Mechanism of gene regulation of HNF4α Item Type Dissertation Authors Guo, Shangdong Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 05/10/2021 14:43:09 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/2053 Mechanism of gene regulation of HNF4α Shangdong Guo A Dissertation in Department of Pharmacology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of State University of New York, Upstate Medical University. Approved__________ Date__________ Acknowledgments First and foremost, I hope to give my greatest acknowledgment to my thesis advisor, Dr. Hong Lu, who is always supportive throughout my Ph.D. career over the years. The path to the science is long and twisted, and it is particularly tough for beginners. I was totally a newbie at the time when I joined the lab, and I would never be able to accomplish all my work without the patient guidance from Dr. Lu. My special thank goes to Lucy Lei, who is more than a colleague. Her patient instructions and excellent technical skills provided me the best training I can earn. I also want to show my appreciation to our past lab members including Lesley Cashon and Qinghao Zhang, for their kind assistance and the great friendship to my Ph.D. life. I would like to thank Dr. Debashis Ghosh and Dr. Guirong Wang for being members of my advisory committee over the years. Their precious advice and suggestions guided me in my adventure of science. I also want to thank all professors who joined my qualify committee and thesis committee. The pharmacology department is a great union that is encouraging and supportive. I would like to express my appreciation to all faculties and staffs in our department for their contribution to creating a fantastic working environment for every student. Last but not least, I hope to thank my family and all my friends, for your endless support and encouragement. Thank you! Shangdong Guo May 2018 i Abstract Mechanism of gene regulation of HNF4A Shangdong Guo Hong Lu Hepatocyte Nuclear Factor 4 alpha (HNF4α) is a master regulator that modulates the liver development and function. The dysfunction of HNF4α causes multiple human diseases, such as hepatocellular carcinoma (HCC) and maturity onset diabetes of the young 1 (MODY1). In contrast, the restoration of HNF4α can inhibit the development of liver cancer and improve the liver function simultaneously. However, HNF4α is an orphan nuclear receptor whose activating ligand remains elusive. Therefore, an alternative approach to enhance the HNF4α activity is to up-regulate the protein expression. While a great progress has been made on the functional study of HNF4α, the mechanistic details regarding the gene regulation of HNF4α are still a vast knowledge gap. The present study was aimed to investigate the mechanism of gene regulation of HNF4α systematically. In chapter 2 and chapter 3, we reported the strong translational inhibition of both human and mouse HNF4α induced by the nucleic acid secondary structure termed “G-quadruplex”(G4) within the 5` untranslated region (UTR). By performing the deletion/mutation studies, we determined the composition of the G4 in HNF4A 5`UTR. We further speculated that this G4 required the adjacent cis-elements, such as the RNA-binding proteins and other secondary structures, to form a conjunction for the strong translational inhibition. We for the first time reported the RNA-G4 induced translational repression within the 5`UTR of a tumor suppressor and highlighted the significance of the “biostability” of G4s in exerting their biological functions. In chapter 4, we conducted a comprehensive study that covered the auto-regulation, transcriptional regulation and transactivation activity of HNF4α. By creating various reporter constructs, we were able to validate the self-stimulation of HNF4α and discovered the strong correlation between HNF4α and its corresponding anti-sense RNA, HNF4A-AS1. Additionally, we identified novel HNF4α mutations such as Q277X that may affect the crosstalk of HNF4α with other transcription factors. Overall, the novel findings from our study shed light on the gene regulation of HNF4α and provide further insights into our ultimate goal that is to up-regulate HNF4α protein expression/activity to treat human diseases. ii Abbreviations AldoB Aldolase B EPO The glycoprotein hormone erythropoietin AF-1 Activation Function domain 1 FACS fluorescence activated cell sorting AF-2 Activation Function domain 2 FMRP Fragile X Mental Retardation Protein AFP Alpha-fetoprotein FXR Farnesoid X Receptor BA Bile Acid G4 G-quadruplex CAR constitutive androstane receptor Gapdh Glyceraldehyde-3-phosphate CD Circular Dischroism dehydrogenase C/EBP CCAAT-enhancer-binding proteins GLUT2 glucose transporter 2 HBV Hepatitis B virus. CNE conserved non-coding element HCC Hepatocellular Carcinoma CRC Colorectal Cancer HNF1α Hepatocyte Nuclear Factor 1 alpha CRISPR-Cas9 Clustered regularly interspaced HNF4α Hepatocyte Nuclear Factor 4 alpha short palindromic repeats, CRISPR-associated hnRNP Heterogeneous Nuclear protein 9 Ribonucleoproteins COUP-TFII COUP-transcription factor 2 LA Linoleic Acid DBD DNA Binding Domains LBD Ligand binding domain DPE Downstream Promoter Element LETF Liver-enriched Transcription Factor DMS dimethyl sulfate LFABP L-type fatty acid-binding protein eIF2 Eukaryotic Initiation Factor 2 MCAD Medium-chain acyl-CoA dehydrogenase EMT Epithelial-Mesenchymal Transition MODY Maturity Onset Diabetes of the Young iii MT3-MMP Matrix Metallopeptidase 16 PDS Pyridostatin NCOR1 nuclear receptor corepressor 1 PXR pregnane X receptor NGS Next Generation Sequencing RBP RNA Binding Proteins NMM N-methy mesoporphyrin IX RHA RNA Helicase A NMR Nuclear Magnetic Resonance RHAU RNA Helicase associated with AU-rich NR Nuclear Receptor element NRAS Neuroblastoma RAS viral oncogene SAR Structure-activity relationship homolog SCARB1 Scavenger Receptor class B type 1 NRD Negatively regulatory domain SNP Single Nucleotide Polymorphisms NTCP Na+-taurocholate cotransporting SPR Surface Plasmon Resonance polypeptide SRSF serine/arginine-rich splicing factor OTC Ornithine transcarbamylase TACE Transarterial Chemoembolization PCR Polymerase Chain Reaction TMPyP4 p21/WAF1 cyclin-dependent kinase inhibitor 1 5,10,15,20-Tetrakis-(N-methyl-4-pyridyl) p300 Histone acetyltransferase porphine PDZK1 PDZ domain containing 1 TRF2 telomeric repeat binding factor 2 PEPCK Phosphoenolpyruvate carboxykinase; TSS Transcriptional start site PMSF Phenylmethylsulfonyl Fluoride TTR Transthyretin PPIX Protoporphyrin IX UTR Untranslated Region P/S Penicillin-Streptoomycin WT Wild Type iv Table of contents Acknowledgements……………………………………..……………………………………………………i Abstract……………………………………..…………………………………………………………….....ii Abbreviations…………………………..………………………………………………………….......……iii List of Figures………………………...………………………………………………………….…………ix List of Tables…………………………………………………...…………………………………………...xi Chapter 1. General Introduction 1.1. The research history of HNF4α……………………………………….………………………………2 1.2. The gene structure and functional domains of HNF4α………………………………..……...………3 1.3. The function and the clinical relevance of HNF4α 1.3.1. Regulation of Hepatocytes differentiation and proliferation…………………...……….......4 1.3.2. The role of HNF4α in HCC………………………………………………………………………….6 1.3.3. The role of HNF4α in MODY1…………………………………………………………….………..7 1.4. The functional difference between P1- and P2-HNF4α 1.4.1. The dynamic switch of HNF4α isoforms during development..…………...……………...…8 1.4.2. The role of P2-HNF4α in carcinogenesis…………………………………………………........…9 1.5. The interaction and regulation of HNF4α 1.5.1. The endogenous ligand of HNF4α……………………………………………………………...…10 1.5.2. The auto-regulatory loops of HNF4α and other Liver-enriched TFs (LETFs)…………….. 11 1.5.3. The regulatory networks of LETFs.……………………………………………………..…11 1.5.4. The interaction of HNF4α with farnesoid X receptor (FXR)………………………………..…13 1.6. An overview of the G-quadruplex (G4) 1.6.1. The topological features of G4………………………………………………………........………15 1.6.2. Current approaches to determine the presence of G4 in vitro and in vivo……………..……16 1.7. The biological functions of RNA G4s 1.7.1. The role of G4s in mRNA transcription and splicing……………………………………...……18 1.7.2. The role of G4s in mRNA translation………………………………………………………..……18 1.8. The interaction and regulation of RNA G4s 1.8.1. Interaction of G4 with small molecules……………………………………………………..……20 1.8.2. Interaction of RNA G4 with RNA helicases and RBPs…………………………………….……21 v 1.9. Summary and research objectives…………………………….……………………………………..22 Chapter 2. Conjunction of potential G-quadruplex and adjacent cis-elements in the 5' UTR of hepatocyte nuclear factor 4-alpha strongly inhibit protein expression 2.1 Abstract…………………………………………………………………………………………………….………31 2.2 Introduction………………………………………………………………………………….……………..………32 2.3 Results 2.3.1 P1-HNF4A 5` UTRs markedly repress the reporter activity of luciferase and the protein expression of HNF4α1……………………………………………………………………………..36 2.3.2 Structure-activity relationship (SAR) studies of P1-HNF4A 5`UTR reveal the importance of the cooperation of the G4 with the potential protein-binding sites…………………………………...37 2.3.3 Protoporphyrin-IX (PPIX)-binding assay and circular dichroism (CD) spectrum further confirm the presence of the G4 in the 5`UTR RNA and deletion/mutation constructs………..…….40 2.3.4 Two regulatory single-nucleotide polymorphisms (rSNPs)