Inosine Monophosphate Dehydrogenase and Transcription: a Mechanism for Retinitis Pigmentosa?

Inosine Monophosphate Dehydrogenase and Transcription: a mechanism for retinitis pigmentosa? Master’s Thesis Presented to the Biochemistry Department Brandeis University Lizbeth Hedstrom, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science by Yuliya Mints May 2011 Copyright by Yuliya Y. Mints 2011 Acknowledgements I would first like to thank Dr. Lizbeth Hedstrom for the opportunity to work in her lab for the past two years. Working in this lab has been an invaluable and rewarding experience. She pushed me to be independent and to think critically. I thank her for her guidance, wisdom, and continued support. I would also like to thank Aimee Butterworth for serving as my mentor. I really appreciate her training and help. Her long discussions with me about science (and life) were very valuable. I would also like to thank Dharia Mcgrew, who was always ready to answer my questions. I am grateful for the rest of the members of the Hedstrom lab who have always helped me and pointed me in the right direction: Xin, Marcus, Minjia, Devi, Corey, Suresh, Kavitha, Greg, Phil, Aleze, James, and other members of the lab in the past and present. Thank you all! I enjoyed coming to lab because of you! I am very thankful for Dr. Michael Marr and members of his laboratory, without whom portions of this project would not have been possible. I thank them for their help and gift of S2 cells and primers. Finally, I would like to thank my family and friends for their continued support and encouragement all of these years. They have always stood by me and I could not have gone through this long journey alone. iii ABSTRACT Inosine Monophosphate Dehydrogenase and Transcription: a mechanism for retinitis pigmentosa? A thesis presented to the Biochemistry Department Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Yuliya Mints Retinitis pigmentosa (RP) is one of the leading causes of retinal degeneration worldwide. Many inherited forms exist, and treatment is limited due to a lack of understanding of the mechanism of the disease. Mutations in the RP10 gene cause the autosomal dominant form of RP. The RP10 gene encodes 5’-inosine monophosphate dehydrogenase type 1 (hIMPDH1), which catalyzes the rate limiting step in de novo guanine nucleotide synthesis. Recently, it was discovered that IMPDH is recruited to actively transcribing genes in Saccharomyces cerevisiae. In this study, chromatin immunoprecipitation experiments in Drosophila melanogaster reveal that IMPDH is enriched at the Actin5C and MtnA promoters, both transcribed by RNA polymerase II, but not at the Ribosomal 28S promoter, which is transcribed by RNA polymerase III. As transcriptional activity of MtnA in induced, IMPDH recruitment to that gene increases two-fold. In addition, NAD(H) do not appear to affect the affinity of IMPDH for nucleic acids. Finally, RP-causing mutants of IMPDH were transfected into HEK293T cells and found to localize to the chromatin. These studies propose a new role for IMPDH in transcription. Since perturbations in transcription of certain genes in photoreceptor cells are known to cause apoptosis, these observations suggest a mechanism for IMPDH-linked hereditary blindness. iv Table of Contents Acknowledgements iii Abstract iv Table of Contents v List of Tables and Figures vi Introduction 1 Materials and Methods 10 Sequence alignment 10 Chromatin immunoprecipitation 10 Chromatin IP analysis by real-time PCR 12 Western blot analysis 13 Transfection of HEK293T cells 13 Filter binding assay of IMPDH with NAD+/NADH 14 Results 16 Human anti-IMPDH antibody recognizes D. melanogaster IMPDH 16 IMPDH associates with genes under RNA polymerase II promoter 20 Act5C downstream affinity for IMPDH 22 IMPDH recruitment increases with transcriptional activity 23 RNA does not mediate interaction between IMPDH and transcribing genes 24 IMPDH in human cell chromatin 25 NAD+/NADH do not affect nucleic acid binding 27 Discussion 29 References 35 Appendix 38 Purification of MBP-tagged extensions of retinal splice variants of IMPDH 38 Filter Binding Assays of IMPDH and MBP-tagged extensions 39 v List of Tables and Figures Tables Table 1 Primer sequences for real-time PCR analysis of ChIPs 12 Table 2 Antibodies used in ChIPs and Western blots 14 Table 3 ChIPs using MTF-1 20 Table 4 MTF-1 ChIPs with transcription activation 24 Table 5 Summary of IMPDH ChIP experiments 25 Table 6 Quantification of filter binding assay 26 Figures Figure 1 Crystal structure of Streptococcus pyogenes IMPDH 3 Figure 2 Map of RP-causing mutations in a monomer of IMPDH 4 Figure 3 Putative binding site of nucleic acids 6 Figure 4 Western blot of S2 using various anti-IMPDH antibodies 17 Figure 5 Standard protein curve 17 Figure 6 Immunoprecipitation of S2 chromatin 18 Figure 7 IMPDH association with Act5C 22 Figure 8 Retinal isoforms of IMPDH in HEK293T chromatin 26 Figure 9 RP-causing isoforms of IMPDH in HEK293T chromatin 26 Figure 10 Filter binding assay with NAD+/NADH 26 vi Introduction Retinal degeneration is a main cause of vision loss that affects over 10.5 million people [1]. Retinitis pigmentosa (RP) is the most common form of inherited retinal degeneration. Its symptoms are a loss of night vision, followed by a loss of peripheral vision, and finally blindness, which are all caused by photoreceptor death. While progression may vary among individuals carrying the same mutation, apoptosis of photoreceptor cells eventually causes blindness. Forms of RP include autosomal dominant, recessive, X-linked, and mitochondrial [2]. No effective treatments for any form of retinal degeneration exist. Insight into the mechanism of retinitis pigmentosa and photoreceptor cell apoptosis may reveal opportunities for treatment and prevention. Over 75 genes have been implicated in RP. Although many of the RP-associated genes encode proteins directly involved in vision, there are some genes that are widely expressed in all tissues [3]. One such gene is RP10, which encodes human inosine monophosphate dehydrogenase type 1 (hIMPDH1). Mutations in this enzyme account for 2% of autosomal dominant RP, however the pathological mechanism is unclear. Retina contains unique splice variants of IMPDH1 Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate limiting step in guanine nucleotide synthesis: it converts inosine 5’-monophosphate (IMP) to 1 xanthosine 5’-monophostphate (XMP) with a reduction of NAD+. Like most mammals, humans have two genes, IMPDH1 and IMPDH2, which encode enzymes that have 84% sequence similarity [4]. IMPDH1 and IMPDH2 have nearly identical catalytic properties and affinities for their substrates and inhibitors [5]. Both IMPDH1 and IMPDH2 localize to the cytoplasm and nucleus of human cells [6]. Most cells express both enzymes, though the relative expression of IMPDH1 and IMPDH2 varies among different tissues [7]. Human retinal cells express significantly more IMPDH1 [8], which may account for the tissue specificity of the effects of the IMPDH1 mutations. In addition, the retina contains two isoforms of IMPDH1: IMPDH546 and IMPDH595 [8]. These isoforms are formed by alternative mRNA splicing. IMPDH546, the major isoform in humans, has a 32 residue C-terminal segment, and IMPDH595 has the 32 amino acids on the C-terminal as well as a 49 residue segment on the N-terminal [9]. Both isoforms IMPDH546 and IMPDH595 are unique to photoreceptor cells. IMPDH has a CBS domain of unknown function While its enzymatic function has been well characterized, IMPDH also appears to have a number of other, poorly understood functions in the cell. It has been suggested that IMPDH is also involved in binding to polyribosomes translating rhodopsin mRNA [10], association with lipid vesicles [11], and most recently in transcription regulation [12]. 2 CBS domain Catalytic domain Figure 1. Crystal structure of Streptococcus pyogenes IMPDH [13].The enzyme is a homotetramer, each monomer made up of a catalytic and CBS domain. S. pyogenes IMPDH is shown because it is the only crystal structure where both domains are ordered. These activities are not surprising because the enzyme contains a domain of unknown function. IMPDH is a homotetramer, each unit of which consists of a main catalytic domain and two cystathionine β-synthetase (CBS) domains attached on the opposite end of the catalytic domain from the active site (Figure 1). The structures of the N- and C-terminal extensions in IMPDH546 and IMPDH 595 are unknown and it is not clear if they interact with the catalytic and CBS domains. Known RP-causing mutations in IMPDH1 (Arg224Pro, Asp226Asn, Arg231Pro) are located in the interface between 3 the catalytic and CBS domains (Figure 2). Other mutations of IMPDH exist that are may also cause RP. Figure 2. RP-causing mutations of IMPDH are located in the junction between the catalytic domain and the CBS domain. Nucleic acids appear to bind in that junction because mutations decrease the affinity for nucleic acids. In this monomer of IMPDH, magenta denotes mutations that are clearly pathogenic; red, likely pathogenic; green, possibly pathogenic. Figure taken from Mortimer et al. [10]. The CBS domain is found in a variety of other proteins, such as voltage-gated chloride ion channels and AMP-activated protein kinases. Certain point mutations in conserved residues in CBS domains are known to cause a number of hereditary diseases, such as homocystinuria [14], idiopathic generalized epilepsy [15], and congenital myotonia [16]. Some CBS domains form interactions with adenosine derivatives, such as AMP and ATP, which then affect the activity of the enzyme [17]. Scott et al. suggest that binding of adenosine derivatives serves as an energy sensor of the cell, so that the enzyme containing the CBS domain is active under appropriate conditions [18]. Disease 4 causing mutations in CBS domains may disrupt ligand binding, and thus cause pathogenesis [18]. The CBS domain in IMPDH is conserved across species.

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