UNIVERSITY of CALIFORNIA, SAN DIEGO The

UNIVERSITY OF CALIFORNIA, SAN DIEGO The Characterization of Enhancer Elements Involved in the Spatial Patterning of the Skin A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Andrew Grainger Committee in charge: Professor Benjamin Yu, Chair Professor James Kadonaga, Co-chair Professor Ella Tour 2014 The Thesis of Andrew Grainger is approved and it is accepted in quality and form for publication on microfilm and electronically: _______________________________________________________________________ _______________________________________________________________________ Co-Chair _______________________________________________________________________ Chair University of California, San Diego 2014 iii Dedication To my parents Jim & Claire, for always providing the support and encouragement needed for my success. iv Table of Contents Signature Page ……………...............................................………………………………………..…………..iii Dedication ……………………………………………………………………………....………………...…..…...iv Table of Contents ………………………………………………………………....……………...….…………..v List of Figures ……………………………………………………………………………....………...…………..vi Acknowledgments ……………………………………………………………………………….……..……...vii Abstract of the Thesis .…………………………………………………………....……………….………...viii I. Introduction …………………………………….…………………………………………………….…………1 II. Results ………………………………….………………..…………………………………………..………...13 III. Discussion ………………………………………………...………………………………………………....37 IV. Materials and Methods ……………………………………...………………………………………….43 References …………………………………………………………………......………………………………....56 v List of Figures and Tables Figure 1. Evolutionary conservation of the Keratin Type II Cluster exons between humans, rhesus macaque, mouse, opossum, chicken, and fugu .....................21 Figure 2: p300 ChIP-Seq data from wild type mouse epidermis, E17.5 (n=1). (A) Initial and filtered read counts, total peaks, and filtered peaks. (B) Total and filtered peak counts at the three keratin cluster loci. .............................................................................22 Figure 3: LICR Histone modification data (h3k4me1, h3k27ac) for multiple tissues at the proposed negative site (qPCR probe in green at top) .................................23 Figure 4: Representative graphs of mice ChIP-qPCR at the Keratin Type II locus exons at E17.5 for h3k4me1 (B) and h3k27ac (C), and at E14.5 for h3k4me1 (D) and h3k27ac (E) .............................................................................................................................................24 Figure 5: Comparison of biological replicate data between e14.5 and e17.5 (n=3 for both) for h3k4me1 (A) and h3k27ac (B). ............................................................................30 Figure 6: CNV loss in psoriasis patients at the gene CDSN ..........................................32 Figure 7: Representative graphs of mice ChIP-qPCR for CDSN at E17.5 for h3k4me1 (A) and h3k27ac (B), and at E14.5 for h3k4me1 (C) and h3k27ac (D) ......33 vi Acknowledgements There are several people I would like to acknowledge for contributions to my research success. First and foremost, I would like to acknowledge my PI, Benjamin Yu, for guiding and mentoring me for the last three and a half years and helping me become a better scientist. Secondly, I’d like to acknowledge Shantanu Kumar, who has been invaluable as a person to troubleshoot with and get advice on experiments and protocols. I owe much of my success to his help. I’d also like to acknowledge Christopher Adase, who has helped me come up with a solid ChIP protocol when neither of us knew much of what we were doing in the beginning. Lastly, I’d like to acknowledge the rest of the Yu lab for making the lab an enjoyable and nurturing environment. vii ABSTRACT OF THE THESIS The Characterization of Enhancer Elements Involved in the Spatial Patterning of the Skin by Andrew Grainger Master of Science in Biology University of California, San Diego, 2014 Professor Benjamin Yu, Chair Professor James Kadonaga, Co-Chair The epidermis is an essential tissue in a large variety of organisms, and differential regulation of keratin genes plays a vital role in maintaining its structural integrity and other various cellular processes. There are a number of diseases associated with changes in keratin gene expression, and so understanding how these keratins are regulated might help in our understanding of their functions in the epidermis. Recent studies have elucidated the large quantities of regulatory elements present in the human genome, many in places we would not have thought for them to exist. Exons of coding genes can contain regulatory elements within them, and might be having regulatory effects on proximal genes. Therefore, the viii keratin genes, which are collected into two distinctive clusters, could potentially contain regulatory elements in their exons. We investigated this possibility using ChIP-seq and ChIP-qPCR, and successfully identified 37 potential enhancer elements residing within the exons of keratin genes in the Keratin Cluster II (KCII). We also identified two novel enhancer elements in CDSN, another gene vital for epidermal structural integrity, and discovered that a loss of copy number of these enhancers could potentially contribute to the skin disease psoriasis. ix I. Introduction 1 2 The human epidermis is a stratified squamous epithelium, composed of proliferating basal and differentiated suprabasal keratinocytes, or the predominant type of cells that act as the body's major barrier against an inhospitable environment (McGrath et al., 2004). The epidermis is differentiated into distinctive layers, the most well studied of which are the basal and suprabasal, which contains the spinous, granular, and stratum corneum; the basal layer is the bottom or closest to the inside of the body and the stratum corneum is the layer exposed to the air. A group of genes identified as the keratin genes fundamentally influence the architecture and mitotic activity of epithelial cells, and are differentially expressed in different layers of the epidermis (Shetty et al., 2012). For example, Keratin 14 (K14) and Keratin 5 (K5) are expressed in the basal layer, while K1, K6, and K10 are expressed in the spinous layer. The original establishment of the different layers was mostly due to the mapping of changes in keratin gene expressions, and many of these keratin genes are used for immunostaining to observe one specific differentiated layer. Keratins are defined as intermediate filament forming proteins with specific physiochemical properties produced in any vertebrate epithelia (Bragulla et al., 2009). Intermediate filaments provide the general scaffold for most cytoskeletons, and in the case of the epidermis provide the structural integrity that maintains intercellular structure. Each keratin gene varies in length and properties, but each works as a means of keeping neighboring cells in a strict orientation. But, outside of this, keratins also perform a multitude of other functions. Aside from providing a 3 scaffold for these epithelial cells to keep a cohesive structure, they also provide a means for the epidermis to sustain various forms of mechanical stress and variations in hydrostatic pressure (Shetty et al., 2012). However, they also play a role in cellular functions as well, involving themselves in cell signaling, transport, compartmentalization, and differentiation (Vaidya and Kanojia, 2007), on top of influencing metabolic processes and cell growth (Coulombe and Wong, 2004; Gu and Coulombe, 2007). The fact that the various keratin genes are differentially expressed in the different layers of the human epidermis implies that these various keratin genes are carrying out varied, albeit not very well known, functions in each of these layers to help enforce differentiation. While the direct effects of keratin genes on differentiation remain unclear, there has been an exponential increase in the number of epidermal stem cell publications over the last 25 years, and our knowledge of how the epidermis proliferates and regulates itself is expanding (Ghadially, 2012). This started with the discovery of hematopoietic stem cells in 1961 and has rapidly expanded in the last decade or two to include the characterization of a multitude of signaling pathways involved in epidermal stem cell maintenance and cell fate determination. One such example is the Notch pathway. It is important in the determination of stem cell self-renewal versus differentiation. Expression of the Jagged 1 and 2 ligands as well as the Notch 1 and 2 receptors increases in differentiating keratinocytes of the suprabasal layers, and this is thought to be important for synchronization of differentiation, or the timing in which these layers differentiate, as well as epidermal 4 border formation (Luo et al., 1997; Rangarajan et al., 2001). But while the pathways involved have been identified, the specific changes in them that give rise to the differentiated layers have yet to be seen. The mechanisms that drive differentiation of the basal layer into the spinous, granular, and stratum corneum are not well known, and those responsible for the observed differential expression of the various keratin genes have yet to be discovered. However, this differential expression of keratin genes is not limited solely to the epidermis. There is differential expression of keratin genes depending on the specific keratinized tissue, as teeth and epidermis show distinctively unique

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