Identification of Genomic Targets of Krüppel-Like Factor 9 in Mouse Hippocampal
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Identification of Genomic Targets of Krüppel-like Factor 9 in Mouse Hippocampal Neurons: Evidence for a role in modulating peripheral circadian clocks by Joseph R. Knoedler A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in the University of Michigan 2016 Doctoral Committee: Professor Robert J. Denver, Chair Professor Daniel Goldman Professor Diane Robins Professor Audrey Seasholtz Associate Professor Bing Ye ©Joseph R. Knoedler All Rights Reserved 2016 To my parents, who never once questioned my decision to become the other kind of doctor, And to Lucy, who has pushed me to be a better person from day one. ii Acknowledgements I have a huge number of people to thank for having made it to this point, so in no particular order: -I would like to thank my adviser, Dr. Robert J. Denver, for his guidance, encouragement, and patience over the last seven years; his mentorship has been indispensable for my growth as a scientist -I would also like to thank my committee members, Drs. Audrey Seasholtz, Dan Goldman, Diane Robins and Bing Ye, for their constructive feedback and their willingness to meet in a frequently cold, windowless room across campus from where they work -I am hugely indebted to Pia Bagamasbad and Yasuhiro Kyono for teaching me almost everything I know about molecular biology and bioinformatics, and to Arasakumar Subramani for his tireless work during the home stretch to my dissertation -I am grateful for the Neuroscience Program leadership and staff, in particular Valerie Smith and Rachel Flaten, for making sure I get paid on time and registered for classes, and Ed Stuenkel for his dedication to keeping everyone on track in their career -Everyone I’ve overlapped with in the Denver Lab (Melissa, Rose, Lilly, Daniel, Preeta, Julia, Christina, Samhitha, Ariel and Cesar stand out) for being unfailingly supportive and pleasant coworkers -I will be forever appreciative of Sue Moenter for funding me on the CTRB fellowship -Eric Horstick and Jeremey Linsley taught me what kind of attitude was conducive to being a good graduate student, although I’m positive they didn’t realize it at the time -My occasional piano teachers Gaye Thomas and Jovanni V. Rey del Pedro for helping keep my hobby alive -My family (Mom, Dad, Andrew, Tim and Molly) for their love and support -My girlfriend Lucy for never stopping believing in me -The Lizardman crew, for keeping the flame of college camaraderie alive -And, last but absolutely not least, my friends in Ann Arbor (those still here and those far away), most particularly Kelly Schwartz, Jessica Adams, Dante Passmore, Julia Kline, Becky Canary- King, and Ben Kazez; the last seven years wouldn’t have been the same without any of you. iii TABLE OF CONTENTS DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT ix CHAPTER 1. INTRODUCTION 1 1. Introduction 1 2. Krüppel-like factors 1 2.1 Krüppel-like factors divide into subfamilies based on shared N-terminal 2 domains 3. Krüppel-like factor 9 is an effector of nuclear receptor action 3 3.1 Structure and biochemical function of Klf9 3 3.2 Klf9 mediates the effects of thyroid hormone on neural development 4 3.2.1 Klf9 is a thyroid-hormone regulated gene in amphibians and 4 mammals 3.2.2 Klf9 mediates the effects of thyroid hormone on neuronal 5 morphology across development 3.2.3 Klf9 promotes myelination in the mammalian central nervous 6 system 3.2.4 Klf9 enhances Trb autoinducion in the frog Xenopus laevis 6 3.3 Role of Klf9 in uterine physiology 7 3.3.1 Klf9 is an accessory transcription factor for the progesterone 9 receptor that promotes progesterone responsiveness in the uterus 3.3.2 A role for Klf9 in estrogen receptor autorepression 9 3.3.3 Klf9 participates in transcriptional networks with Klf13 during 10 pregnancy 3.4 Summary of the role of Klf9 in nuclear receptor signaling 11 4. Evidence for a role for Klf9 in circadian regulation of gene expression 12 4.1 A transcription-translation feedback loop marks time in mammalian cells 12 4.2 Modulation of peripheral clocks by glucocorticoids 14 iv 4.2.1 Regulation of adrenal steroid secretion and production by the 14 clock 4.2.2 Influence of glucocorticoids on the cellular circadian oscillator 15 4.3 Known roles of Klfs in circadian physiology 16 4.3.1 Klf15 is a clock-output gene that orchestrates circadian variation 16 in metabolism and physiology in the heart and liver 4.3.2 Klf9 mediates circadian variations in cell proliferation in human 17 keratinocytes 4.3.3 Klf10 is a clock-output gene that contributes to circadian 18 regulation of lipid homeostasis 5. Summary of the dissertation 18 References 21 2. KRÜPPEL-LIKE FACTOR 9 FUNCTIONS PREDOMINANTLY AS A TRANSCRIPTIONAL REPRESSOR IN MOUSE HIPPOCAMPAL NEURONS 30 Abstract 30 Introduction 32 Materials and Methods 34 Results 43 Discussion 55 References 63 3. KRÜPPEL-LIKE FACTOR 9 IS A NOVEL NEGATIVE REGULATOR OF THE CLOCK OUTPUT THAT PLAYS A ROLE IN MODULATION OF PERIPHERAL CLOCKS BY ADRENAL STEROIDS 96 Abstract 96 Introduction 97 Materials and Methods 100 Results 106 Discussion 115 References 120 4. CONCLUSIONS AND FUTURE DIRECTIONS 145 Klf9 acts as a repressor of gene transcription in hippocampal neurons 145 Evidence for a role for Klf9 in the cellular circadian oscillator 147 Klf9 is a novel modulator of the clock-output 148 Predicting the consequences of Klf9 disruption on the core circadian clock 151 A Klf network for fine-tuning gene expression 153 Concluding remarks 154 References 154 APPENDIX 157 v LIST OF TABLES Table 2.1 Top 10 up- and down-regulated genes in HT22[TR/TO-Klf9] cells +/- 8 hours of dox treatment 88 Table 2.2 Motifs enriched in Klf9 peaks 89 Table 2.3 All GO:PANTHER pathways enriched in set of genes repressed by Klf9 91 Table 2.4 Top ten GO:PANTHER pathways enriched in genes with Klf9 associated 92 in their genomic region Table 2.5 Top GO: Panther pathways associated with genes associated with Klf9 peaks of different clusters 93 Table 3.1 E-box motifs discovered in Klf9 peaks by HOMER 144 Supplemental Table 2.1 Oligonucleotide sequences 157 Supplemental Table 2.2 All Klf9-regulated genes 159 Supplemental Table 2.3 Klf9 ChSP peaks 170 Supplemental Table 2.4 Additional GC-rich motifs identified in Klf9 peaks 249 Supplemental Table 2.5 All GO:PANTHER pathways enriched in genes with Klf9 251 peaks associated vi LIST OF FIGURES Figure 2.1 Stably transfected HT22 cell lines expression functional Klf9 protein that associates in chromatin 67 Figure 2.2 Klf9 functions predominantly as a transcriptional repressor 69 Figure 2.3 Peak calling and classification paradigm 71 Figure 2.4 FLBIO-Klf9 associates near transcription sites and is more likely to be associated with repressed than upregulated genes 71 Figure 2.5 Klf9 associates with peaks identified by ChSP-seq in vitro and in vivo 73 Figure 2.6 Endogenous Klf9 protein associates with peaks in parent HT22 cells 75 Figure 2.7 ChSP and ChIP at negative control regions 76 Figure 2.8 Recruitment of Sin3a to Klf9 peaks 77 Figure 2.9 GC-rich motifs correlate with sequencing tag density within peaks and with overall peak height 78 Figure 2.10 Peak clustering parameters 79 Figure 2.11 Klf9 peaks group into shape clusters that show different likelihood of association with repressed genes 80 Figure 2.12 Forced expression of Klf9 represses transcription from sequences corresponding to Klf9 peaks in a GC box-dependent manner 82 Figure 2.13 Klf13 5’ upstream region cloned for functional analysis 84 Figure 2.14 Klf9 target genes are dysregulated in Klf9 HT22 knockout cells and in Klf9 knockout mice 85 Figure 2.15 Dysregulation of Klf9-regulated genes (with no associated peaks) in Klf9-null mouse hippocampus. 87 Figure 3.1 Klf9 associates in chromatin in close proximity to E-box motifs at several clock-output genes 124 Figure 3.2 Klf9 associates with clock-output genes in mouse hippocampus 126 vii Figure 3.3 Klf9 is a Clock/Bmal1-regulated gene whose mRNA level oscillates in CORT-synchronized HT22 cells 127 Figure 3.4 Klf9 mRNA is circadian in liver and is phase-advanced compared with Klf9 129 target genes Figure 3.5 Klf9 shows circadian recruitment to CLOCK-output genes in mouse liver 131 Figure 3.6 Klf9 represses Dbp mRNA level and shows circadian recruitment to an evolutionarily conserved E-box motif that is required for circadian Dbp expression 133 Figure 3.7 Klf9 represses transcription of the Dbp promoter and antagonizes CLOCK/Bmal1 transactivation of Dbp in HEK293 cells 135 Figure 3.8 Forced expression of Klf9 represses transcription for Dbp, Tef, and Wee1 promoters. 137 Figure 3.9 Klf9 does not inhibit CLOCK/Bmal1 transactivation of Tef and Wee1 promoters 138 Figure 3.10 Klf9 interferes with CORT induction of Per1, Wee1 and Klf9 139 Figure 3.11 Inhibition of protein synthesis enhances upregulation of Per1, but not Per2, in response to CORT 141 Figure 3.12 Klf9-null mice show impaired Per1 induction in response to acute restraint 142 stress viii ABSTRACT Krüppel-like factor 9 (Klf9) is a transcription factor that has diverse roles in development and physiology. Earlier work showed that Klf9 functions in neuronal differentiation, but nothing was known of its target genes in neurons. I used the mouse hippocampus-derived cell line HT22 to identify Klf9 genomic targets. I engineered HT22 cells to express Klf9 under the control of the tet repressor, and used RNA sequencing to identify genes modulated by Klf9.