CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1 a Thesis S

CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1 A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology By Loni M Hands August 2016 Copyright by Loni Hands 2016 ii The thesis of Loni Hands is approved by: _____________________________________________ _______________ Kerry K. Cooper, Ph.D. Date _____________________________________________ _______________ Rheem D. Medh, Ph.D. Date _____________________________________________ _______________ Cheryl L. Van Buskirk, Ph.D. Date _____________________________________________ _______________ Cindy S. Malone, Ph.D., Chair Date California State University, Northridge iii ACKNOWLEDGMENTS I would like to thank my P.I., Dr. Cindy Malone, for her constant, strong support throughout my time as a masters student. I would like to thank my committee for their help and challenging questions during my proposal and defense. I would like to thank Dr. Daniel Odom for always being available to talk science, reason through protocols, and help me with my lab techniques. Finally, I would like to thank my family for always supporting me no matter what. iv TABLE OF CONTENTS Signature Page iii Acknowledgments iv Abstract vii Chapter 1: Introduction 1 1.1 Importance of gene regulation 1 1.2 Consequences of gene dysregulation 1 1.3 Gene regulation – Overview 2 1.4 Gene regulation – Core promoters and transcription initiation 3 1.5 Gene regulation – Proximal promoter elements (PPEs) 5 1.6 Gene regulation – Enhancers and silencers 6 1.7 Gene expression in mantle cell lymphoma vs. small lymphocytic 8 lymphoma 1.8 Nucleosome assembly protein 1-like 1 (NAP1L1) 9 1.9 Characterizing the NAP1L1 promoter region and enhancer element 10 Chapter 2: Methods 12 2.1 Cloning the NAP1L1 promoter region into pGL3 Basic 12 2.2 Cloning a larger fragment of the NAP1L1 promoter region into pGL3 14 Basic 2.3 Cloning a region of NAP1L1’s intron 1 into pGL3N-1273 and pGL3 16 Basic 2.4 Boiling lysis plasmid purification 17 2.5 Restriction digests 18 2.6 Transcription factor consensus sequence identification 18 2.7 Designing initial deletion constructs of the NAP1L1 promoter region 19 2.8 Designing smaller deletion constructs of the NAP1L1 promoter region 21 2.9 Mutating the myc binding site within the first intron of NAP1L1 24 2.10 Transfection of promoter constructs into human embryonic kidney 27 (HEK293T) cells 2.11 Harvesting transiently transfected HEK293T cells 28 2.12 Dual-Luciferase Assay of transfected pGL3 constructs 29 v Chapter 3: Results 30 3.1 Bioinformatics analyses of the NAP1L1 promoter region 30 3.2 Cloning the NAP1L1 promoter region into pGL3 Basic 32 3.3 Removing the intron from pGL3 Basic containing the NAP1L1 promoter 34 region 3.4 Cloning the intron into the enhancer region of pGL3 Basic and 35 pGL3N-1273 3.5 Transient transfections of NAP1L1 promoter deletion constructs 36 revealed regions between -442 bp and -297 bp that may contain a positive regulatory element and -297 bp and -183 bp that may contain a negative regulatory element 3.6 Preliminary transient transfection results from the smaller NAP1L1 39 promoter deletions between -442 bp and -297 bp as well as between -297 bp and -183 bp relative to the TSS 3.7 Transient transfections of the NAP1L1 promoter and intron regions 41 3.8 Transient transfections of NAP1L1 promoter and intron regions with the 42 mutated c-Myc consensus sequence Chapter 4: Discussion 45 4.1 Transient transfections of initial deletion constructs revealed a 45 regulatory element located within the region between -442 bp and -297 bp relative to the transcription start site (+1) and smaller deletions of this region suggest that the element lies between -367 bp and -335 bp relative to the TSS 4.2 Transient transfections of deletion constructs revealed a regulatory 46 element located within the region between -297 bp and -183 bp relative to the transcription start site (+1) and smaller deletions of this region suggest that there is an element that lies between -297 bp and -254 and a second element that lies between -254 bp and -215 bp relative to the TSS 4.3 Transient transfections of initial deletion constructs revealed a potential 48 element between -183 bp and -56 bp relative to the TSS 4.4 Transfections of intron constructs revealed a functional c-Myc binding 48 site in the first intron of NAP1L1 4.5 Future directions 53 References 55 vi ABSTRACT Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1 By Loni Hands Master of Science in Biology The precise regulation of gene expression within a cell determines the structure and function of a given cell type. Dysregulation of normal gene expression may lead to diseases such as cancer. Differences in gene expression among cancers of similar origin may explain the differing phenotypes between more aggressive cancers and less aggressive cancers. Mantle cell lymphoma (MCL), an aggressive cancer, expresses a higher level of the nucleosome assembly protein 1-like 1 (NAP1L1) than small lymphocytic lymphoma (SLL), a less aggressive cancer of similar origin. NAP1L1 is involved in cell cycle and gene regulation, making it a candidate for involvement in cancer progression. In order to understand the mechanisms behind NAP1L1 expression, the putative regulatory region of NAP1L1 from -1273 bp to +387 bp relative to the transcription start site (TSS, +1) was cloned into the luciferase reporter vector pGL3 Basic. After transient vii transfections of this construct into human embryonic kidney (HEK293T) cells confirmed the ability of this region to regulate gene expression, deletions of the promoter region were made and revealed the location of a positive regulatory element between -442 bp and -297 bp and a negative regulatory element between -297 bp and -183 bp. Preliminary data from transfections of smaller deletion constructs within each of these regions narrowed down the location of the positive regulatory element to between -367 bp and - 335 bp and potentially two negative regulatory elements between -297 bp and -254 bp and between -254 bp and -215 bp. Further research will be necessary to determine the exact identities of these elements. In order to determine whether there is an enhancer element within the first intron of NAP1L1, as there appears to be in mice (Wu et al., 2008), a region from +382 bp to +1915 bp containing the beginning of the first intron was cloned into the pGL3 construct containing the full isolated promoter region of NAP1L1 (-1273 bp to +387 bp). Transient transfections of this construct with the intron in the “forward” direction resulted in increased luciferase activity over 2-fold compared to the construct containing only the full NAP1L1 promoter region. Interestingly, and unexpectedly, the construct containing the full promoter region of NAP1L1 and the intron region in the reverse direction resulted in luciferase activity that was comparable to the construct with the full promoter and no intron. When the transcription factor binding sequence for c-Myc within the intron region was mutated in the construct containing the full promoter region and intron in the forward direction, the results were also similar to those of the construct containing only the full promoter, suggesting a potential role for c-Myc in regulating NAP1L1 in a location- independent, orientation-dependent manner. viii CHAPTER 1: INTRODUCTION 1.1 Importance of gene regulation Proper gene regulation is essential to the normal functions of the cell. Coordinating the development of an organism, ensuring that genes are up regulated or down regulated at the right time during each stage is the responsibility of gene regulatory processes. Essentially, gene regulatory programs maintain the normal function of a given cell type. Characteristics like cell shape and cell type-specific functions such as immunoglobulin production and secretion by B lymphocytes are determined by the regulated gene expression of that cell type. For example, in the case of immune cells, differences in cell surface receptors and markers between cell types and cell signaling pathways that lead to regulatory responses are important to an appropriate immune system response to an infection. When this regulation is disrupted or prevented in any way, diseases such as cancer or abnormalities in development can result. 1.2 Consequences of gene dysregulation Dysregulation of certain genes or classes of genes can lead to diseases or problems in development. Cancer, as an example, is a disease that results from the dysregulation of apoptotic and cell cycle genes such as proto-oncogenes. Proto- oncogenes are drivers of the cell cycle; they are directly involved in pushing the cell cycle forward (Vermeulen et al., 2003). When mutated or dysregulated, these oncogenes will cause a cell to continue inappropriately dividing, eventually resulting in the formation of a tumor (Vermeulen et al., 2003). One such example is the oncogene MYC, 1 which encodes the transcription factor c-Myc, which regulates genes involved in moving the cell cycle forward and whose overexpression has been correlated with multiple cancers (Tu et al., 2015; Wu et al., 2008). This transcription factor binds an enhancer box along with the regulator Max to increase expression of cell cycle genes, such as cyclin D1, and drive proliferation (Tu et al., 2015). Uncontrolled cell divisions will result in an accumulation of mutations that will allow for tumor formation and even for the cancer to become metastatic. 1.3 Gene regulation – Overview In order to understand why the dysregulation of certain genes leads to diseases such as tumor formation, the normal genetic regulation within various cell types must be studied. The genetic composition within a cell is generally the same between cell types of an organism, but it is the differences in genetic regulation that distinguishes one cell type from another.

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