IDENTIFICATION OF NEUROGENIC DIFFERENTIATION FACTOR AND NEUROGENIN HOMOLOGS IN Schistosoma mansoni BY SHIKHA TANDON Submitted in partial fulfillment of the requirements for the degree of Master of Science Thesis advisor: Dr. Emmitt R. Jolly Department of Biology CASE WESTERN RESERVE UNIVERSITY May 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of SHIKHA TANDON candidate for the MS BIOLOGY degree*. (signed) Dr. Roy Ritzmann (chair of the committee) Dr. Emmitt R. Jolly Dr. Christopher Cullis Dr. Claudia Mizutani January 18, 2012 *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS 1. INTRODUCTION 1 1.1 Schistosomiasis 1 1.2 bHLH Transcription factors 2 1.3 NeuroD and Neurogenin 3 1.4 Evolution of NeuroD and Neurogenin 5 1.5 NeuroD and Neurogenin in different organisms 6 1.5.1 Caenorhabditis elegans 6 1.5.2 Xenopus laevis 7 1.5.3 Homo sapiens 7 1.6 NeuroD and Diabetes Mellitus in Humans and Mice 9 1.7 NeuroD in the Mammalian Retina 11 1.8 NeuroD and Neurogenin in Schistosomes 12 2. MATERIALS AND METHODS 13 2.1 Identification and Cloning of NeuroD/Neurogenin homolog 13 2.2 Sequencing of NeuroD/Neurogenin homolog 15 2.3 cDNA synthesis and Real-Time Polymerase Chain Reaction (Absolute qPCR) 16 2.4 Yeast One Hybrid system to test for transcriptional activation 16 2.5 Protein expression, purification and quantification 18 2.6 Electrophoretic Mobility Shift Assay (EMSA) to test DNA-Protein binding 20 2.7 Cloning, protein expression, protein purification and EMSA of Smp_125400 DNA Binding Domain 22 3. RESULTS 24 4. DISCUSSION 30 5. FIGURES 35 6. BIBLIOGRAPHY 47 LIST OF FIGURES Figure 1: Life cycle of Schistosoma mansoni Figure 2: BLASTp search using Smp_125400 full transcript Figure 3: Multiple sequence alignment of HLH domain of Smp_125400 with a) Human neurogenic differentiation factors and neurogenins and b) HLH domain of multiple organisms Figure 4: Expression of Smp_125400 during different stages of S.mansoni life cycle via RT-PCR Figure 5: Nucleotide and protein sequence of Smp_125400 splice variant Figure 6: Expression of Smp_125400 during different stages of S.mansoni life cycle via qPCR Figure 7: Modified Yeast-One-Hybrid system to test whether Smp_125400 can activate transcription Figure 8: Results of modified Yeast-One-Hybrid system reporter assays Figure 9: Growth assay to test for Smp_125400 reporter activity by Spot Test Figure 10: Electrophoretic Mobility Shift Assay to test for the DNA binding ability of a) Smp_125400 full transcript and b) Smp_125400 DNA Binding Domain protein Figure 11: Orthogonal type of nervous system in S. mansoni Identification of Neurogenic Differentiation Factor and Neurogenin Homologs in Schistosoma mansoni Abstract By SHIKHA TANDON Schistosomiasis is a parasitic disease that is caused due to an infection by the parasitic worm, Schistosoma mansoni. S. mansoni has a complex life cycle transitioning from free swimming larval forms to an intermediate snail host or to a human definitive host. However, little is known about neural development in this parasite, nor how gene function and expression is regulated within the life cycle of S. mansoni. Neurogenic differentiation factor (neuroD) and neurogenin are basic helix- loop-helix transcription factors that play a role in the development and maintenance of the peripheral and sensory nervous system. They also have the ability to bind to and induce expression of the Insulin gene in beta cells of the pancreatic islet. Here we describe the identification of a neuroD/neurogenin homolog in S. mansoni and demonstrate that it functions as a transcriptional activator. We also test its ability to bind to DNA. 1. INTRODUCTION 1.1 Schistosomiasis Schistosomiasis is a parasitic disease that infects 200 million people worldwide with an estimated 779 million at risk of infection [1] [3]. It infects people of all ages from 76 countries within Asia, Africa, South America and the Middle East [2]. Schistosomiasis is caused by the helminth parasite of the genus Schistosoma, namely Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium [4]. These three species can infect humans. Infection due to S. mansoni and S. japonicum causes intestinal schistosomiasis whereas urinary schistosomiasis is caused by S. haematobium [5]. Symptoms of an acute infection include fever, headache, diarrhoea and respiratory symptoms. Chronic infection is due to host immune responses to the eggs of the parasite [6]. The life cycle of S. mansoni involves a series of morphological and biochemical transitions from two free swimming larval forms, an intermediate snail host and a human definitive host [2] [Figure 1]. Cercariae, infect the human host by penetrating through the skin. Upon infection, they shed their tail and transform into schistosumula. These schistosomula travel to the liver via the lungs and develop into adult worms. The mature adult worms pair and females produce almost 300 eggs per day [7]. These eggs are excreted into fresh water through the feces. Upon contact with fresh water, the eggs hatch into free swimming miracidia which in turn infect the snail intermediate host. Inside the snail, miracidia transform into mother and daughter sporocysts. The daughter sporocysts produce cercariae, which is a larval form capable of infecting humans [2]. 1 Praziquantel is the drug of choice to treat schistosomiasis, but it does not prevent reinfection. There are concerns that schistosomes may become resistant to praziquantel as some reports have shown selection of resistance in a laboratory setting [1]. Thus, it is paramount that ongoing research be aimed at understanding gene expression and regulatory function within the life cycle of S. mansoni. Transcription factors are proteins that are capable of binding to certain DNA sequences and thereby regulating expression of genes by controlling transcription of DNA to mRNA [22]. They play key roles in developmental processes in all organisms. In this regard, understanding schistosome basic biology, particularly the function of regulators of gene expression during development, will elucidate how development is controlled in parasites and may help to identify potential therapeutic targets. 1.2 bHLH transcription factors Basic helix-loop-helix (bHLH) proteins are a family of transcription factors. The bHLH structural motif contains two α- helices that are amphipathic and are connected by a loop of variable length [8]. Of the two α- helices, one is smaller than the other and the larger helix usually contains the DNA binding region. DNA binding is facilitated by the presence of basic amino acid residues in the larger helix [9]. Overall, the HLH regions of these transcriptional factors are hydrophobic and are composed of approximately 50 amino acid residues. Proteins containing bHLH structural motifs form functional dimers and are capable of binding to other bHLH proteins [8]. There are currently over 400 proteins that are known to contain the bHLH domain [8]. These complex structures are functionally heterogeneous and play a role in essential 2 developmental processes including neurogenesis, myogenesis, cell proliferation and tissue differentiation [8]. Evolutionarily, sequencing of the non bHLH domains between clades, revealed very little sequence similarity [8]. Between clades, although a high degree of similarity is seen in the bHLH domain, the location of this domain within the overall structure varies. The dissimilarity between non bHLH domains, suggests that these clades must have diverged a long time ago [8]. It is hypothesized that, the bHLH and non bHLH domains, which we now appreciate as the bHLH group of transcriptional factors, were shuffled around during evolution [8]. bHLH transcription factors are known to bind to a specific consensus DNA sequence (CANNTG), known as E-box. These E-box sequences are present in the promoter region of the target genes and binding occurs via the DNA binding domain of the transcription factor [10]. 1.3 Neurogenin and neuroD Neurogenins are a subfamily of bHLH transcriptional factors that are involved in neuronal differentiation [11]. They are related to the Drosophila atonal gene family. They play an essential role in the development of the dorsal root ganglia as part of the sensory lineage in the neural crest cells of Drosophila [11]. There are three types of neurogenins; neurogenin 1 (encoded by the NEUROG1 gene), neurogenin 2 (encoded by the NEUROG2 gene) and neurogenin 3 (encoded by the NEUROG3 gene) [11]. 3 In humans, neurogenin 1 interacts with cAMP response element binding protein or CREB [12]. CREB protein is encoded by the CBP gene which acts as a coactivator for many transcriptional factors and is involved in embryonic development, growth control, and homeostasis [13] [14]. Neurogenin 2 is expressed in neural progenitor cells and is involved in the development of the central and peripheral nervous systems [15]. Neurogenin 3 is expressed in the pancreas and intestine and is essential for endocrine cell development [16]. Neurogenic differentiation factor or neuroD is a bHLH transcription factor that plays an important role in neuronal differentiation [10]. It is also known as beta2 (β-cell E- box transactivator 2) and is also involved in development of the pancreas [10]. NeuroD is expressed in the brain, pancreatic beta cells, enteroendocrine cells and neuroendocrine cells [10]. NeuroD is capable of forming heterodimers with ubiquitous bHLH factors such as E47 and E12 [31]. This NeuroD/E47 complex is capable of binding to the E-box sequence within the promoter region of the insulin gene [31]. There are five major members of the neuroD family, not all of which are seen in all species. NeuroD1 (encoded by the NEUROD1 gene) is capable of forming heterodimers with other bHLH proteins and is also responsible for activating transcription of the Insulin gene in humans. Mutations in this gene cause Type II Diabetes Mellitus in humans [33]. NeuroD2 is encoded by the NEUROD2 gene and is involved in the determination and maintenance of neural cells [35].