Epigenetic reprogramming byCytomegalovirus infection

Marjan Mehrab Mohseni

Degree project in biology, 2007Examensarbete i biologi, 20 p, 2007Biology Education Center, Uppsala University, Department of Clinical Neurosience, Center forMolecular Medicine, Karolinsks Institute,StockholmSupervisors: Prof. Tomas Ekström and Mohsen Karimi

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Table of Contents Summary………………………………………………………………….…………………..3

Abbreviations……………………………………………………………………………...….4

Introduction…………………………………………………………………………………...5

DNA methylation…………………………………………………………………..…..5

Histone modification……………………………………………………………..…….6

Cytomegalovirus………………………………………………………………...……...7

Cytomegalovirus and cancer…………………………………………………...……….8

Cytomegalovirus and Epigenetics……………………………………………………....8

Confocal microscopy…...……………………………………………………………....9

Aim of study…...……………………………………………………………….……...10

Results.......................................................................................................................................11

Human cytomegalovirus effects on the methylation…………………………………………..11

Quality and quantity control of the pIE-86 plasmid…………………………………………...14

Discussion…………………………………………………………………………….……….15

Materials and methods.............................................................................................................17

MRC5 cell culture and CMV infection………………………………………………...17

Macrophage cell culture and CMV infection……………......………………………....17

Staining .…………….……………………………………………………..............…...17

Confocal microscopy…….….………...…………...………………………….………..18

Bacterial culture and plasmid amplification.......……………………………………….18

Plasmid purification…………………………………………………………………….19

Agarose gel electrophorasis…………………………………………………………….19

Restriction enzyme analysis…………………………………………………………….19

Acknowledgment……………………………………………………………………………….20

References....................................................................................................................................21

The picture on the cover is adapted from (http://pasteur.crg.es) with permission from the copy-

right holder.

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Summary

Epigenetics denotes heritable changes affecting gene expression that do not involve changes in primary DNA sequence. Studies of the molecular basis of epigenetics have largely focused on DNA methylation and histone modifications. All of these mechanisms act in concert to provide stable and heritable control of gene expression in higher eukaryotic genomes. DNA methylation is performed by adding a methyl group from a methyl donor to the fifth position of cytosine bases by DNA methyltransferase enzymes (DNMTs).

Viruses like Human Cytomegalovirus (HCMV) exploit the genomic machinery of the host for its own replication. HCMV infection is also associated with different types of cancer, like neuroblastoma, glioblastoma, colon carcinoma, prostate adenocarcinoma, cervical carcinoma and Wilms tumor.

This study was aimed to identify epigenetic alteration in the host by HCMV. In order to achieve this, immediate early (IE) proteins of HCMV (IE-1 & IE-2) and DNMT1 were analyzed in CMV- infected cells by immunofluorescense, and cells were observed by confocal microscopy to monitor the amount and intracellular localization of DNMT1 before and after infection.

I found that HCMV infection caused DNMT1 to move out of the nucleus, the movement of DNMT1 out of the nucleus suggests reduced DNA methylation of the host genome in both HCMV infected Human embryonic lung fibroblast cells (MRC5) and macrophage cells. Abnormal methylation patterns such as global hypomethylation are observed in tumorogenesis. In addition there is some evidence showing association between certain types of viruses and cancer. Therefore, according to this result, it could be speculated that a change of intracellular localization of DNMT1 resulting from HCMV infection provide a possible mechanism for abnormal DNA methylation observed in tumors. Understanding the exact mechanism of abnormal methylation through viral infection will have important consequences for the development of epigenetic therapies against cancer, as well as better understanding of tumor biology.

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Abbreviations BSA Bovine Serum Albumin DMEM Dulbeccos Modified Eagle Medium DNMTs DNA methyltransferase enzymes FBS Fetal Bovine Serum HAT Histone acetyltransferases HCMV Human cytomegalovirus HDAC Histone deacetylases (HDAC) HLA Human Leukocyte Antigen IE Immediate early IMDM Iscove’s Modified Dulbecco’s Medium LUMA LUminometric Methylation Assay MBD Methylated DNA-binding proteins Mecp2 Methyl-CpG-binding protein 2 MOI Multiplicity of infection PBMCs Peripheral blood mononuclear cells SAM S-adenosylmethionine WT Wild Type

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Introduction

Epigenetics can be defined as changes in gene expression or cellular phenotype that occurs without changes in DNA sequence. Epigenetics could be considered as a bridge between genotype and phenotype, and is in fact a phenomenon that changes the final outcome of expression from a locus or chromosome without changing the underlying DNA sequence (Goldberg et al., 2007). Since histone modification and DNA methylation patterns affect gene expression and are heritable through mitosis, DNA methylation and histone modifications are referred to as epigenetic mechanisms (Lu et al., 2006).

DNA methylation

DNA methylation is perhaps the most investigated chemical modification of chromatin. In vertebrates, DNA methylation entails transferring of a methyl group from the methyldonor S-adenosylmethionine (SAM) to cytosine bases at position C5 by a DNA methyltransferase (DNMT). In mammals, most DNA methylation occurs on cytosine residues in the CpG dinucleotide sequence. These sequences frequently occur in CpG islands, which are regions of high CpG densities and are associated with genes, particularly housekeeping genes. The human genome has revealed the presence of about 29,000 CpG islands (Lander et al., 2001) and it has been shown that about 60% of the human genes have associated CpG islands. These CpG islands are located in or near the promoter of genes, which remain non-methylated in normal cells. DNA methylation of these islands correlates with transcriptional repression (Yamashita et al., 2005).

In mammals there are five known DNMT family members. DNMT 1, 2, 3a, 3b, and 3l. DNMT1 is the main abundant enzyme of the family and has high affinity for a hemimethylated DNA, it is therefore called the main maintenance DNMT (Santos et al., 2005). Maintenance methyltransferases add methyl groups to hemi-methylated DNA during DNA replication, whereas the activity of de novo DNMTs does not depend on DNA replication. DNMT3a and DNMT3b are mainly involved in de novo methylation. They are therefore important for the establishment of new methylation patterns of the genome during differentiation and in cancer. Weak DNA methylation activity of DNMT2, another DNA methyltransferase, was recently shown, but its biological role remains elusive (Hermann et al., 2003).

Aberrant DNA methylation patterns, i.e. global hypomethylation and local hypermethylation, are commonly found in cancerous cells (Jones et al., 2002). Promoter CpG island hypomethylation can result in increased gene expression of oncogenes (Attwood et al., 2002). In addition, hypomethylation is believed to result in genomic instability (Feinberg & Vogelstein 1983)

In addition to hypomethylation, aberrant hypermethylation of CpG islands can lead to inappropriate gene silencing. For instance, methylation of tumor suppressor gene CpG islands leads to their silencing, and may predispose to carcinogenesis (Attwood et al., 2002). Hypermethylation of MLH1 (a DNA mismatch repair gene) also causes transcriptional inactivation, which results in genomic instability (Mukai &Sekiguchi, 2002).

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Figure 1. The effect of DNA methylation on gene regulation. Transcription is suppressed in promoter regions containing methylated CpGs that are bound by MeCP2 as shown in the left panel. MeCP2 binds methylated DNA and recruits chromatin-remodelling complexes that contain SIN3A (a transcriptional co-repressor), and HDACs. This leads to chromatin condensation owing to histone deacetylation, which results in a limited accessibility of the transcriptional machinery to promoter regions. When MeCP2 is not bound to methylated DNA (right panel), the complex is not recruited. Therefore, histones remain acetylated and the DNA at the promoter remains in an open conformation, allowing transcription factors to bind DNA and initiate transcription. (Adapted From Bienvenu & Chelly, 2006 with permission from the copy-right holder). Histone Modification

Chromatin organization has an important role in gene regulation by modifying the tertiary structure to an open or accessible (euchromatin) status, or to a tighter and inaccessible configuration (heterochromatin). Chromatin structure plays a key role as a mediator between DNA methylation and the transcriptional silencing of genes observed in cancer.

The basic building block of chromatin is the nucleosome, consisting of 146 bps of DNA wrapped around a protein core. This protein core consists of eight subunits, two each of histones H2A, H2B, H3, and H4. Adjacent nucleosomes are connected by a short stretch of DNA, and resemble beads on a string. The DNA in chromatin assembled in vitro is tightly compacted and in its native configuration it is inaccessible to transcription initiation complexes (Lu et al., 2006). However, the N-terminal tails of the histones are extensively modified by acetylation, methylation, phosphorylation, and ubiquitination. Among these, acetylation and deacetylation are very important to determine accessibility of DNA to transcriptional factors. Acetylation is catalyzed by histone acetyltransferases (HAT), and deacetylation is catalyzed by histone deacetylases (HDAC). In general, increased level of histone acetylation (hyperacetylation) is found in euchromatin, and decreased levels of acetylation (hypoacetylation) are found in the tightly compacted chromatin (heterochromatin) (Herranz et al., 2007).

A correlation has been found between the DNA methylation state and histone modification. Usually, methylated genes are associated with deacetylation and hence inactive chromatin. DNA methylation recruits a group of proteins called methylated DNA-binding proteins (MBD) such as methyl-CpG-binding protein 2 (Mecp2). This protein in turn recruits histone deacetylase and other proteins that interfere with binding of transcription factors and RNA polymerase to the promoter region and subsequent gene silencing. In the hypomethylated form of DNA which is associated with hyperacetylation of histones, transcription factors and RNA polymerase can

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easily bind to the promoter sequence and this leads to gene transcription, as shown in figure 1 (Turek-Plewa & Jagodzinski, 2005). Cytomegalovirus

Human cytomegalovirus (HCMV) is a beta-herpes virus that causes widespread, persistent human infection. Other members of the herpes virus family cause chickenpox, infectious mononucleosis, fever blisters (herpes I) and genital herpes (herpes II). Herpes viruses have a unique four-layered structure (Figure 2). A core containing the large, double-stranded DNA genome, with an approximate size of 200-250 kbp, is enclosed by an icosapentahedral capsid which is composed of capsomers. The capsid is surrounded by an amorphous protein coat called the tegument. It is encased in a glycoprotein-bearing lipid bilayer envelope like other herpes viruses (Whitley 1996). CMV infection can become dormant for a while and may reactivate at a later time. HCMV does not usually cause clinically obvious disease upon primary infection of an immunocompetent individual, but clinical disease is much more prevalent in immunosuppressed patients especially patients suffering from AIDS (Sinclair& Sissons, 2006).

Three classes of viral genes are expressed in a permissive cultured cell infected by human cytomegalovirus; immediate early (IE), early, and late. There are two IE proteins (IE1 & IE2) that have overlapping sequence. The immediate early 72-kDa IE1 and 86-kDa IE2 proteins are the first proteins that are expressed at the start of infection.

Figure 2.Schematic view of cytomegalovirus (Adapted from http://www.biosciences.bham.ac.uk/labs/sweet/cytomegalovirus.htm) with permission from the copy-right holder)

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Expression of IE genes is required for progression into the early phase of the infectious cycle and the subsequent replication of viral DNA. Early genes encode enzymes for replicating viral DNA while late genes encode structural proteins.

An interesting biological aspect of HCMV is the ability of the virus to establish persistence within the host following asymptomatic infection, which is called latency stage. This strategy, used by all herpes viruses to persist in the infected individual, is the establishment of cellular sites of viral latency: virus persists at specific sites in the host, but in the absence of any detectable production of infectious virus (Sinclair& Sissons, 2006).

Human cytomegalovirus uses different ways to escape the immune system. One of these mechanisms is down-regulation of expression of surface Human Leukocyte Antigen (HLA) class I. HCMV also can block the presentation of the immediate early proteins, one of the first viral proteins to be produced, to hide itself from immune system (Michelson et al., 1999). These ways permit the cytomegalovirus to persist in the host, but apart from the immune evasion mechanisms, HCMV exploits the expression and replication machinery of the host for its own replicative purposes.

Cytomegalovirus and cancer

Detection of viral DNA, mRNA and/or antigens in tumor tissues as well as seroepidemiologic evidence, has shown the importance of HCMV infection in the etiology of several human malignancies like colon carcinoma, prostate adenocarcinoma, cervical carcinoma, Wilms tumor, neuroblastoma and glioblastoma (Cinatl et al., 2003).

HCMV-mediated inactivation or down-regulation of tumor suppressor proteins controlling the cell cycle pathways has already been shown. For instance IE2 has been shown to interact with p53, which leads to the down-regulation of p53 transactivation function (Speir et al., 1994; Tsai et al., 1996). Also HCMV infection has been reported to induce degradation of p21Waf1 (Chen et al., 2001), which is an inhibitor of the G1/S phase of the cell cycle.

Interactions between HCMV and cellular tumor suppressor proteins as well as stimulation of S-phase genes that are important for HCMV replication result in the inactivation of checkpoints in the cell cycle, giving rise to uncontrolled cellular DNA replication and transformation that can lead to cancer (Jault et al., 1995; Bresnahan et al., 1996; Castillo et al., 2000). HCMV has positive effects on cell proliferation proteins. Among many factors that are up-regulated during HCMV infection, the proto-oncogenes c-myc, c-fos, and c-jun are rapidly activated in infected cells (Boldogh et al., 1990).

As HCMV infection is observed in tumor tissues, it is suggested that treatment of HCMV-positive cancer patients with antiviral drugs abolishing HCMV-infection may be beneficial to suppress tumor progression.

Cytomegalovirus and Epigenetics

There is still not much known about the epigenetic factors of host cells that could be

altered by HCMV, but viral proteins may regulate epigenetic modifications in virus-infected cells. Once delivered to cell nuclei, the genomes of all herpes viruses, including HCMV, are packed into nucleosomes. Therefore, it is logical to propose that histone modifications also may play a critical role in the regulation of viral gene expression. Indeed, it was shown that

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transcribed regions of genomes of different herpes viruses become associated with acetylated histones (Ioudinkova et al., 2006).

Some studies have shown that HCMV IE1, and probably IE2 as well, promote viral transcription by antagonizing histone deacetylation. The major IE promoters were found to be hypoacetylated in IE1 deficient mutant virus as compared to Wild Type (WT) virus-infected cells. In addition the expression of viral mRNA was reduced in mutant infected-cells, and it was found that HDAC inhibitors enhanced the replication of an IE1-deficient mutant HCMV, but did not enhance the replication of WT virus (Nevels et al., 2004).

Virus infection, especially with DNA viruses and retroviruses, that may cause insertion of viral DNA sequence into the host genome, often triggers host defense mechanisms, particularly the DNA methylation machinery, to cause methylation of the foreign viral genome. DNA methylation of these viral DNAs is an effective method to silence viral gene expression (Li et al., 2005). Therefore it is reasonable to consider viral mechanisms to inhibit DNA methylation machinery as part of their replication process.

Confocal microscopy

Confocal microscopy is a fluorescence microscopy technique that combines different technologies like laser, computer and microelectronics to produce very high resolution images. In a conventional fluorescence microscope, signals come from the full thickness of the specimen where most of it is out of focus. Confocal microscope is able to eliminate out-of-focus light (light from above and below the plane of focus of the object). A pinhole is located in front of the detector (see figure 3) which acts as a spatial filter and allows only the in-focus portion of light to pass.

In the confocal microscope, lasers are used as light sources instead of the conventional mercury arc lamps because they produce extremely bright light at very specific wavelengths for fluorochrome excitation. Another useful advantage of confocal microscope is the ability to show colocalizations of signals from different fluorochromes.

Figure 3. Schematic view of a simple laser scanning confocal microscope. (Adapted from http://www.microscopyu.com with permission from the copy-right holder)

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In specimens doubly-labeled for different molecules or structures, different channels can collect different fluorochromes to make color images; there is also the possibility to make three dimensional pictures by confocal sectioning.

Aim of Study

Previous results in this laboratory had shown that HCMV infection induces global hypomethylation of DNA. The main objective of this study was to investigate the mechanisms of this hypomethylation. Thus, changes of DNMT1 were assayed by fluorescence immunocytochemistry both in human MRC5 fibroblast cell lines and in human macrophages.

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Results Human cytomegalovirus effects on the methylation

In order to investigate the effect of HCMV on epigenetic factors (here I mostly focused on methylation), MRC5 fibroblast cell lines and macrophage cells were infected with HCMV. The nucleus, DNMT1, and IE86 were fluorescently labeled with an immunohistochemical staining method using different fluorophores. Different dyes had different emission and excitation wavelengths. Using confocal microscopy, I was able to observe the effect of CMV infection on DNMT1 localization in the cell.

Figure 4 shows result from an experiment where DNA was stained blue with DAPI (panel A), and DNA methyl transferase DNMT1 was stained red with a Texas Red coupled antibody (panel B). When the two images were superimposed (panel C), a pink color shows the areas where both dyes were present. This shows that DNMT1 is a nuclear protein, as expected since that is where the DNA is located. Figure 5 shows result from infected MRC5 fibroblast cells. DNA was stained blue with DAPI (panel A), IEs were stained green with an Alexa488 coupled antibody (panel B), and DNMT1 was stained red with a Texas red coupled antibody (panel C). When the three images were superimposed (panel D) it was observed that in the CMV-infected cells (as observed as green nuclear staining of IE) the nucleus was empty of DNMT1 (red). These results were confirmed in CMV infected macrophages as well (Figure 6), except that infection rate in macrophages was much lower than infection rate in MRC5. A. B. C.

Figure 4. Nuclear localization of DNMT1 in uninfected MRC5 cells. A) The nuclei of MRC5 cells were stained with DAPI (blue). B) The same MRC5 cells stained using an antibody against DNMT1 (red). C) superimposed image of A and B, Observed at 400 x magnification.

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A. B.

C. D.

Figure 5. Changes in DNMT1 localization caused by CMV infection in MRC5 cell – MRC-5 cells were infected with HCMV. At day 3 post-infection cells were stained and observed by confocal microscope. A) The nuclei of MRC5 cells were stained by DAPI (blue color). B) The same MRC5 cells stained using an antibody directed against IEs (green). C) The MRC5 cells stained using an antibody directed against DNMT1 (red). D) Superimposed image of A, B, and C observed at 400x magnification.

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A. B.

C. D. Figure6. Changes in DNMT1 localization caused by CMV infection in human macrophges: Human macrophages were infected with HCMV. At day 3 post-infection cells were stained and observed by confocal microscope. A) The nuclei of macrophages were stained by DAPI (blue color). B) The same macrophages stained using an antibody directed against IEs (green). C) The macrophages stained using an antibody directed against DNMT1 (red). D) Superimposed image of A, B, and C observed at 400x magnification. Two nuclei at the bottom of images belong to uninfected cells.

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Quality and quantity control of the pIE-86 plasmid

Since high quality of plasmid is essential for cell culture transfection, the quality and quantity of pIE-86 plasmid were analyzed with different methods.

The results showed an A260 OD 36.75, and a ratio of A260/A280 OD 1.88. The concentration of the DNA was 1800 ng/ul.

Figure 7 shows the result of the digested and undigested purified plasmid DNA. Two bands were observed for purified plasmid; supercoiled, and circularly closed forms of IE-86 plasmid.

There is one restriction site in pIE86 for EcoRI and BamHI therefore pIE86 is linear after digestion with EcoRI or BamHI. After double digestion with BamHI + EcoRI, two bands are observed one of them corresponds to plasmid backbone, and the small one is IE-86 coding sequence. This plasmid is going to be transfected in MRC5 cells for further study to investigate the specific role of IEs in DNMT1 translocation. We are planning to do co-transfection studies of pIE-86 with pDNMT1-red in MRC5 to prove if change of DNMT1 localization is the direct effect of immediate early protein.

Figure 7. Electrophoresis of purified IE86 plasmid and restriction enzyme digestion

Bam

HI+E

coRI

EcoR

I B

amH

I U

ncut plasmid

MW

M

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Discussion

Impact from the environment can, and should, influence gene expression through alterations of the epigenetic mechanisms. Pathogens, like viruses, likely influence the host cell in order to activate replication, transcription and translation of their viral genome. In addition, virus may need to evade the host immune system. In order to achieve this, the host epigenome needs to be reprogrammed to serve the virus, and it needs to be altered to facilitate virus production. DNA methylation is a part of host defense mechanisms, and also serves as regulator of gene expression (reviewed by Li et al., 2005).

In my experiments, it was found that wherever cells were infected with HCMV, and IE2 was observed in the nuclei, the nuclei of the cells were empty of DNMT1. This result was observed in both infected MRC5 cells and infected machrophage cells.

This result supports the hypothesis that infection of cells with HCMV is associated with changes in intracellular localization of DNMT1 suggesting global hypomethylation. This is also in accordance with previous result from the group that HCMV infection was associated with reduced level of global DNA methylation measured by LUminometric Methylation Assay (LUMA) (Karimi et al, 2006). Since DNMT1 is a critical enzyme responsible for maintenance of correct methylation patterns during DNA replication, it is suggested that HCMV affects cellular mechanisms of methylation.

Gene regulation mediated by DNA methylation has been reported for both host cells and viruses. Latent viral genomes may be affected by DNA methylation in their host (Takacs et al., 2001). Genomes of DNA viruses, similar to their host genomes, are subject to chromatin-based repression and regulation. During lytic infection, the genomes of papovaviruses, adenoviruses, and herpesviruses are assembled into chromatin to various degrees of regularity of the nucleosome repeat structure (Huang et al., 2006). Herpes simplex virus recruits various histone modification enzymes, including HATs and ATP-dependent remodeling enzymes (Herrera et al., 2004), coordinately with the activation of the IE genes during acute infection. Histone acetylation is likely accompanied by a DNA hypomethylation state, which might indicate a similar mechanism to what was observed in my study as well.

In a study with murine CMV, it was found that treatment of a spleen explant model with the DNA methylation inhibitor 5-aza-2-deoxycytidine results in more rapid induction of immediate early gene expression, and reactivation of the virus (Hummel et al., 2007). This result suggests a role for methylation in the latency of the virus. Therefore, comparing with my results, it could be concluded that virus affects the cellular DNA methylation machinery by changing intracellular localization of DNMT1 to increase its own replication. Like the cellular genome, HMCV DNA is associated with histones (Nevels et al., 2004), and the possible reduction in level of methylation might be necessary for virus to counteract hypermethylation of CpG islands in the promoter region of some of its own genes, and some of the genes in the host genome which are induced and necessary for viral infection .

Abnormal methylation patterns have been observed that provide cells with a growth advantage, resulting in cancer (Jones & Baylin, 2002). Previous results are also indicative of abnormal methylation patterns after HCMV infection. It was found that active infection by CMV cause host genome hypomethylation with potential far going consequences, which might include pathway to neoplasia. As several tumor types are associated with viral infections, e.g. 100% association of glioblastoma cells with HCMV (Cobbs et al, 2002), association of cervical

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carcinoma with HPV, association of lymphoma with EBV, and association of hepatocellular carcinoma with HBV (Li et al., 2005), identification of mechanisms by which HCMV, and likely other viruses, disrupts the normal DNA methylation machinary in host cells might be helpful to find new therapeutic approaches, considering the availability of epigenetic drugs.

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Materials and methods MRC5 Cell culture and CMV infection

Human MRC5 embryonic lung fibroblast cells were cultured in Dulbeccos Modified Eagle Medium (DMEM) with 580 mg/l L-glutamine, 1000 mg/l D-glucose, containing 110 mg/l sodium pyruvate (Cat.No.31885 Gibco BRL). The medium was supplemented with 10% Fetal Bovin Serum (FBS), 1% streptomycin-penicillin. Cells were grown at 5% CO2 and 37° C in a humid incubator. Cells were cultured in eight –well culture slides (BD Falcon, Bcdford, MA). At 70% confluency cells were infected with CMV endothelial cell-adapted clinical isolate VR1814 (kindly provided by Professor Giuseppe Gerna, University of Pavia, Italy). Infected cells were incubated for 3 days before staining. Macrophage Cell culture and CMV infection

Peripheral blood mononuclear cells (PBMCs) from healthy blood donor were isolated by density gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). Lymphocytes were washed and reconstituted twice in sterile Hanks’ Balanced Salt Solution (Sigma-Aldrich Gmbh, Stein, Germany) and reconstituted in Iscove’s Modified Dulbecco’s Medium (IMDM), supplemented with L-glutamine (Gibco), 25 mM HEPES, 1% streptomycin-penicillin and 10% heat- inactivated human AB-serum. Cell were plated in eight-well chamber slides and incubated for 2h. Non-adherent cells were removed; remaining cells were stimulated during the next 24h for macrophage differentiation as described previously (Soderberg-Naucler et al., 2001), this part of experiment was done with kind support from Sodenberg-Naucler group in CMM , Karolinska Institute. After 24h, cells were washed in 1X Phosphate Buffered Saline ( PBS)(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) repeatedly and “60/30/10” medium (60% of Serum Free Lymphocyte Medium AIM V (Gibco), 30% of IMDM medium (Gibco) with the addition of L-glutamine, 25 mM HEPES, 1% streptomycin-penicillin, and 10% human AB-serum) was added to the cells which were cultured for 7 days at 37° C and 5% CO2. Macrophages were infected at a multiplicity of infection (MOI) of 10 for 96h with the HCMV (Strain VR1814) or non-infected. After incubation, cells were washed in 1 x PBS and fresh “60/30/10” medium was added to the cell. Staining (immunofluorescence)

Cells on eight-well slides were fixed with ice cold methanol:acetone 1:1 for 10 min and were air dried. After blocking with 1% bovine serum albumin (BSA) in PBS for 20 minutes, slides were incubated for 1 h at room temperature with a monoclonal mouse antibody specific for HCMV immediate early proteins (1/150 dilution in 1% BSA in PBS), and polyclonal rabbit antibodies specific for DNMT1 (1/50 dilution in 1% BSA in PBS) as shown in table 1. Following 3 washes in PBS for 5 min, antibody binding was visualized after incubation at room temperature for 60 min with donkey antimouse IgG antibodies conjugated to Alexa Flour 488 (1/500 dilution in 1% BSA in PBS), and a donkey antirabbit antibody conjugated to Texas Red (1/500 dilution in 1% BSA in PBS). Slides were washed 3 times for 5 min with PBS. Then, the cell nuclei were stained with DAPI (Sigma, USA), prior to mounting the slides with fluorescent

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anti fade mounting medium (Dako). Negative control slides were processed in the same manner, but omitting the primary antibodies. Slides were analyzed by fluorescence microscopy and confocal microscopy.

Table 1. Different antibodies used in immunofluorescence staining

Name Antigen Conjugated Dye

Company Dilution

Monoclonal mouse antibody

IEs - Argene Parc 1/50 Primary Antibodies

Polyconoal rabbit antibody

DNMT1 - H-300, Santa Cruz 1/50

Donkey anti-mouse antibody

mouse Ab

Donkey-Alexa 488 Molecular probe 1/500 Secondary Antibodies

Donkey anti-rabbit antibody

rabbit Ab Donkey –Texas red

Molecular probe 1/500

Confocal microscopy

In my investigation, I used a Leica TCS SP5 confocal laser scanning microscope with a 40 x, HCX PL APO NA plan-Apochromt oil immersion objective (Leica Germany). For Alexa 488 visualization, a 488-nm Ar laser was used as an excitation light, and green color was detected at the range of 494-567nm wave length from fluorescent specimens. For Texas red visualization, a 594-nm HeNe laser was used as an excitation light, and red color was detected at the range of 596-691nm wave length from fluorescent specimens. For nuclear visualization by DAPI, a 405 nm laser was used as an excitation light, and blue color was detected at the range of 420-476 nm wave length from fluorescent specimens. The pinhole was set to 67.9 µM. Bacterial culture and plasmid amplification

Expression vector pIE86-GFP (kindly provided by Dr. J. Nelson, Oregon Health and Science University, Portland, Oregon) was transformed to E.Coli competent cells by electroporation as described previously (Dower et al., 1988).Briefly one microliter of pIE86-GFP was added to 50 µl competent (E. coli, DH5α ) on ice, samples were mixed by pipeting up and down and then transferred to an electroporation cuvette. The cuvette was placed in electroporation instrument (Gene Pulser II, Biorad) and condition was adjusted for E. coli (25 µF, 200Ω) as recommended by manufacturing company. The cuvette was transferred to ice immediately, and 950 µl LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.0) was added.

The whole suspension (containing transformed E. coli) was transferred to a 10 ml tube, and incubated at 37 ˚C for one hour on a shaker. The cells were spread on LB agar plates (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar pH 7.0) + kanamycin (50 µg/ml), and incubated at 37 ˚C overnight. The following day, a single colony of transformed bacteria was incubated in 50 ml LB containing 50 µl/ml kanamycin at 37 ˚C on a shaker overnight.

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Plasmid Purification

Plasmids were purified using the Purelink HiPure Plasmid Filter Purification Kit (K2100-14, Invitrogen) according to the manufacturer’s protocol.DNA pellet was resuspended in 200 µl TE Buffer (10 mM TrisHCl pH 8, 1 mM EDTA).

Agarose gel electrophorasis

One microliter of purified plasmid was mixed with 8 µl dH2O and 1 µl loading buffer (25% bromophenol blue, 30% glycerol in H2O) and was loaded on 1% agarose gel in TAE buffer (40 mM Tris-acetate, 1Mm EDTA). Samples were electrophoresed for 80 min at 100 Volt.

The absorbance of purified plasmid DNA was measured by a Nanodrop spectrophotometer (Saveen Werner AB, Sweden) at 260 nm (A260), and 280 nm (A280), and the A260/A280 ratio was determined. An absorbance ratio A260/A280 > 1.8, when samples are diluted in TrisHCl pH 7.5, indicates that the DNA is reasonably free from proteins. Restriction Enzyme Analysis: Plasmid DNA (1 µg) was digested with restriction enzymes in 2µl 10x NEB2 buffer (New England Biolab) and dH2O up to 20 µl under condition recommended by the manufacture. 10 unit each of the following enzymes were added to three separate tubes; EcoR1, BamH1, and EcoR1+BamH1. Tubes were incubated at 37°C for 3 hours. Ten microliter of each reaction was mixed with 1µl loading buffer and was loaded on a 1% agarose gel in TAE buffer. DNA was visualized by ethidium bromide staining (0,5µg/ml etidium bromide in dH2O) for 20 min. 1 kb plus DNA Ladder (Fermentas AB, Sweden) was used as marker.

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Acknowledgment

I would like to express my special gratitude to people who directly or indirectly helped me during my studying and my research: Tomas Ekström, my supervisor for giving me chance to work with his group. Always being kind and friendly to me. Also for his excellent comments and great guiding during the research and for final report. Mohsen Karimi, my tutor and one of my best friends, for his outstanding help during my project, for giving me confidence and being an excellent teacher not only in lab sciences but also for matters of real life. Also for honestly sharing his excellent knowledge with me and others. I wish I would become a perfect molecular biologist like him one day. Karin Carlson, our scientific coordinator, our hard working, strict but kind teacher that I have learned a lot from her. My dear husband, Shahin, I know that I can never appreciate him for all nice and superb helps that he did to me. I found this chance that his love to be proved to me one more time, after thousands of times. I am sure without his help, it was almost impossible to finalize my study with this level of knowledge and experience. My friends in CMM, Tzvety for her nice and kindly behavior, and for teaching me western blot technique, Ghada for guiding me with my lab problems and answering my questions patiently. Monira for sharing her solutions and materials with me. Zahidul for his sense of humor. Anna maria for her kind behavior. Janos Geli, for his great helps from the first day that I came to the CMM until the last moment, for his all nice and optimistic sentences that always made me hopeful and optimistic about future. I am so happy that I had this chance to find such a great friend during my project. Rebecca Braden, for her kindness and sharing beautiful moments in Uppsala. Atosa, for always being kind and helpful. Reza and Sakineh, for always being there for my problems supporting me emotionally and for their nice advice for life. The last but not the least, my parents for their great supports and encouragement .My kind sister Mahtab to call me frequently in the days that I was really lonely and encouraged me especially in the exam days. My brother Majid who supported me emotionally in all the moment that I really need to be supported .My big sister Mojgan. My father in low for his encouragements and enormous support during my study.

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