................................................................................... 53 Supplementary Figure 2.2.2: WBM from aged mice shows depletion of functional repopulating HSCs ........................................................................................................ 54 Figure 2.3: Kdm6b is required for HSC self-renewal ..................................................... 56 Figure 2.4: Kdm6b is required for self-renewal of leukemia-initiating cells .................... 58 Supplementary Figure