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2017 The Role of CREG1 as a Master Regulator of Liver Function Abdulrahman Siran Aldaghmi Eastern Illinois University This research is a product of the graduate program in Biological Sciences at Eastern Illinois University. Find out more about the program.

Recommended Citation Aldaghmi, Abdulrahman Siran, "The Role of CREG1 as a Master Regulator of Liver Function" (2017). Masters Theses. 2719. https://thekeep.eiu.edu/theses/2719

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Date

Please submit in duplicate. THE ROLE OF CREG1 AS MASTER REGULATOR

OF LIVER FUNCTION

(TITLE)

BY

ABDULRAHMAN SIRAN ALDAGHMI

THESIS

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

Master of Science

IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS

2017

YEAR

I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE

.7/7//11 �I..f/W/Y �r· THESIS CO�ITTEE CHAIR DATE DEPARTME�]SCHOOLCHAIR DATE OR CHAIR'S DESIGNEE �8'/1r

THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER DATE

SLrtf 'J-0\ I THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER DATE The Role of CREG 1 as a Master Regulator

of Liver Function

Abdulrahman Aldaghmi

Department of Biological Sciences

Eastern Illinois University

Charleston, IL-61920 © 201 7 by Abdulrahman Aldaghmi

ii COMMITTEE IN CHARGE OF CANDIDACY

Professor Dr. Gary A. Bulla, Advisor

Professor Dr. Britto P. Nathan

Instructor Dr. Antony 0. Oluoch

iii ABSTRACT

The liver is known as the chemical factory of the body because it performs a wide range of biochemical functions required for life. Since the liver has such an important role in regulation of normal physiological processes, liver diseases cause a high rate of morbid ity and mortality. Therefore, understanding the mechanisms of liver development will shed light on the causes of liver disease. In this study, a cell line model that utilizes rat hepatoma cells (Fg14) and hepatoma variant cells (Hll) was used to identify master regul ators of liver expression. Whole genome expression studies identified the gene CREGl (Cellular repressor of ElA-stimulated -1) as a potential master regulator of liver .

The ability of CREGl to restore liver gene expression in the Hll variant cell line was determined by forced overexpression of this gene via transfection of Hll cells with a CREGl expression vector. Rescue of the liver-specific gene SERPINAl (which produces a that plays a role in regulatory functions in the inflammatory, complement, coagulation, and fibrinolytic cascades and whose expression is used as a marker for liver function) was tested using RT-PCR. Results show that SERPINAl gene expression was fully rescued. Importantly, genes encoding liver-specific transcription factors were also rescued, including Hepatocyte Nuclear Factors HNFla and HNF4.

In subsequent experiments, it was found that CREGl expression also activated several additional genes located in the SERPIN locus which have been suggested to be controlled by the SERPIN Locus Control Region (LCR). This was surprising considering most serpin genes have not been shown to be activated by the HNF4/HNF1 pathway. Because HNF6 (Onecut) has been previously shown to bind to the serpin LCR, we measured its expression and found it to be rescued as well with CREGl overexpression. We next asked whether HNF6 alone could rescue serpin locus gene expression by transfecting Hll cells with an HNF6 expression vector. The H11- HNF6 cells showed rescue of many of the serpin locus genes, although only partial rescue was observed. Likewise, a partial rescue of HNF4 and HNFl genes was observed in the H11-HNF6 cells, but not to the level observed in the H11-CREG1 cells.

We conclud e that CREGl can fully rescue expression of several liver-specific genes, including transactivation genes, suggesting its role as a master regulator of liver function. CREGl action appears to act at least partially through HNF6 gene activation as well as through activation of the HNF4/HNF1 pathway, both of which act to increase expression of the serpin locus through both LCR activation (through HNF6) and individual serpin gene activation (through HNF4/HNF1).

iv Dedicated to Fandia Alrowily and Siran Aldaghmi

v Acknowledgments

I would like to express the deepest appreciation to everyone who helped and encouraged me until completion of my Master's thesis project. Without their support and assistance this work would not have been possible. I would like to acknowledge those who gave me deep insight and expertise that also extremely assisted the project.

I submit my heartiest gratitude to my respected advisor Dr. Gary A. Bulla, for his detailed instructions, advice, suggestion, support, and constructive criticism that led me to complete my thesis. He gave me worthy information in my research filed that would not have been easy to comprehend. I am thankful for keeping your office door always open for me and all students. It was a pleasure working as member in his lab crew.

Actually, there are no words that sufficiently describe how thankful I am for your kindness.

I would like to thank my committee members, Dr. Britto P. Nathan and Dr. Antony 0. Oluoch I am deeply grateful for your comments and suggestions. They were keen to give important advice to finish my research properly. It was a pleasure to stand in front of you and hear your guidance that helped me a lot in supporting my research.

A very special thanks to my parents for their absolute encouragement, I could not reach what I want without their determination and persistence to achieve my goals. I would also like to express my gratitude to all of my family who urged me and put their faith in me to achieve my ambitions. I am deeply indebted to my brother Ahmed who did not go for higher stud ies due to his responsibilities to take care of my family.

Last but not least, I would like to take this opportunity to record my sincere thanks to Al Jouf

University for p roviding a full scholarship that fully supported me through tuition and living expenses. I am grateful to be one of its faculty members. Lastly, I am hugely indebted to Eastern Illinois University for giving me the opportunity to be a graduate student here and to fulfill my dreams.

vi Table of contents

Abstract...... iv

Acknowledgments ...... vi

Table of contents ...... vii

List of Figures ...... ix

List of Tables ...... x

Chapter 1: Introduction ...... 1

1.1. Liver Development...... 1

1.2. Transcriptional Regulation of Gene Expression ...... 2

1.3. Liver-specific Transcription Factors (e.g. HNF4, HNF6) ...... 5

1.4. Locus Control Regions ...... 7 1.5. The Serpin Locus ...... 8

1.6. Cell Culture Models Systems ...... 10 1.7. Monitoring Whole Genome Expression with DNA

M icroarrays ...... 10

1.8. Cregl as a Candidate Gene for a Liver Master Regulator...... 12 1.9. Hypothesis ...... 14

Chapter 2: Materials and Methods ...... 15

2.1. Cell Culture...... 15

2.2. Primer Design ...... 16

2.3. Cell Transfection ...... 18

2.4. RNA Extraction...... 19

2.5. Complementary DNA (cDNA) ...... 20

2.6. Quantitative Reverse Transcription PCR (qRT-PCR) ...... 20

Chapter 3: Results ...... 22

3.1. Overexpression of Cregl activates the Serpinal and the SERPIN Locus ...... 22

vii 3.2. Rescue of Transcription Factors HNFla and HNF4 ...... 24

3.3. Rescue of HNF6 ...... 25

3.4. HNF6 Rescues Serpinal and Partially Rescues Serpin Locus

Expression ...... 25

Chapter 4: Discussion ...... 40

4.1. Cregl as a Candidate Gene for a Liver Master Regulator...... 41

4.2. HNF6 as an Activator of Liver function ...... 43

4.3. Conclusions ...... 45

4.4. Future Directions ...... 46

References ...... 48

viii List of Figures

1. Serpin family genes located on rat 6 ...... 9

2. CREGl was one of the candidate genes that repressed in variant cells...... 13

3. Expression of CREGl in transfected hepatoma variant Hll cells ...... 27

4. CREGl over-expression effects in transfected Hll cells...... 28

5. H11-CREG1 cells show partial rescue of serpin locus genes...... 30

6. H11-CREG1 transfected cell line shows activation of transcription factor HNFla ...... 31

7. H11-CREG1 transfected cells over-express the transcription factor HNF4.; ...... 32

8. CREGl rescues expression of HNF6 in H11 cells...... 34

9. HNF6 is expressed in pooled H11-HNF6 transfectants...... 35

10. HNF6 rescues SERPINAl expression in Hll cells...... 36

11. HNFG rescues expression of several serpin locus genes in Hll cells ...... 37

12. Expression of SerpinAl and SerpinA3n in H11-HNF6 transfectant cells...... 38

13. HNF6 partially rescues expression of HNF4 and HNFl in Hll cells ...... 39

ix List of Tables

1. Primers used in the quantitative Polymerase Chain Reaction (qRT-PCR) in the current

study...... 17

2. AAT selection of candidate gene: Failure of SERPINAl-AAT rescue by CREGl

overexpression in Hll cells...... 29

3. Expression levels of Serpin genes in hepatoma and hepatoma variant cell lines by

microarray analysis ...... 33

x Chapter 1

Introduction

The mammalian liver is an essential organ (gland). Therefore, much research been done to understand how the liver functions and how its function might be restored or regenerated in liver disease. More research is required to understand the molecular foundations of liver function including regeneration, growth and maintenance, as well as cell proliferation once the liver has reached its completed mass. Addressing these points will provide essential insight into understanding liver tissue growth as well as different aspects of liver disease that would benefit from liver regeneration, included liver transplantation, viral hepatitis, and toxic damage (Taub,

1996).

The liver is described as a chemical factory of the body because it performs a wide range of biochemical functions required for life. Therefore, many model systems have been developed to explore liver development and regeneration. One of these models is the use of dedifferentiated liver cells to identify hepatic genes and the transcriptional pathways used to drive expression of these genes.

1.1. Liver Development

The liver is the largest gland in the body that has both endocrine and exocrine properties. Endocrine functions include the excretion of many hormones such as insulin-like developmental factors, angiotensinogen, and thrombopoietin, while the major exocrine excretion is in the form of bile (Si-Tayeb et al, 2010}. Moreover, metabolism of dietary

1 compounds, detoxification, organizing of glucose levels through glycogen storage and control of blood homeostasis by excretion of clotting factors and plasma such as albumin are among liver functions (Zorn, 2009). Because the liver has a role as an important regulator of normal physiological processes, liver diseases including hepatic fibrosis, cirrhosis, hepatitis, and hepatocellular carcinoma result in high rates of morbidity and mortality, with liver disease representing the fourth leading cause of death among middle-aged adults in the United States

(Si-Tayeb et al, 2010).

Hepatocytes have similar functional units and make up 90% of the mamma lian liver mass, the rest of the cells being termed non-parenchymal cells and including Kupffer cells,

Stellate or Ito cells, and Sinusoidal Endothelial Cells (Briers, 2012). A large number of liver­ specific genes have been identified in the mammalian liver that respond to metabolic and catabolic needs of the body (Cheng et al, 2006).

1.2. Transcriptional Regulation of Gene Expression

Gene expression in eukaryotic cells is regulated at transcriptional, post transcriptional and translational levels (Tomizawa et al, 1997). However, tra nscriptional control is the primary means of regulating gene expression. Euka ryotic transcriptional control functions at three levels: modulation of the activities of activators and repressors; variations in chromatin structure; and through the effect of activators and repressors on assembly of initiation complexes (Lodish et al, 2000).

Regulation of transcription initiation is the most widespread form of gene control in euka ryotes. However, transcription in some cases can attenuate and regulate subsequent

2 steps, such as during or after the transcription process occurs (Cooper, 2000). Eukaryotic genes are regulated by multiple transcription control elements, including promoter-proximal elements and enhancers. In addition, elements including the TATA box and initiator sequence

(Lodish et al, 2000} are required to initiate transcriptional activity.

Transcription is controlled by trans-acting proteins, called transcription factors, which bind at cis-acting regulatory DNA sequences. Transcription factors are equivalent to the repressors and activators in bacteria, which control the transcription of operons. Cis-acting DNA control elements are often located tens of thousands of base pairs away, either upstream or downstream, from the promoter they regulate. In that way, transcription from a single promoter may be regulated by the binding of multiple of transcription factors to alternative control elements, inducing a complex control of gene expression (Lodish et al, 2000).

Transcription factors bind to the specific regulatory sequences and modu late the activity of

RNA polymerase, which is required during the tra nscription process. Another aspect in control of eukaryotic gene expression is the packaging of DNA into chromatin and its modification by methylation, with chromatin structure playing an important role in the process of gene expression regulation (Cooper, 2000).

In mammalian livers, many genes can respond reversibly to external stimuli such as noxious chemicals. Genes are reversibly induced and repressed by transcriptional control in order to adjust the cell's enzymatic machinery to its immediate nutritional and physical environment, which means genes are controlled in response to environmental variables, as occurs in bacteria. In general, in multicellular organisms, only a small fraction of genes respond

3 to environmental changes compared with single-cell organisms such as bacteria (Lodish et al,

2000).

The regulation of specific gene expression in the liver is an active process and includes transcriptional control, post-transcriptional regulation and cell-cell contact (Panduro et al,

1987). Cell type-specific gene expression is controlled primarily at the level of transcription and involves several factors, such as CCAAT/enhancer binding proteins (C/EBPs) and some hepatocyte nuclear factor (HNFs), some of which from autoregulatory loops. For example, the gene encoding HNFla is controlled by HNF-4a (White, 2009). Promoters and enhancer regions of liver genes are composed of multiple cis-acting DNA sequences, binding different HNFs to regulatory regions of gene and providing a synergy of transcriptional activation. These interactions play an important role in tissue-specific gene expression maintenance by the manifestation of distinct hepatocyte-specific target genes. Specific amino acid sequences make up structural motifs in the DNA-binding domain of a transcription factor mediating the specific recognition of the DNA sequence. This specificity allows the recognition of DNA sites by the transcription factor, either in a proximal promoter or in distal enhancer sequences of hepatocyte-specific genes, defining the hepatocyte-specific genes regulated by a particular tra nscription factor (Costa et al, 2003).

Maintenance of liver-specific gene transcription is related to the cooperation of liver­ enriched factors with the ubiquitous transactivating factors (Schrem et al, 2002). In general, the stimulation of hepatocyte-specific gene transcription is associated to the binding of the liver­ enriched transcription factors (C/EBP, HNFl, HNF3, HNF4, and HNF6) to multiple

promote/enhancer sites, interacting in a synergistic manner (Costa et al, 2003).

4 1.3. Liver-specific Transcription Factors (e.g. HNF4, HNF6)

Transcription factors are trans-acting DNA binding proteins that enable selective gene

expression and regulation. These factors bind to a specific cis-acting DNA sequence in a

regulatory element of a gene, interacting with the tr