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The Transcription Factor PU. 1 Is Enriched at Inflammatory Bowel Disease Risk Loci in CD56+ Cells

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The Transcription Factor PU. 1 Is Enriched at Inflammatory Bowel Disease Risk Loci in CD56+ Cells The Transcription Factor PU.1 Is Enriched At Inflammatory Bowel Disease Risk Loci in CD56+ Cells A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of Master of Science In the division of Immunology of the College of Medicine By: Fazeela Yaqoob M.Phil. Government College University Lahore, Pakistan, 2011 August 2017 Committee Chair: Stephen Waggoner, Ph.D. Jonathan Katz, Ph.D. Leah Kottyan, Ph.D. Abstract Inflammatory bowel disease (IBD) affects the well-being of 1.6 million people in the United States. The etiology of IBD is strongly linked to genetic risk loci and to a dysregulated immune response against the intestinal microbiome. Genome-wide association studies identified more than 200 discrete genetic loci associated with risk for IBD, but mechanistic understanding of how these sites collectively promote disease is lacking. We hypothesize that altered binding of transcription factors (TFs) at IBD risk loci is a mechanism to globally promote gene expression changes associated with IBD. We used an innovative algorithm to assess intersection between known IBD genetic risk loci and transcription factor binding (ChIP-Seq) in a variety of cell types to reveal that PU.1 binding in human CD56+ cells (highest scoring dataset) overlaps variants at more than half of the IBD risk loci assessed (62 of 112, 3.7-fold enrichment, p<10-30). The majority (but not all) of human CD56+ cells are natural killer (NK) cells, which are implicated in mouse models of IBD pathogenesis and observed to accumulate in the inflamed intestines of IBD patients. In this thesis, we optimized conditions for PU.1 ChIP in the human NK cell line, KHYG1, and observed successful PU.1 enrichment of sites known to be bound by PU.1. These results position our lab to assess allele-dependent PU.1 binding to IBD risk loci in primary human NK cells, which we hypothesize is a mechanism contributing to genetic risk for IBD. ii Acknowledgements I first and foremost would like to thank my mentor Stephen Waggoner for his sincere efforts and thoughts in developing me a scientist. I always learned something new under his guidance and I will keep idealizing him as a scientist. I am also thankful to all members of the Waggoner lab, a group of talented researchers. I would say a big thanks to my co-mentor Stacey Cranert. I found myself lucky that I got a chance to meet her and work with her. Undoubtedly, I found her amazing in every scientific discipline and technique. I am highly thankful to David Ochayon who was around whenever I needed him. He trained me to think like a researcher and act accordingly. iii iv Table of Contents 1. Introduction 1 1.1. Inflammatory Bowel Disease: An autoimmune disorder with a genetic component 1 1.2. Translating genetics of disease risk to functional genomics 2 1.3. Regulatory Element Locus intersection (RELI) 3 1.4. ILCs regulation of intestinal homeostasis and inflammation 8 1.5. PU.1 regulates CD56+ cells differentiation and functioning 10 1.6. ChIP-Seq: A method for identification of gene regulatory elements 11 2. Materials and Methods 14 3. Results 3.1. PU.1 is expressed in Human Natural Killer cell lines KHYG1 and NK-92 20 3.2. KHYG1PU.1 ChIP-PCR reveals that PU.1is enriched at CD11b Promoter in NK cells 22 3.3. PU.1 binds to LIMD1 promoter 27 4. Discussion 31 5. Conclusion 37 6. References 39 Appendix 50 v List of Tables and Figures Figure 1 RELI (Regulatory Element Locus Intersection) working scheme: A new computational approach to discover gene regulatory mechanisms 5 Figure 2 Global view of RELI results – all diseases versus all TFs 6 Figure 3 Steps involved in Chromatin Immuno-precipitation (ChIP) 17 Figure 4 Western blots for endogenous expression of PU.1 in KHYG1 and NK-92 cell lines 21 Figure 5 Sonication Optimization in KHYG1 and NK-92 24 Figure 6 PU.1 is enriched in KHYG1 at CD11b promoter 25 Figure 7 PU.1 is enriched in KHYG1 at CD11b promoter immuno- precipitated with monoclonal antibody 26 Figure 8 PU.1 is enriched in KHYG1 at LIMD1 promoter through standard PCR 28 Figure 9 PU.1 is enriched in KHYG1 at LIMD1 promoter as evident by qPCR 29 Figure 10 TRC1, non-targeting shRNA control vector 38 Table1 PU.1 binds to IBD risk loci more often than expected by chance 7 Table2 List of positive (+) and negative (-) primer sets used to amplify IPs and input DNA 18 Table3 Percent Input Method to calculate PU.1 enrichment 19 Table4 Fold enrichment Method to calculate PU.1 enrichment 19 Table5 Calculation of PU.1 fold enrichment at LIMD1 and GAPDH promoter regions through ΔΔCt method 30 vi List of Abbreviations Name Description IBD Inflammatory Bowel Disease TF/TFs Transcription Factor/Transcription Factors NK Cells Natural Killer Cells ILCs Innate Lymphoid Cells DSS Dextran Sodium Sulphate ChIP Chromatin Immunoprecipitation U6 U6 Promoter cppt Central polypurine tract hPGK Human Phosphoglycerate tract puroR Puromycin resistance gene for mammalian selection SIN/3” LTR 3’ self-inactivating long terminal repeat f1 ori F1 origin of replication ampR Ampicillin resistance gene for bacterial selection pUCori pUC origin of replication 5’ LTR 5’ long terminal repeat Psi RNA packaging signals RRE Rev response element vii viii 1. Introduction: 1.1. Inflammatory Bowel Disease: An autoimmune disorder with a genetic component Inflammatory bowel disease (IBD) encompasses chronic inflammation of the gut as categorized into two anatomically similar but physiologically distinct debilitating conditions, Ulcerative Colitis (UC) and Crohn’s disease (CD)[1]–[4]. Although IBD is not fatal in nature, its relapsing and remitting nature, along with a range of symptoms, makes this disorder a significant burden on human health[5]–[8]. Regardless of many shared clinical features, UC and CD exhibit distinct histopathology, localization, and incidence [1], [9]. There is currently no known cure for IBD and treatment is limited to life-long symptomatic care through anti-inflammatory and immunosuppressive drugs [10]. The prevalence of IBD is highest in Europe and North America, and it is rapidly increasing in other parts of the world. Currently, 1.6 million Americans are suffering from IBD, while worldwide prevalence approaches 4 million individuals[11]. IBD is more common among Ashkenazi Jews, who are five to eight times more likely to develop IBD compared to non-Jews[12]. Moreover, populations earlier thought to be at “low risk”, such as Indians and Japanese, are experiencing an increased prevalence in IBD[2], [13]. The intestinal immune compartment is the largest in the body with a continuous exposure to antigens from both diet and the symbiotic bacteria [14]. It also serves as an opening to large numbers of pathogenic microbes. The integrity of the gut immune system is maintained by a balance between eradication of pathogenic microbes and a tolerance to symbiotic ones, and this is crucial to maintain homeostatic conditions [15]. In IBD, tolerance to intestinal and environmentally acquired antigens is disrupted due to an excessive and abnormal immune response in a genetically susceptible host. This results in a breakdown of the intestinal epithelial 1 barrier and gut malfunction [1], [16]. Due to the complex nature and multifactorial etiology ofIBD, it is thought that many factors are involved in disease course. Among these, three main factors are genetics, the environment (including microbes, diet, and drugs), and the host immune system[4], [17]. 1.2. Translating genetics of disease risk to functional genomics: The present theories for the etiology of IBD highlight the significance of genetics in the context of defective gut immune responses to microbes. Twin studies, although few in number, are important sources to determine the role of a genetic component in both UC and CD etiology[18].In one large European study, CD concordance rate in monozygotic twins was found to be high (20-50%) [19], [20]. While they shared the same environment, the concordance for CD was low (less than 10 %) among dizygotic twins[21]. The risk of IBD among first- degree relatives of patients with CD and UC is also 10 times higher than the control population[18].These data combined with many other genetic epidemiological studies provide strong rationale for the role of genetics in IBD development. Genome-wide association studies (GWAS) have provided new insight into IBD etiology by the identification of 215 IBD risk loci and 99 confirmed association studies [22]–[24]. A third of these risk loci are shared between UC and CD [1]. Over 90% IBD risk loci are located in non-coding regions of the genome and are enriched in gene regulatory elements[25]. Many of these regions also serve as expression quantitative risk loci (eQTLs) for nearby genes by being tightly linked to them [26]; however, GWAS do not convey much information about casual variants underlying these associations [24]. Therefore, robust molecular work is required to understand how IBD variants collectively promote disease in a genotype-dependent manner [27], [26]. 2 Single nucleotide polymorphisms (SNPs) are the most common form of variations in the human genome[28]. Trait-associated non-coding variants are enriched in regulatory elements and are categorized into rare, common, coding, non-coding, and functional variants. Although it is hard to identify the rare variants associated with common diseases, a possible approach for annotating non-coding variants is to ascertain which of them are linked with transcription factor binding. This concept is quite significant as TF binding is largely determined by local sequences, thereby decreasing the search areas for eQTLs. Rare functional variants with allele specific transcription factors binding could be responsible for genetic effects that make the host susceptible to certain diseases, including autoimmune diseases[29].
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