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

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.

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

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

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

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].

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

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

causing mutations in CBS domains may disrupt ligand binding, and thus cause pathogenesis [18]. The CBS domain in IMPDH is conserved across species. According to

Scott et al., binding of ATP to the CBS domain causes an increase in IMPDH activity

[18]. RP-causing mutations of IMPDH disrupt ATP binding to the CBS domain [18].

However, other groups have not observed a change in enzymatic activity of IMPDH1

[19] or IMPDH2 [5] in the presence of adenosine derivatives. In fact, deletion of the CBS domain has no effect on enzymatic activity [20]. It remains unclear what function the

CBS domain in IMPDH serves.

IMPDH binds to nucleic acids in vivo

Previous work has found that in addition to controlling the guanine nucleotide pool, IMPDH also binds to DNA and RNA in vivo [6]. The nucleic acid binding site is approximately 100 bases per tetramer, and data suggests that the nucleic acid interacts with all four monomers [6]. This interaction with nucleic acids does not affect the enzyme’s catalytic activity, implying that the interaction occurs away from the active site.

Evidence suggests that nucleic acids bind in between the CBS domain and the catalytic domain (Figure 3) because both are required for nucleic acid association. Furthermore, deletions of the CBS domain do not affect the catalytic activity of the enzyme, but significantly reduce affinity for nucleic acids [19]. RP-causing mutations also significantly perturb the interaction with nucleic acids [19]. The ability of IMPDH to bind to RNA and DNA suggests that the enzyme is involved a process that requires this type of interaction between protein and nucleic acid to occur, such as in translation and/or

transcription in the cell. A perturbation of these processes may be the underlying cause of

RP.

In fact, IMPDH has been found to be involved in translation in both human embryonic kidney (HEK293T) and bovine retinal cells. Mortimer et al. 2008 found that

IMPDH1 associates with polyribosomes translating mRNA in human and bovine retinal cells. The mRNA was identified to be rhodopsin in retinal cells [10]. Both retinal splice variants of IMPDH also exhibited this activity. Deletions of the CBS domain caused a decrease in the enzyme’s association with polyribosomes, providing support for the hypothesis that the CBS domain mediates the interaction between IMPDH and translating polyribosomes. Further, the most common RP-causing mutation in IMPDH595,

Asp226Asn, blocks association with polyribosomes [10]. Slight changes in rhodospin expression result in blindness [21, 22]. It is possible that changes in IMPDH association with polyribosomes translating rhodopsin affect protein levels, which leads to blindness.

Figure 3. A monomer of IMPDH, which contains the catalytic domain and the CBS domain. The putative nucleic acid binding site is located in the junction between these two domains.

IMPDH is involved in transcription

A recent study in Saccharomyces cerevisiae implicates IMPDH in transcription as well [12]. Park and Ahn found that IMPDH is recruited to genes actively transcribed by

RNA Polymerase II [12]. Additionally, IMPDH travels along with the transcription complex as long as serine 2 on the C-terminal domain of RNA polymerase II is phosphorylated by Ctk1 kinase. This new finding indicates an unsuspected role of

IMPDH in transcription regulation. Perturbation of this function could be the pathological mechanism of RP.

RNA transcription regulation is a complicated process. It requires the careful coordination of several types of molecules and enzymes. These enzymes include kinases, acetylases, ubiquitinases, methylases, and many other categories of enzymes that cause epigenetic modifications or signal various phases of transcription. Transcription is divided into 5 stages: pre-initiation, initiation, promoter clearance, elongation, and termination. Each stage has its own set of factors that must be present in order for transcription to occur successfully. Because of the number of molecules involved in the process, there are many opportunities for regulation. RNA polymerase II, which is responsible for transcribing most protein coding genes, has sites for various transcription factors. Additionally, RNA polymerase II has up to 52 tandem heptad repeats of Tyr-Ser-

Pro-Thr-Ser-Pro-Ser on the C-terminal domain (CTD) of its largest subunit, RpII215. The residues in these heptad repeats are prone to modifications by transcription factors, such as phosphorylation. Modifications to the heptads correlate with different stages during transcription. For example, when the serines, and to a lesser extent, threonines and

tyrosines, are hyperphosphorylated, RNA polymerase II is in a form that is associated with RNA elongation. Transcription factors can bind or modify the CTD to affect the enzymatic activities of RNA polymerase. Researchers are constantly uncovering new molecules and factors that are critical to transcription.

IMPDH is not the first dehydrogenase discovered to play a role in transcription.

C-terminal binding protein (CtBP) directly interacts with histone deacetylases, methyl transferases, and demethylases to aid in chromatin remodeling that is necessary for transcription to occur. CtBP protein catalyzes the NAD-dependant conversion from pyruvate to lactate (although the activity is weak and considered “ancestral”) [23]. Mani-

Telang and colleges show that the dehydrogenase function is not a requirement for normal cell transcription levels [23]. However, NAD(H) binding does induce a conformational change in CtBP required to recruit E1A, which causes transcription repression [24]. Perhaps IMPDH is performing a similar function.

IMPDH’s role in transcription is especially interesting because of its connection to the autosomal dominant form of retinitis pigmentosa. It is not clear why certain mutations in RP10 cause apoptosis exclusively of photoreceptor cells. A role in transcription regulation presents an attractive model for the mechanism of the disease, especially since photoreceptor cells are among the most actively transcribing cell types

[25]. Further, retinal cells contain two splice forms of hIMPDH1, IMPDH546 and

IMPDH595, which contain extra residues on the N- and C-termini. As mentioned above, it is unknown what the function of these additional residues in retinal IMPDH is. These

isoforms are enzymatically identical to the canonical form, however were initially believed to be unable to bind nucleic acids like canonical IMPDH [26]. Subsequent modification of the purification method produced enzyme that does associate to nucleic acid with high affinity [A. Butterworth and D. McGrew, unpublished]. This suggests that the N- and C-terminal extensions have another function specific to the retina which may be involved in transcription.

The goal of this study is to determine whether IMPDH is involved in transcription in higher eukaryotes, and whether this function is linked to the underlying cause of retinal degeneration. First, the results of Park and Ahn will be confirmed in Drosophila melanogaster, which can better mimic a human disease than yeast. D. melanogaster also have retinas and are more likely to contain other factors required for the visual cycle.

Second, the effect of NAD+ and NADH on IMPDH affinity for nucleic acids will be measured. Although it does not appear that CtBP directly binds to nucleic acids, the cofactors are necessary for normal transcription function. Finally, human isoforms of

IMPDH1 and RP-causing mutants of IMPDH1 will be investigated in tissue culture to determine their association with chromatin.

Materials and Methods

Sequence Alignment

Sequence identities of various IMPDH enzymes were determined from global alignments of pairs of sequences generated with MUSCLE using the default parameters

[27].

Chromatin immunoprecipitation

Drosophila S2 cells were grown in Schneider’s media (10% FBS, 1x penicillin/streptomycin) in a flat bottom flask to a density of 5 × 106 cells/mL. The cells were diluted and 2 mL and plated in three wells of a six-well plate at a final concentration of 1 × 106 cells/mL. For experiments involving induction of MtnA transcription, the cells were incubated with 2 mM copper (II) sulfate for 4 hours at 27oC. Formaldehyde was added for a final concentration of 1.0% and incubated with cells at room temperature for

15 minutes. The crosslinking reaction was quenched with 1 M Tris-Cl pH 7.4 (final concentration 100 mM). Cells were collected by centrifugation at 1000g for 5 minutes at

4oC, and then resuspended in 1 × PBS/0.1 M Tris-Cl pH 7.4. The cells were collected again and resuspended in 1 × PBS/0.5% Triton X-100 to lyse the cells and incubated on ice for 10 minutes with periodic mixing. Lysed cells were centrifuged at 15000g for 10 minutes, and pelleted nuclei were resuspended in 500 μL of cold ChIP Lysis Buffer (1×

PBS, 50 mM HEPES/K+, pH 7.6, 2 mM EDTA, 1% Triton X-100, 0.1% Na-

deoxycholate, 0.2 mg/mL RNase A, Protease Inhibitors Cocktail (Sigma Aldrich P8340).

Sarkosyl was added to the sample for a final concentration of 2%. The samples were sonicated with a Fisher Scientific Sonic Dismembrator 550 at setting 3, remote setting and pulsed 30 times. Sonicated samples were then centrifuged at 1000g for 2 minutes.

Sonication and centrifugation were repeated two more times. The supernatant was transferred to a new eppendorf tube and diluted with 1 mL of ChIP Lysis Buffer to be used for immunoprecipitation.

For IMPDH ChIPs, 5 µL of polyclonal anti-IMPDH antibody (Abcam 70045, 0.5 mg/mL) or polyclonal anti-MTF-1 antibody (purified by M. Marr) were added to 200 µL of chromatin. The volume was brought up to 1 mL with ChIP Lysis Buffer and incubated overnight at 4oC. An additional 200 µL was saved as “input” and stored at -20oC.

Sepharose Protein A beads (Sigma Alrich P3391) or Protein G Dynabeads (Invitrogen

100-03D) were washed three times with 1 × PBS and blocked with 0.1 mg/mL yeast tRNA and 1 mg/mL BSA overnight. The preblocked beads were incubated with the chromatin and antibody mixture for 4 hours at 4oC and then washed once with 1.5 mL

ChIP Wash Buffer (1× PBS, 50 mM HEPES/K+ , pH 7.6, 1 mM EDTA, 1% Triton X-

100, 0.1% Na-Deoxycholate, 0.1% Sarkosyl, 0.1% BSA, 0.5 M KCl, Sigma Protease

Inhibitors). Next, the IPs were incubated in ChIP Wash Buffer for 30 minutes at room temperature, and then washed one more time with ChIP Wash Buffer. The beads were then washed once with Li Wash Buffer (10 mM Tris/HCl at pH 8.0, 0.25 M LiCl, 0.5%

NP-40, 0.5% Na-Deoxycholate, 1 mM EDTA) and once with TE. After all of the supernatant was removed, the beads were resuspended in 150 μL of Elution Buffer (50

mM Tris/HCl at pH 8.0, 10 mM EDTA, 1% SDS, 1 mM DTT, 0.1 mg/mL Proteinase K) and incubated for 2 hours at 37oC. The supernatant was transferred to a fresh tube. Both the immunoprecipitated sample and original 200 µL input were incubated overnight at

65oC to reverse the cross-links. The DNA was purified using the PCR Purification Kit

(Qiagen).

Chromatin IP analysis by real-time PCR

Real-time PCR was performed on the immunoprecipitated and input DNA in a

Bio-Rad Chromo4 thermocycler. The following primers were used:

Table 1: Primer sequences for real-time PCR analysis of ChIPs. Primer names are in parentheses. Primer Sequence MtnA promoter forward (Mtn 5’-GCAAGTAAGAGTGCCTGCGCATGC-3’ 584) MtnA promoter reverse (Mtn 5’-TAGGCCTTTAGTTGCACTGAGATG-3’ 585) 28S promoter forward (Mtn 5’-GAGTAGGAAGGTACAATGGTATGC-3’ 636) 28S promoter reverse (28S rev) 5’-GAACCGTATTCCCTTTCGTTCAA-3’ Act5C promoter forward 5’-ACCCAATCGGCGAACAATTCATACCC-3’ (YM5) Act5C promoter reverse (YM6) 5’-GACGACTGCTGGCTGATGGAG-3’ Act5C downstream forward 5’-GTGCCCATCTACGAGGGTTA-3’ (YM7) Act5C downstream reverse 5’-GCCATCTCCTGCTCAAAGTC-3’ (YM8)

20 µL reactions were set up using 1.5 µM of each primer, 1× GoTaq qPCR Master Mix

(Promega), and 5 µL of DNA. Cycling parameters were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, and

78°C for 1 second. Fluorescence intensities were plotted versus the number of cycles by using an algorithm provided by the manufacturer.

Western blot analysis

Chromatin from growing cells was prepared as described above (6 × 106 cells) and resolved by electrophoresis on a 12% polyacrylamide gel, which was then electroblotted onto PVDF. The membrane was incubated with a 1:800 of mouse monoclonal anti-IMPDH1 (Antibody Solutions AS37-P), 1:500 of mouse polyclonal mouse anti-IMPDH2 (Abcam, ab70045), or 1:500 of rabbit anti-IMPDH1 (Abcam 33039,

0.4 mg/mL) overnight at 4oC. Following three 5-minute washes, the membrane was incubated with 1:5000 goat anti-mouse HRP conjugate (Upstate 12-349) or goat anti- rabbit HRP conjugate (Sigma Aldrich). Blots were exposed using ECL+ detection system

(GE Healthcare).

Transfection of HEK cells

Human embryonic kidney 293T (HEK293T) tissue culture cells were grown as described [26] in DMEM (5% FBS, 1x penicillin/streptomycin) in a flat bottom flask to

~90% confluency. The cells were split 1:3 and allowed to grow overnight before transfection. The cells were transfected with 5 µg of plasmid containing the appropriate

GFP-tagged IMPDH splice variant or mutant as previously described [26]. Transfectin

(10 µL) and DMEM (490 µL) were mixed with an equal amount of plasmid in DMEM and incubated at room temperature for 20 minutes. The transfection mix was then applied to the growing cells and incubated overnight. The following day, 6 × 106 cells were

plated onto a circular 10 cm plate. The cells were treated with formaldehyde for a final concentration of 1.0% for 15 minutes, followed by quenching with 1M Tris pH 7.4 (860

µL). The same chromatin preparation protocol was followed as described above for S2 cells until sonication to shear crosslinked DNA fragments. After sonication, 100 µL of chromatin was added to a clean tube and incubated with 150 µL of Elution Buffer without Proteinase K at 65oC overnight.

To analyze the chromatin, a Western blot was preformed with monoclonal α-

IMPDH antibody (Antibody Solutions AS37-P, 1 mg/mL) at a dilution of 1:800. The blot was exposed with ECL+ detection system.

Table 2. Antibodies used in ChIPs and Western blots. Antibody Source Concentration mouse polyclonal α-hIMPDH2 Abcam (ab70045) rabbit polyclonal α-hIMPDH1 Abcam (ab30339) 0.4 mg/mL mouse monoclonal α-hIMPDH1/2 Antibody Solutions (AS37-P) 1 mg/mL Purified by A. Butterworth from serum rabbit polyclonal α-hIMPDH2 produced by Cocalico Biologicals using 0.25 mg/mL 100 µg/rabbit of IMPDH2 as the antigen Purified by M. Marr from serum α-MTF-1 nd produced in rabbit

Filter Binding Assay of nucleic acid binding by IMPDH with NAD+/NADH

IMPDH-binding experiment with single stranded DNA was performed as described in [6]. The experiment was performed in Filter Binding Buffer (10 mM Tris, pH 8.0, 50 mM KCl, 1 mM DTT). The sequence of DNA used was

5′-

GGGAATGGATCCACATCTACGAATTCN30TTCACTGCAGACTTGACGAAGCTT-

3′, where N30 denotes a random sequence 30 bases long. 175 µL reactions were set up 14

with 10 nM of human IMPDH1 or IMPDH2 (purified by A. Butterworth), 500 µM NAD+

(Roche 10127973001) or NADH (Sigma Aldrich N6879), and 5 µL of 5′-32P-labelled ssDNA (2 nM). The reactions were mixed and incubated at room temperature for 20 minutes. The protein bound nucleic acid was separated from free nucleic acid by filtration on a vacuum manifold (Schleicher and Schuell) containing a nitrocellulose membrane

(Bio-Rad) and PVDF membrane (Amersham Biosciences). The nitrocellulose membrane binds protein and associated nucleic acids while the PVDF membrane binds free nucleic acids. The well in the vacuum manifold were washed once with 100 µL of cold Filter

Binding Buffer. The membranes were transferred to a storage phosphor screen and the radioactivity bound to each filter was quantified using ImageQuant v2005.

Results

Human anti-IMPDH antibody recognizes D. melanogaster IMPDH

In order to study D. melanogaster IMPDH, appropriate antibodies needed to be identified for chromatin immunoprecipitation. Since antibodies against the insect IMPDH are not available, human polyclonal anti-IMPDH2 antibodies were tested to determine if they recognize the D. melanogaster enzyme. The IMPDHs of the two species exhibit

65% sequence identity, so it was reasonable to expect that antibodies against the human protein would recognize D. melanogaster IMPDH. The total cell lysate of S2 tissue culture cells was separated on a polyacrylamide gel and probed with various polyclonal antibodies against human IMPDH1 or IMPDH2. All three antibodies detect a protein slightly above the 50 kD mark in a protein ladder, and another protein just below in the

Western blot (4). According to the standard curve constructed from Figure 4A, the weight of the upper band is 58 kD and the lower band is 47 kD (Figure 5). These two proteins are consistent with the two isoforms of D. melanogaster IMPDH (also known as Ras for

Raspberry). The major isoform is a 58 kD protein that has been characterized [28].

Another 47 kD isoform has been predicted computationally [29]. Therefore human anti-

IMPDH antibodies recognize D. melanogaster IMPDH. Mass spectrometry should be preformed to further confirm that the bands are IMPDH.

Figure 4. Western blot of total S2 cell lysate probed with various anti-IMPDH antibodies. Lane 1 contains lysate, and lane 2 contains protein ladder (Bio- Rad Precision Plus Protein) from the same blot; intervening lanes have been removed for clarity. A) Probed with rabbit anti-IMPDH2 (Abcam 70045). B) Probed with rabbit α-hIMPDH2 purified by A. Butterworth from serum produced by Cocalico Biologicals. C) Probed with rabbit anti-IMPDH1 (Abcam 30339).

Figure 5. Standard curve of protein migration distances from Figure 4A.

Even though anti-hIMPDH recognizes to D. melanogaster IMPDH in a Western blot, an immunoprecipitation needed to be performed to determine if the antibody recognizes properly folded protein. S2 chromatin was crosslinked and immunoprecipiated with mouse polyclonal anti-hIMPDH2 antibodies (Abcam 70045) and using Protein G

Dynabeads. The IP was analyzed by Western blot (Figure 6) using rabbit polyclonal anti-

IMPDH1 (Abcam 30339) to prevent cross reaction with the heavy chain of the antibody used in IP. This blot shows that antibodies against the human protein can immunoprecipitate D. melanogaster IMPDH.

Figure 6. Immunoprecipitation of S2 total lysate using mouse polyclonal anti- hIMPDH antibodies (Abcam 70045) using Protein G Dynabeads (Invitrogen 100-03D). Visualized using rabbit polyclonal anti-IMPDH (Abcam 30339) to prevent cross reaction with immunoglobulin chains. The IMPDH IP contains four times as much IMPDH as the no antibody control. 5% of the total IMPDH in the input was recovered in the IP. The thick band at the bottom of the IP lanes is consistent with Protein G from resin. 18

The IMPDH IP precipitates four times as much IMPDH than the no antibody control. Only the 58 kD band is isolated by immunoprecipitation. The IMPDH that appears in the control IP may be due to nonspecific binding to resin. The 47 kD band is not visible in either IP lanes, however there is such a small amount of protein in the no antibody control that it would not be visible if it were present in the same ratio as the input. The IMPDH IP lane has a darker band at 58 kD and the 47 kD band is expected to be visible if it were present. While all three polyclonal antibodies recognize the 47 kD band in a Western blot of total lysate, the lower band disappears after immunoprecipitation. In fact, all other bands that appear in the total cell lysate are not present after IP, indicating that the human antibodies selectively precipitate IMPDH.

Thus, antibodies against hIMPDH2 are suitable to use in chromatin IP experiments in S2 cells.

Before using anti-IMPDH antibodies, an immunoprecipitation was performed using anti-MTF-1 antibodies as a positive control. The chromatin of S2 cells was isolated and sonicated to obtain 450-500 base pair DNA fragments. The chromatin was then immunoprecipitated with polyclonal anti-IMPDH antibodies (Abcam 70045) and using magnetic resin, Protein G Dynabeads. After crosslinks were reversed, the DNA that crosslinked to IMPDH was analyzed by real-time PCR. The primers that were used recognized 200 base pair sequences in the promoter of Metallothionien A (MtnA) and

Ribosomal 28S (28S). To quantify the data from real-time PCR analysis, the percent input

-∆Ct for each gene was calculated by using the 2 method [30]. The Ct refers to the cycle number at which the SYBR Green fluorescence signal, and thus DNA concentration,

reaches a set minimum value. To normalize the IP for the amount present in the input, the

∆Ct is calculated by subtracting input Ct and the natural log of the dilution factor of the input from the IP Ct:

∆Ct = Ct (IP) – Ct (input) – ln(dilution factor of input)

Then, the percent of DNA that is bound by IMPDH and immunoprecipitated is calculated by:

percent input = 2-∆Ct × 100%

The percent input represents the percent of a given segment of DNA that is bound by IMPDH during crosslinking and immunoprecipitated. The results of real-time PCR are summarized in Table 3. The IP was repeated, this time using Sepharose Protein A resin

(Table 3). Real-time PCR analysis reveals that the magnetic Dynabeads have a larger background signal, as determined by the percent input of 28S. MTF-1 is a specific inducer of metallothionien genes and therefore is not expected to associate with 28S. In addition, 28S is a multicopy gene (200-250 copies) and serves as a stringent control.

Also, the Dynabeads bind a lower percent of MtnA from the input. Based on the results in

Table 3, Protein A Sepharose beads were used for all further IPs.

Table 3. Percent input of 28S or MtnA bound by either Protein G Dynabeads (Invitrogen) or Protein A Sepharose beads (Sigma Aldrich). The immunoprecipitation was performed using anti-MTF-1 antibodies. Protein G Dynabeads Protein A Sepharose Beads 28S promoter 0.74% 0.13% MtnA promoter 5.86% 8.49%

IMPDH associates with genes under RNA polymerase II promoter

Since the previous experiment indicates that human anti-IMPDH antibodies can immunoprecipitate D. melanogaster IMPDH, the next aim was to probe the association of 20

IMPDH with actively transcribing genes. Since Park and Ahn report that IMPDH associates with any gene transcribed by RNA polymerase II, the constitutively transcribed Actin 5C (Act5C) was studied. The recruitment of the enzyme to Act5C was compared to Ribosomal 28S (28S), another constitutively transcribed gene. 28S is transcribed by RNA polymerase III and serves as a stringent negative control because it is a multicopy gene. It is estimated that the D. melanogaster genome contains 200-250 copies of 28S [31]. Since previous work in yeast identified IMPDH recruitment to genes under RNA polymerase II promoters, a much greater enrichment of IMPDH is expected at Act5C than at 28S.

After ChIP and real-time PCR, the percent inputs of Act5C were normalized to the percent input of 28S for those samples and adjusted for copy number (assuming a copy number of 200 for 28S). The results show that the IMPDH is significantly enriched at the

Act5C promoter (Figure 7).

Figure 7. Results of the Act5C ChIP analyzed using real time PCR with primers for the promoter of Actin5C as well as an exonic region approximately 2kb downstream of the promoter. 28S is used as an internal control. Values for 28S are adjusted assuming a copy number of 200. For the ChIPs, Sepharose Protein A resin was used with Abcam 70045 antibodies.

Act5C downstream affinity for IMPDH

To further explore the interaction between actively transcribing genes and

IMPDH, another region within Act5C was studied. While the previous ChIPs demonstrated Drosophila IMPDH is present at the promoter of genes transcribed by RNA polymerase II, it is unclear if IMPDH also travels with the transcription machinery down the gene as it does in yeast [12]. Another ChIP experiment was preformed, this time using primers in real-time PCR that correspond to an exonic region approximately 2 kb downstream of the promoter in Act5C. The enrichment at each region was calculated using the 2-∆Ct method as before. It was found that, similar to the promoter, IMPDH is enriched at the 2 kb downstream region (Figure 7). The amount of enrichment between 22

trials is the same within the error of the experiment, however the variability between trials is high. The Ct of the input (i.e. the cycle number at which the concentration of total

DNA passes the threshold) varies between biological replicates. This suggests that the efficiency of crosslinking and shearing the DNA fluctuates. Regardless, it is clear that

IMPDH is at both sites in an actively transcribing gene, indicating that it may play a role in initiation and travels with the RNA elongation complex.

IMPDH recruitment increases with transcriptional activity

While Park and Ahn demonstrated that IMPDH is recruited to constitutively transcribing genes, it was unknown whether an increase in transcription causes an increase in IMPDH recruitment to the gene. In order to determine the effect of an increase in transcriptional activity on the enrichment of the enzyme at a gene, the copper- inducible Metallothionein A (MtnA) gene was studied. Upon a four hour incubation with

500 µM Cu2+, transcription levels of the gene increase 100-fold in response to the metal

[32]. Since IMPDH appears to play a role in transcription, an increase in activity should result in an increase in IMPDH recruitment. The enrichment at the MtnA promoter of the copper treated and untreated cells was normalized to the enrichment of the 28S promoter for those samples. From three biological replicates, IMPDH is two-fold enriched at MtnA upon transcription activation with copper (Table 5). This result is similar to the increase in enrichment of MTF-1, a known transcription factor that induces transcription of MtnA upon exposure to metal. When the ChIP was performed using anti-MTF-1 antibodies, the increase of MTF-1 recruitment to MtnA upon copper induction was also approximately

two-fold (Table 4). This suggests that IMPDH recruitment is similar to that of a well established transcription factor.

Table 4. MTF-1 ChIP experiment. S2 cells were treated with Cu2+ for 4 hours, and ChIP was performed using 5 µL of anti-MTF-1 antibody purified by M. Marr. This data is normalized for copy number of 28S, assuming a copy number of 200.

untreated = 13000

Cu2+ induced = 29600

fold change = 2.3

RNA does not mediate interaction between IMPDH and transcribing genes

Both in vivo and in vitro studies show that IMPDH associates with RNA [6, 10].

Mortimer et al. showed that adding RNase disrupts the association between IMPDH and polyribosomes, implying that RNA mediates the interaction. To determine if the association of IMPDH and transcribing genes is also mediated by RNA, RNAse was omitted from the chromatin preparation. If RNA were to play an important role in the interaction between the enzyme and DNA, an increase in the amount of immunoprecipitated gene is expected. The above experiment was repeated in S2 cells that were not treated with RNase in the chromatin preparation or IP. Similar results were observed as previously; there is approximately a two-fold enrichment of IMPDH at MtnA upon transcription activation (Table 5). Thus, RNA does not mediate the interaction between IMPDH and actively transcribing genes.

Table 5: IMPDH ChIP experiments. S2 cells were treated with Cu2+ for 4 hours, sufficient to induce a 100-fold increase in transcription of MtnA. This data represents the average of 3 biological replicates and is normalized for copy number of 28S, assuming a copy number of 200.

with RNase without RNase

untreated = 178 ± 180 266 ± 140

Cu2+ induced = 420 ± 260 560 ± 300

fold change = 2.4 ± 1.2 2.1 ± 0.8

IMPDH in human cell chromatin

The above experiments demonstrate that the canonical IMPDH is located in chromatin and plays a role in transcription in D. melanogaster cells. However, it is not yet clear whether IMPDH is involved in transcription in human cells as well. In order to begin to answer this question, the chromatin of human tissue culture cells was probed for the presence of IMPDH. First, plasmids containing GFP-tagged IMPDH546 and

IMPDH595 were transfected into HEK293T cells. The chromatin was isolated and analyzed by Western blot with antibodies for IMPDH (Abcam 30339). Since the transfected IMPDH contains a GFP tag, it is easily discernable from the endogenous

IMPDH. Both GFP-tagged retinal isoforms and endogenous IMPDH co-purified with the chromatin, indicating that human IMPDHs also associate with chromatin (Figure 8). This association provides support for transcriptional activity of IMPDH in human cells, in addition to yeast and D. melanogaster.

Figure 8. Retinal isoforms IMPDH546 and IMPDH595, with GFP tags were transfected into HEK293T cells. Chromatin was crosslinked and isolated, and then analyzed by Western blot, probing for IMPDH (Abcam 30339).

Next, the experiment was repeated for three mutants of IMPDH546, two of which cause retinitis pigmentosa (H372P and T116W [33]) and one which causes Leber congential amaurosis, LCA, (N198K [34]), a more severe form of hereditary retinopathy. All three mutants were also found to also be located in the chromatin of HEK cells (Figure 9), suggesting that they may also be involved in transcription.

Figure 9. Mutants of IMPDH546 that are known to cause RP or LCA were transfected into HEK cells and the chromatin extract was probed for IMPDH (Abcam 30339).

Two of the mutants, N198K and T116W, contain another band between the transfected

IMPDH and endogenous IMPDH. It is not clear what that band is, but other members of the laboratory have noted the appearance of degradation products of retinal splice variants.

NAD+/NADH do not affect nucleic acid binding

Kumar et al. suggest that for CtBP, another protein recruited to actively transcribing genes with weak dehydrogenase activity, the binding of NAD+ or NADH induces a crucial conformational change for normal transcriptional activity. In general, it has been shown that NAD(H) play an important role in transcription regulation [35].

Perhaps NAD+ or NADH also affect IMPDH’s transcriptional activity, by changing its affinity for nucleic acids. A filter binding assay was performed to calculate the affinity of

IMPDH for a random pool of single stranded DNA in the presence of NAD+ or NADH.

IMPDH1 was used in assays because it is the most abundant form in the retina. The

+ concentration of NAD and NADH in the assay was 500 µM, which is ten times the Km of NAD+ and IMPDH1, to ensure that the enzyme is fully saturated [5]. The results of the filter binding assay are shown below (Figure 10). The fraction of nucleic acid bound was calculated by dividing the volume of the dot on the nitrocellulose membrane by the total volume on the nitrocellulose and PVDF membranes (Table 6).

Figure 10. Filter binding assay of 10nM IMPDH incubated with 500 µM NAD+/NADH and a pool of 2nM random ssDNA. A) Nitrocellulose membrane blot, which traps protein complexes. B) PVDF membrane, which traps free nucleic acid.

Table 6. Quantification of filter binding assay. Intensities were calculated using ImageQuant v2005 and the fraction bound was determined by dividing the volume on the nitrocellulose membrane by the total volume (nitrocellulose and PVDF) for that well. The data represents the average of duplicates. Fraction Bound no protein (negative control) 1.3 ± 0.5 % hIMPDH2 (positive control) 30 ± 17 % hIMPDH1 87 ± 4 % IMPDH1 + NAD+ 91 ± 2 % IMPDH1 + NADH 88 ± 3 %

As shown in the table above, the fraction bound of nucleic acid is the same within error when NAD+ or NADH are added to IMPDH1. However, all of these enzymes are almost saturated with nucleic acid, so small changes in affinity may not be detected in this assay. To further determine whether NAD+ or NADH cause an increase in affinity of

IMPDH1 for nucleic acid, the filter binding assay should be repeated varying the concentrations of NAD+ and NADH or by lowering nucleic acid concentration. Even without the addition of NAD+/NADH, the enzyme binds most of the ssDNA pool. By titrating NAD+ and NADH, it would be easier to see if the cofactors affect nucleic acid binding. An increase in NAD(H) should cause a higher percentage of DNA bound if the cofactors affect affinity.

Discussion

This study was able to extend the finding by Park and Ahn that IMPDH is recruited to genes that are actively transcribing by RNA polymerase II in a higher organism, Drosophila melanogaster. Previous work in yeast suggests that IMPDH associates with all genes constitutively transcribed by RNA polymerase II. Yeast IMPDH and D. melanogaster IMPDH show 58% sequence identity, which suggests that all

IMPDHs will exhibit this activity. To confirm the finding, two representative genes,

Actin 5C and Ribosomal 28S, which are transcribed by RNA polymerase II and III, respectively, were tested in a chromatin immunoprecipitation using anti-IMPDH antibodies. The ChIP results indicate that IMPDH is at least 200 times more enriched at the Act5C promoter than the 28S promoter, when the copy number of 28S is taken into account. Therefore, IMPDH is preferentially recruited to genes transcribed by RNA polymerase II.

Park and Ahn also suggested that IMPDH associates with downstream regions of active genes, in addition to promoters. To test this in D. melanogaster, ChIP experiments were preformed and analyzed at the Act5C promoter and an exon 2 kb downstream of the promoter. While the relative amount of IMPDH at each location was not determined, it is clear that it is enriched in both places. This evidence supports the model that IMPDH travels down the gene with the transcription complex and plays a role in elongation.

While Act5C experiments demonstrated that IMPDH associates with a gene that is constitutively transcribed, it is not known if IMPDH levels increase with transcriptional activity. Park and Ahn examined yeast genes that are constitutively active or repressed.

This study analyzed an inducible gene, Metallothionien A (MtnA). Upon transcription activation with metal, the promoter region of the gene demonstrated a two-fold enrichment of IMPDH. This can be compared to recruitment of MTF-1, a known transcription factor. Upon transcription induction with copper, the enrichment of MTF-1 at the MtnA promoter increases three-fold. Since IMPDH recruitment is comparable to that of a known transcription factor, it provides further support that IMPDH plays a role in transcription.

Interestingly, RNA does not appear to play a role in the interaction between

IMPDH and the transcription complex. Previous studies have shown that IMPDH binds to RNA in vivo. It is reasonable to suspect that IMPDH also interacts with transcriptional machinery through mRNA, and perhaps even carries the mRNA to the cytoplasm.

However, ChIP experiments suggest that the association between IMPDH and transcribing genes in the nucleus is not mediated by RNA. When ChIP experiments were repeated with RNase treatment to degrade the RNA, the extent of IMPDH enrichment at the promoter did not change. While it is known that IMPDH binds to nucleic acids in vivo, the role it plays in transcription does not appear to take place through an interaction with RNA. Park and Ahn suggest that IMPDH interacts with the CTD of RNA polymerase in yeast, so it is possible that IMPDH does not directly bind to transcribing genes. In fact, since IMPDH is enriched at the promoter and downstream regions of actively transcribing genes, the simplest model is that it associates with RNA polymerase

and moves with the transcription machinery. However based on current data, it cannot be excluded that IMPDH binds to nucleic acids directly because nucleic acids co-purify with

IMPDH.

In addition to association with D. melanogaster chromatin, IMPDH is also found in the chromatin of human cells. The fly IMPDH is most identical to canonical IMPDH1

(65% identity) and does not appear to have segments corresponding to the additional N- and C-terminal extensions in the retinal splice variants. Both retinal splice variants localize to chromatin when transfected into HEK293T cells. RP- and LCA-causing mutants of IMPDH546 also appear to have no effect on the association of IMPDH with chromatin, however subtle perturbations would not have been detected in the present experiments. Two of the mutants are known to cause retinitis pigmentosa (H372P and

T116W [33]), but one of the mutants causes LCA (N198K [34]), a more severe form of retinopathy. Future studies should repeat these experiments for RP- and LCA-causing mutations in IMPDH595, which cause a larger perturbation in polyribosome binding

[10]. It is interesting to note that of the three mutants, H372P is the only one that is not located in between the catalytic and CBS domains – it is found entirely on the catalytic domain. Yet, it is still associated with chromatin. Further studies should investigate this observation to verify this finding. In addition, the percent of each IMPDH localized to chromatin was not determined in this study, and future studies will compare the chromatin extracts to the total cell to determine the percent associated with chromatin.

This may provide some clues as to whether RP-causing mutants affect this association, and thus serve as a mechanism of retinitis pigmentosa. While HEK293T cells are better for studies than D. melanogaster S2 cells because they are human cells, they are not an

ideal model to investigate a retinal disease. Photoreceptor cells express vision specific proteins and have unique transcriptional activity to those cells. Of the approximately

1390 transcription factors in humans, 25% of them exhibit tissue-restricted expression patterns [36]. While there are no exclusively eye-specific transcription factors, several vision diseases result from a loss-of-function of a transcription factor [36]. While

HEK293T cells are not the perfect model for photoreceptor cells, they are still suitable for studies of various effects of IMPDH isoforms and mutants in transcription association. The basic studies will provide a model to then test in retinas.

The evidence strongly suggests that IMPDH is involved in transcription, although its exact role remains unclear. Since the enzyme’s main catalytic function is the production of guanine nucleotides, it is possible that it travels with the transcription complex to serve as a local source of nucleotides for actively transcribing genes.

However, since there are several more steps required to convert xanthosine 5’- monophosphate to GTP, it is unlikely. Other dehydrogenases found to play a role in transcription, such as CtBP, regulate transcription by recruiting other factors, in this case, corepressors that modify chromatin structure. The dehydrogenase function and transcriptional activity appear to be separated, and that NAD(H) binding cause a necessary conformational change to allow transcription regulation to occur. It is possible that IMPDH functions in a similar way, with catalytic activity not being a requirement for transcriptional activity. To probe for the role of enzymatic activity, an inhibitor of

IMPDH (such as mycophenolic acid) could be added to growing cells and ChIP experiments repeated to determine if association changes. The cell media should also be supplemented with guanosine to ensure that changes in the cell do not arise from the

depletion of guanine nucleotides. The overall transcription levels could also be measured to determine if IMPDH inhibition changes the rate of transcription in the cell. After mycophenolic acid treatment, the total RNA should be isolated and converted to cDNA by RT-PCR. The resultant cDNA should be analyzed by PCR to determine if transcription levels in the cell decrease. Another way to determine the function of the active site in transcription is to transfect catalytically inactive mutants, and then characterize the association to actively transcribing genes. To differentiate between the transfected mutants and endogenous IMPDH, the mutants should have a tag (i.e. a GFP tag) or the endogenous IMPDH should be knocked down with double stranded IMPDH.

These experiments would elucidate whether IMPDH’s catalytic properties are involved in its recruitment to actively transcribing genes.

In addition to the unknown function of IMPDH at transcriptionally active genes, it is also not clear how the protein interacts with the DNA and transcription machinery.

Future studies should further explore the physical interaction between IMPDH and transcribing genes. Endogenous IMPDH will be knocked down and structural mutants will be introduced into cells and the enrichment of the enzyme at actively transcribing genes will be measured. Since previous studies show that IMPDH most likely binds nucleic acids between the CBS domain and the catalytic domain, it is possible that the interaction with transcribing genes occurs in that region as well. Mutants to be tested include deletions of the CBS domains, active site mutants, and RP-causing mutants. ChIP experiments should be carried out and enrichment of the transfected IMPDH protein will be compared to endogenous IMPDH. Also, total mRNA levels in the cell should be quantified in order to determine the effect of these structural mutants on the overall rate

of transcription. These experiments will shed light on the structural components of the enzyme that are involved in its transcription activity, as well as the effect of disease causing mutants on that activity. This insight may reveal the mechanism of IMPDH- linked retinitis pigmentosa. Since photoreceptor cells are among the most actively transcribing cells, IMPDH’s transcription activity serves as an attractive model for pathogenesis. A better understanding of the transcription function of IMPDH will help further elucidate the role it plays in the cell, thus potentially allowing for necessary treatment for IMPDH-linked retinitis pigmentosa.

References

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Appendix

I. Purification of MBP-tagged extensions of retinal splice variants of IMPDH

The N- and C- terminal extensions of retinal splice variants of IMPDH, exon A and exon 13b, respectively, are cloned into pMAL-c2X vectors (NEB N8706). The vectors are ampicillin resistant. Each contains a maltose binding protein (MBP) tag on the N-terminal of the extension, connected by a linker with a cleavage site by Factor Xa. The plasmids are stored in the -20oC freezer in a boxed labeled “Julia”. The plasmids are labeled “pMAL-exA” and “pMAL-ex13b”. An additional plasmid exists that contains just the MBP tag, and is stored in the same location with the label “pMAL”. a) Expression: Transfect into competent cells and plate on ampicillin plates. Innoculate a colony for an overnight starter culture (5 mL) at 37oC. Add 1 mL of starter culture to 500 mL o of LB with ampicillin. Grow at 37 C until A600 reaches 0.6 and induce with 1 mM IPTG. Shake for another 3 hours. Harvest cells by centrifugation. b) Purification: Purify MBP-tagged extensions on the FPLC using the 5 mL MBP column (stored in Fridge 1). Resuspend pellet from 500 mL of culture in 20 mL of MBP Wash Buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM DTT). Sonicate in the cold room and pellet cell debris. The supernatant can be ultracentrifuged for 30 minutes at 80,000 rpm (4oC) to remove membrane lipids and keep the MBP column from clogging up. The supernatant should then be passed through a 0.22 µm filter.

After washing the FPLC and column (milliQ and then MBP Wash Buffer), turn on the HiTrap MBP protocol under the “Guest” login. Prepare Elution Buffer (MBP Wash Buffer + 10 mM maltose). Place tube B1 into Elution Buffer.

HiTrap MBP Protocol: 1. equilibrate with 2 CV 2. empty loop 3. wash with 15 CV (1.5 mL/min) 4. elute with 5 CV (2 mL/min)

The protein mainly elutes into fractions B5 and B6.

To clean the MBP column, wash with 15 mL of 0.4 NaOH and store in 20% ethanol.

II. Filter Binding Assay of IMPDH and MBP-tagged extensions

Follow the same filter binding assay protocol as described in Materials and Methods. Various filter binding assays can be found in Notebook 1:

a. Add both extensions to IMPDH1 in trans (page 34). [IMPDH1] = 40 nM (5 times the Kd) [MBP-ex] = 160 nM or 400 nM of each

Fraction Bound 40 nM IMPDH only 0.6% 160 nM of each MBP-extension 0.7% 40 nM IMPDH + 160 nM each MBP-extension 0.7% 40 nM IMPDH + 400 nM each MBP-extension 0.6%

b. Repeat experiment (a) with a higher concentration of IMPDH1 (page 47). [IMPDH1] = 90 nM [MBP-ex] = 360 nM or 900 nM of each

Fraction Bound no protein 1.2% 90 nM IMPDH1 only 5.4% 900 nM of each MBP-extension 1.1% 90 nM IMPDH + 360 nM each MBP-extension 3.7% 90 nM IMPDH + 900 nM each MBP-extension 3.9%

c. Test each extension individually for nucleic acid binding (page 64). [IMPDH 1] = 100 nM [MBP-ex] = 400 nM

Fraction Bound no protein nd 100 nM IMPDH1 only 5.6% 400 nM MBP-ex13b 1.3% 400 nM MBP-exA 1.2% 400 nM MBP 0.6% 400 nM MBP-ex13b + 400 nM MBP-exA 0.8% 100 nM IMPDH1 + 400 nM MBP-extensions 3.2% 100 nM IMPDH1 + 400 nM MBP 3.3%