Analysis of Chromatin Structure Indicates a Role for Fluoxetine in Altering Nucleosome Distribution Ely Gracia

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Honors Theses The Division of Undergraduate Studies

2012 Analysis of Chromatin Structure Indicates a Role for Fluoxetine in Altering Nucleosome Distribution Ely Gracia

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THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS & SCIENCES

ANALYSIS OF CHROMATIN STRUCTURE INDICATES A ROLE FOR FLUOXETINE

IN ALTERING NUCLEOSOME DISTRIBUTION

By:

ELY GRACIA

A Thesis submitted to the Department of Biological Sciences in partial fulfillment of the requirements for graduation with Honors in the Major

Degree Awarded: Spring, 2012

The members of the Defense Committee approve the thesis of Ely Gracia defended on April 17, 2012.

______Dr. Jonathan H. Dennis Thesis Director

______Dr. Debra A. Fadool Committee Member

______Dr. Joab Corey Outside Committee Member

Dedication:

To my mother and father for all their love and support. To Nicole Cordero for always believing in me and pushing me to meet and exceed my potential. To Dr. Jonathan Dennis for allowing me the opportunity to challenge myself.

Abstract

An interesting stimulus for chromatin structural changes is the generic and popular anti-depressant drug Fluoxetine, commonly known as Prozac. Generally accepted as a Selective Serotonin Reuptake Inhibitors (SSRI’s), recent work has emerged suggesting that this antidepressant also functions as a Histone Deaceylase

Inhibitors (HDIs). Studies have also come out indicating that Fluoxetine acts as an immunosuppressant drug. Treatment with Fluoxetine is believed to reduce the over- activation of the immune system associated with depression.

We have used an innovative microarray technology to measure changes in nucleosomal positioning that stem from Fluoxetine treatment. With the use of the microarray, we were able to show that Fluoxetine regulated chromatin structure, that

Fluoxetine induced nucleosomal changes show time-dependent kinetics, and targeted genes responsible for the regulation of immune system processes. These results give new and important insights into non-SSRI roles of this highly prescribed class of drugs.

Table of Content

Approval Page 2

Dedication 3

Abstract 4

Introduction 6

Materials and Methods 16

Results 20

Discussion 29

References 33

Appendix A 36

Introduction

According to the Center for Disease Control, depression affects one in ten adults in the United States. Depression costs Americans close to $100 billion dollars a year in treatment-related expenses and decreased productivity (Wittchen et al., 1994).

Depression also takes a toll on human lives. It has been estimated that 70% of people who commit suicide suffered from depression (Kirino et al., 2011). Although certain types of depression run in families, anyone can develop this disease even without genetic predisposition.

Depression is characterized as a mental illness that involves both the mind and the body and affects how we feel, think, and behave. Depression is a disease recognized by the American Psychiatric Association (APA) that hampers daily activities, relationships, and moods, and can have a negative effect on people’s health. The APA established a set criterion of symptoms and scenarios that must be met for the diagnoses of major depression. To be diagnosed with major depression, patients must demonstrate five of the following symptoms for a period of two weeks and these symptoms must represent a change from a previous level of functioning: depressed mood nearly every day during most of the day; marked diminished interest or pleasure in almost all activities; significant weight loss (while not dieting), weight gain, or a change in appetite; insomnia or hypersomnia; psychomotor agitation or psychomotor retardation; fatigue or loss of energy; feelings of worthlessness or inappropriate guilt, impaired ability to concentrate or indecisiveness, or recurrent thoughts of death or suicide (APA, 2000).

Depression is caused by a chemical imbalance or deficit of specific neurotransmitters, such as norepinephrine and serotonin, within the neurons of the brain (Ehlers et al., 1988).These neurotransmitters serve as the vehicle of communication between neurons; carrying chemical impulses. Generally, high levels of neurotransmitters are associated with good mood, whereas patients suffering from depression exhibit low levels of neurotransmitters.

People battling depression not only deal with mental complications, but also suffer from weakened immunity. Depression has a deleterious effect on the immune system by impairing the body’s ability to fight off infection and by potentially lessening the ability to deal with the progression of potential diseases. The immune system of a depressed individual has a high level of corticosteroids, which are immune suppressing hormones released during periods of elevated stress, along with hyperactivation of the immune system and high levels of proinflammatory cytokines (Antonioli et al., 2012).

This hyperactivity of the immune system leaves the body susceptible to complications when battling illnesses.

Currently, there is a variety of treatments available to help individuals control their depression. Some of these treatments include changes in lifestyle, such as increasing exercise, and administering therapy, such as psychotherapy and electro- convulsive shock therapy. The most common method used is through the use of antidepressant medication, with the most popular drug being Fluoxetine, commonly known as Prozac. These anti-depressant drugs work by selectively binding to reuptake channels on neurons and inhibiting the removal of the specific neurotransmitter.

Since its approval in the late 1980s, Fluoxetine has become widely

acknowledged as the breakthrough drug for depression. With total sales over $22

billion, Fluoxetine has been prescribed to over 40 million patients worldwide (Wong et

al., 2005). The development of Fluoxetine is due to evidence discovered in the early

1970s showing the role of serotonin in depression. From these studies came the

hypothesis that an effective way to treat depression was to use antidepressants to

modulate the serotonin mechanism by preventing the removal of serotonin.

Fluoxetine functions as a selective serotonin reuptake inhibitor (SSRI). These

compounds target the serotonin transporter located on presynaptic neurons. The goal of

SSRIs is to increase the amount of serotonin active in the synaptic cleft by causing a

negative allosteric modulation of the presynaptic serotonin transporter (Stahl, 1998).

The serotonin transporter is composed of the enzyme Na+/K+ ATPase and binding sites for sodium, serotonin, and SSRIs (Stahl, 1998).The binding of sodium to the transporter allows the transport of serotonin out of the synaptic cleft and into the neuron, through positive allosteric modulation. With the binding of the SSRI to the serotonin transporter, the affinity of the transporter for serotonin decreases, thus inhibiting the binding of serotonin to the transporter. By preventing the removal of serotonin from the synaptic cleft, serotonin is able to continue its interaction with the postsynaptic neuron.

Since Fluoxetine is administered orally, it is a systemic drug that interacts with various systems in the body. It is well absorbed in the gastrointestinal tract and excreted by the kidneys. Fluoxetine also has a half life of 1 to 3 days The Fluoxetine inhibition of the serotonin uptake is strong and concentration dependent, having an IC-50 of 17.6 nM

(Iceta et al., 2006).

Recent studies have illustrated that histone deacetylase inhibitors (HDI), which

modify chromatin within cells, have anti-depressant activity very similar to fluoxetine in

mice (Covington et al., 2009). HDIs have common features to their structures, such as a

hydrophobic chain and a polar region. Some HDIs, such as suberoylanalidehydroxamic

acid (SAHA), possess benzene rings as part of their structure. The structure of SAHA

similar to that of Fluoxetine, with both having benzene rings and a non-polar

hydrocarbon chain. Since SAHA is one of the HDIs used in the study that had anti-

depressant activity, I wanted to determine if the reverse logic may be true, that

fluoxetine, because of its similarities to HDIs, may alter chromatin structure.

Comparison of Histone deacetylase Inhibitor (HDI)

Figure 1. A comparison of suberoylanalidehydroxamic acid (SAHA) and Fluoxetine

Chromatin plays an essential role in the organization of DNA within cells. With

over 2 meters of DNA within cells, chromatin facilitates the DNA in packing into a 5 m nucleus. While chromatin allows the efficient packaging of DNA into the nucleus, vital cell functions require the de-condensing of the chromatin in order to have access to the vital information kept within DNA. Chromatin alters the packaging of DNA, modifying the

9 accessibility to the genes vitally necessary to specific cells while repressing other, less important, genes.

Chromatin’s structure consists of about 150 base pairs (bp) wrapped 1.7 times around an octameric group of basic histone proteins (Fig.2). These eight histones consist of two copies of each of the following histone cores, H2A, H2B, H3, and H4.

This “spool” of DNA wrapped around the histone octamer forms the basic structural unit of chromatin called the nucleosome. The nucleosome, along with 10 to 60 bp of DNA, forms the 10 nanometer fiber of chromatin and can be visualized as “beads on a string”

(Peterson et al., 2004).

Figure 2. Repressentation of chromatin structure consisting of repeating nucleosomal units made up of about 150 base pairs of DNA wrapped around a histone octamer.

The nucleosome is a very fluid protein-DNA complex. Although it is a very stable complex, it is not static and has the ability to rearrange its structures into different positions. The underlying DNA strand has the ability to dictate how and where it wraps itself around the histone octamer (modifications in cis). The position the DNA helix adopts in regards to the histone core is predetermined by a “DNA sequence

10 preference.” This is due to the particular DNA sequence having a higher affinity for the histone core, which would require less mechanical work to wrap the DNA around the histone proteins (Thastrom et al., 1999).

Chromatin is also modified by regulatory elements working in trans. The repositioning of chromatin takes place via the “sliding” of nucleosomes within the strand, which generates regions free of nucleosome about 200 bp upstream of the transcription start site that is flanked by well positioned nucleosomes on both sides (Fu et al., 2008).

Chromatin can be altered by the binding of protein transcription factors to the nucleosome. The binding of these transcription factors can cause the displacement or recruitment of nucleosomes within the chromatin (Teif et al., 2009).

Covalent modifications are a type of chromatin modification that takes place on the “tails” of histones. These histone tails are defined as the regions of the histone protein sequences that reach outside of the nucleosomal disk defined by the flat DNA superhelix. These tails extend to both sides of the histone-DNA region and are amino- terminal to the histone-fold domains. In addition, histone tails are extremely basic due to the high proportion of lysine and arginine amino acids they contain (Luger et al., 1998).

There are many post-translational modifications and they include acetylation, methylation, phosphorylation, ubiquitination, and glycosylation. Most except ubiquitination occur on the N-terminal basic tail domain. These histone modifications are predicted to affect transcription (Allfrey et al., 1964). Recent studies indicate that these modifications alter the histone-DNA and the histone-histone interactions (Ye et al.,

2005).

Histone acetylation takes place at lysine residues found on the N-terminal tail

domain of histones. Histone acetyltransferases (HATs) are enzymes that directly link

chromatin modification to gene activation (Marmorstein et al., 2011). HATs target ε- amino groups of specific lysine residues on the histone tails and add an acetyl group to them (Wolffe, 1992). There are 20 members in the HATs family including CREB binding protein (p300/CBP), General Control Nonderepressible 5 (Gcn5), and Steroid receptor coactivator-1 (SRC-1). These HATs act as transcription factors via interaction within the transcription activation complex (Peterson et al., 2004).

Histone deacetylases (HDACs) are enzymes that remove acetyl groups present on the ε-amino group of histone tails and render a compacted chromatin structure. The effects of HDACs are generally to decrease gene expression (Pacheco et al., 2012).

The HDAC family is comprised of 11 members that share a zinc-dependent catalytic domain with high degree of homology. This family is further organized into three classes based on their structural similarity to homologous proteins in yeast (Pacheco et al.,

2012).

A way to counteract the actions of HDACs is with the administration of compounds known as histone deacetylase inhibitors (HDIs). HDIs have a long history of usage for the treatment of many psychiatric and neurologic disorders, functioning as mood stabilizers and anti-epileptics. Studies have also shown that HDIs have been able to alleviate the symptoms associated with neurodegenerative diseases, such as

Huntington’s and Alzheimer’s disease (Hahnen et al., 2008).Some of the most common

HDIs available today are sodium butyrate, valproic acid, and suberoylanilidehydroxamic acid (SAHA). HDIs, in theory, induce the hyperacetylation of nucleosome core histones

in chromatin. This hyperacetylation affects only a small subset of genes that leads to

activation of some genes and equal repression of others (Vigushin, 2004). In addition,

many HDIs, such as SAHA and sodium butyrate, have the ability to cross the blood

brain barrier (BBB) (Hockly et al., 2003).

HDIs have a specific role in the modification of chromatin. Once inside the

nucleus, HDIs have been shown to bind directly to the active site of HDACs, blocking

access of the substrate. Because of this inhibition of HDACs, there is an accumulation

of acetylated histones.

Recent studies have revealed that HDI may have new potential uses in the

treatment of Major Depressive Disorders, such as depression and bipolar disorder.

When administered to mice in a state of chronic depression, treatment with HDIs

produced results similar to those seen after treatment with popular antidepressants,

such as Fluoxetine (Covington et al., 2009). The mice were monitored for total time

spent in the “interaction zone.” The results of this experiment demonstrated that socially

defeated mice fitted with a vehicle spent considerably less time in the interaction zone

than non-defeated control mice. In contrast, the socially defeated mice receiving 100 M of either MS-275 or SAHA resumed the typical interaction of non-defeated, control mice.

In addition to social interaction, similar results were seen in sucrose preference and the forced swim tests. Overall, treatment with HDIs produced similar results to treatment with antidepressants when administered to mouse models of depression.

This experiment demonstrated a new potential use of HDIs for the treatment of depression due to the generated data showing the similarities of HDI treatment to those

13 caused by Fluoxetine. What my experiment is trying to determine if there is a further connection between Fluoxetine and chromatin remodelers and whether Fluoxetine may have a secondary off-target effect on chromatin structure. Since Fluoxetine is a systemic drug, it circulates throughout the body, interacting with other systems, such as the excretory and immune system, before reaching its intended target within the brain.

Besides the side effects associated with this drug, secondary, off-target effects of

Fluoxetine are poorly understood.

Covington et al. linking HDIs to anti-depressant activity, there was a clear connection between the effects generated by treatment with HDIs and treatment with

SSRIs, specifically Fluoxetine. One of the HDI that was used, SAHA, has a structural composition that is similar to that of Fluoxetine (Fig.1). Both have a benzene ring, an amine group, a hydrophobic carbon chain, and a polar region within their overall structure. Because of the structural similarities between Fluoxetine and an HDI,

Fluoxetine may play a role in modifying the structure of chromatin.

The goal of my experiment is answer a few vital questions. Does Fluoxetine regulate chromatin structure? Do the specific targets belong to a particular gene function? Do these changes reflect an underlying mechanism? By understanding how

Fluoxetine works, we can potentially identify the off target, secondary effects it may have and identify the mechanism, generating a new, potential use for this powerful drug.

Materials and Methods

Tissue Culture

This experiment was conducted using Jurkat cells, a human T-lymphocyte cell line. The cells were cultured in full growth media (RPMI 1640 enhanced with 10% Fetal

Bovine Serum, 50 M β-mercaptoethanol, and 100g/ml Gentamicin). Approximately

0.5x106 cells/ml were plated in 25ml of full growth media and given a treatment consisting of 12.5l of Fluoxetine at a concentration of 50 mM for a total concentration of 10 M. Cells were harvested at approximately 1x106 cells/ml after a time period of 12 hours and 24 hours.

Harvest/Micrococcal nuclease Cleavage of Chromatin

Approximately 2x106 cells/ml were harvested by centrifugation at 1000 x g for 5 minutes. Media was removed and the cells were gently resuspended in 10 ml of phosphate-buffered saline. The cells were cross-linked by adding formaldehyde to a final concentration of 1% and incubated for 10 minutes at room temperature with gentle rocking. Glycine was added to a final concentration of 125 mM, quenching the formaldehyde. Cells were collected by centrifugation at 1000 x g for 10 minutes. The cell pellets were resuspended in 15ml of nucleus-isolation buffer (0.3 M sucrose, 2 mM

MgOAc2, 1 mM CaCl2,1% Nonidet P-40, 10 mM HEPES pH 7.8). The solution was then centrifuged at 1000 x g for a period of 10 minutes. Next, the pellets were resuspended in 500 l MNase cleavage buffer (4 mM CaCl2, 25 mM KCl, 4 mM MgCl2, 12.5% glycerol, 50 mM HEPES pH 7.8) with 1.25 U/ml, 2.5 U/ml, 5 U/ml, or 10 U/ml of MNase.

The reactions were incubated at 37°C for 5 minutes and stopped using a final

15 concentration of 50 mM EDTA. Proteinase K was then added to a final concentration of

0.2 G/L. Next, sodium dodecyl sulfate was added to a final concentration of 1%. The reactions were incubated at 65°C overnight to reverse the cross-linking reaction.

Isolation of MNase digested DNA

One volume of Tris-saturated phenol, pH 8, was added to sample reactions, which were then vortexed. The samples were centrifuged at 14,500 x g in a tabletop microcentrifuge for 10 minutes. The aqueous phase was removed and transferred to a new 1.5ml microcentrifuge tube. Next, one volume of phenol:chloroform was added, samples were vortexed, and then centrifuged at 14,500 x g for 10 minutes. The aqueous phase was again removed and transferred to a new 1.5ml microcentrifuge tube.

One volume of chloroform was added to each sample. The reactions were vortexed and then centrifuged at 14,500 x g for 10 minutes. The aqueous phase was again removed and 1/10 of a volume of 3 M sodium acetate was added and mixed by inversion.

Two volumes of 100% ethanol were added to each sample and then vortexed.

Next the samples were incubated at room temperature for a period of 30 minutes, followed by centrifugation at 14,500 x g for 10 minutes. The supernatant was then discarded and the DNA pellet retained. One ml of 70% ethanol was added to each sample to wash away excess salt. The samples were centrifuged again at 14,500 x g for

10 minutes. The supernatant was discarded after centrifugation.

The remaining pellets were dried in a vacuum for 5 minutes to remove any

residual alcohol. The pellets were then resuspended in 100 l of Tris-EDTA and the

DNA was allowed to dissolve completely. The remaining DNA was quantified using a spectrophotometer and the MNase digestion was verified on a 2% agarose gel to assess the level of cleavage to the chromatin (Fig.3).

Isolation of mononucleosomally protected DNA

Approximately 20 g of each DNA sample was loaded in a 1% gel. After electrophoresis, mononucleosomal bands were cut out and gel slices were placed in dialysis tubing. The mononucleosomally protected fragments were then isolated using

gel electrophoresis. These fragments were extracted from the dialysis tubing using 20

g/ml glycogen and then purified using a standard ethanol precipitation.

Fluorescent labeling of DNA

The protocol outlined by NimbleGen for labeling material was successfully used to hybridize mononucleosomally protected fragments against a bare genomic control to in-house designed microarrays. A fluorescently labeled random 7-mer oligonucleotide primer was used in a Klenow fill-in reaction to label the mononucleosomally protected and bare genomic reference DNA samples. The mononucleosomally protected DNA sample was labeled with Cy3 primer and the reference bare genomic DNA was labeled with Cy5 primer. One microgram of sample was used for each labeling reaction. The microarray used in this study was a custom-designed nucleosome positioning array containing 2 kilobases surrounding the transcription start sites of 505 immunity-related genes. The 60-mer probes on the arrays are tiled at an average of 13bp spacing with a

47bp overlap.

Data Analysis

Microarray data were analyzed using a custom designed suite of tools in the R software environment developed by the Dennis lab, including the “drawGff” program.

The log2(nucleosomal/bare genomic) signal intensities were plotted to determine

regions of increased or decreased occupancy. The X-axis indicates genomic position

and the Y-axis indicates hybridization signals, with higher values indicating greater

nucleosomal protection (Fig.4A and 4B).

Results

Measured changes in nucleosomal distribution in response to Fluoxetine treatment

Fluoxetine induces changes in chromatin structure. In order to track changes in the positioning of nucleosomes around the transcription start site, we used a systematic and unbiased approached to measure the amount of change between treated and untreated samples. We used a sliding window of 3 probes to perform a paired t-test determining if pre and post treatment with Fluoxetine represented similar nucleosome distribution. Because the probes are 60 to 75 base pairs in length, spaced approximately 13 base pairs apart, the window we generated was 80 to 100 bases in length. Using this criterion, we assigned a cutoff value to determine how substantial a change is, based on the discrimination ability of each threshold (Fig. 5). Different thresholding gives different levels of discrimination for the genes with altered nucleosomal distribution at each time point. Fig.5A and 5B represent cutoff values of 0.2 and 0.3, respectively. These two cutoff values show no discrimination between the two time points, whereas Fig.5F shows the most discrimination between time points. We used 0.4 as the discrimination value because this threshold contains the greatest number of loci who regulation by Fluoxetine is confirmed in the literature [e.g. CCL2

(Riksen et al., Chen et al.), IL2RA (Kovaru et al.), C3 (Laudenslager et al.), STAT1

(Tsao et al.), TGFB1 (Lee et al.), NOS2 (Ha et al.), IL10 (Kubera et al., Song et al.,

Maes et al.)]. Of the 505 immunity-related genes tested, 224 genes (44%) had a substantial change in nucleosomal distribution induced by treatment with Fluoxetine after 12 hours. In contrast, when treated with Fluoxetine for 24 hours, 305 genes (60% of the 505 genes) had a substantial change (Fig.5C). An example of the changes induced by treatment can be seen with the gene MAPK14. At the 12 hour time point,

MAPK14 had areas around the transcription start site with a signal intensity that differed from the control sample (Fig. 6A). Just upstream of the TSS, there is an increase in signal intensity followed by a decrease. These results suggest a shift in nucleosomal positioning. In the 24 hour time point, the treated sample had a more substantial change when compared to the 12 hour time point. In addition, upstream of the TSS there is a steep drop in signal intensity (Fig.6B). These results suggest either the removal of a nucleosome or the shift of a nucleosome at this position.

5A 5B 5C

5D 5E 5F

12 24

Fluoxetine induced nucleosomal changes show time dependent kinetics

Fluoxetine’s effects on chromatin structure exhibits time-dependent kinetics in altering the positioning of nucleosomes. Using the 0.4 cutoff (Fig.5C), we see that there is a 20% increase in the number of genes showing substantial change in nucleosome distribution at the 24 hour time point. There were 305 genes altered at the 24 hour time point (60%) versus 224 at the 12 hour time point (44%). Using smaller cutoff values, such as 0.2 or 0.3, we saw that the discrimination between the two time points was minimal (Fig.7A and 7B). These results suggest that at these cutoff values, Fluoxetine’s effects are primarily time independent. As we increased the cutoff value, the results become more time dependent, with greater discrimination between the specific time points. At cutoff values of 0.5, 0.6, and 0.7, the discrimination is greatest, with almost all

22 the substantial changes in nucleosomal distribution belonging exclusively to either time point (Fig. 7D-F).

The effect Fluoxetine had on CCL2 appeared to be time dependent with two regions of decreased nucleosomal occupancy after 12 hours occurring in different regions compared to after 24 hours. After 12 hours, there is a decrease in occupancy

500 base pairs upstream and 300 base pairs downstream of the transcription start site

(Fig.8A). After 24 hours, there was a decrease in nucleosomal occupancy at the transcription start site and about 800 base pairs downstream of the transcription start site (Fig. 8B).

In contrast, treatment with Fluoxetine caused IL2RA to exhibit changes in nucleosomal distribution that were time independent. Both the 12 and 24 hour samples showed changes in nucleosomal occupancy in the same regions, such as 800 base pairs upstream, 300 base pairs upstream, and 300 base pairs downstream of the transcription start site (Fig. 9A and 9B).

Treatment with Fluoxetine also caused changes in the nucleosomal distribution in

STAT1 that were both time dependent and time independent. After both 12 and 24 hours (Fig.10A and 10B), STAT1 had a decrease in nucleosomal occupancy at the transcription start site, representing time independent changes in distribution. STAT1 also showed a change in occupancy exclusive to the 12 hour time point 600 base pairs upstream of the transcription start site (Fig.10A).

IL10 showed time-dependent changes exclusive to each time point. After 12 hours, there is an increase in nucleosomal occupancy 800 base pairs upstream and a

23 decrease in occupancy 400 base pairs upstream of the transcription start site (Fig.11A).

After 24 hours, there is a decrease in occupancy at the transcription start site and an increase in occupancy 600 base pair downstream of the transcription start site

(Fig.11B).

Overall, we see greater changes at the 24 hour time point than the 12 hour time point. Based on these results, we conclude that Fluoxetine induced changes show time dependent chromatin structure kinetics.

Fluoxetine targets genes responsible for the regulation of immune system processes.

To determine if Fluoxetine had any specific targets that were linked to cellular processes, we searched for enrichment of particular ontologic classes of genes using the program gene ontology enrichment analysis and visualization tool (GORILLA).

GORILLA allowed us to investigate if these subsets of genes were enriched for any specific biological processes. The genes that exhibited substantial changes at the 12 hour time point were enriched for the negative regulation of immune system processes and regulation of angiogenesis, while those treated with Fluoxetine for 24 hours were enriched for the negative regulation of protein metabolic process and negative regulation of cellular protein metabolic processes (Fig.12). Each of the genes listed had a specific response to treatment with Fluoxetine consistent with down-regulating immune responses. Fluoxetine attenuated the activity of immunostimulant genes, such as CCL2 and STAT1, and increased the production of the negative immunoregulator

IL10.

Discussion

Depression is a powerful disease affecting almost 20 million people each year.

People battling depression not only deal with mental complications, but also suffer from weakened immunity. Depression is also associated with impairing the body’s immune system. The immune system of a depressed becomes hyperactive, causing a release of corticosteroids and proinflammatory cytokines (Antonioli et al., 2012). This hyperactivity of the immune system leaves the body susceptible to complications when battling illnesses. Taking Fluoxetine to treat depression may also work to alleviate the overstimulation associated with depression.

In the beginning of this report we set out to answer three questions:

1. Does Fluoxetine regulate chromatin structure?

2. Do the specific targets belong to a particular gene function?

3. Do these changes reflect an underlying mechanism?

Does Fluoxetine have an effect on chromatin structure? Yes, Fluoxetine does affect chromatin structure with the number of genes that experienced a substantial change in nucleosomal positioning totaling 349 of 505 genes across both time points

(70% of genes). It was clear that when compared to the untreated, bare genomic DNA, the samples treated with Fluoxetine differed in the positioning of the nucleosomes.

These alterations in chromatin structure may help to highlight a connection between

Fluoxetine and its chromatin regulatory properties such as a histone deacetylase inhibitor.

Does Fluoxetine target specific loci? Yes, a number of genes that exhibited substantial changes between treated and untreated samples are associated with certain biological processes such as the negative regulation of immune system processes and the regulation of angiogenesis. We interpret these results to mean Fluoxetine has anti- inflammatory effects on the immune system, alleviating over activation by inhibiting immunostimulant genes, such as CCL2, STAT1, and IL2RA and by stimulating IL10, an immunosuppressant gene.

After generating the results of the experiment, we began to see that there was a link between the immune system’s over active response to depression and how the effects of treatment with Fluoxetine may counteract the hyperactivity of the immune system. One of the genes that showed substantial change at both the 12 and 24 hour time point was CCL2. This is a gene that is involved in immunoregulatory and inflammatory processes by acting as a chemotactic factor that attracts monocytes and basophils and is stimulated by chronic stress associated with depression. When cells were treated with Fluoxetine, there was a down-regulation of CCL2 secretion through transcription inhibition (Riksen et al., 2010). CCL2 showed a decrease in nucleosomal occupancy in the region where the SP1 and c-jun are located. It is interesting to speculate that these regions’ activity may be affected by treatment with Fluoxetine and that these results suggest Fluoxetine may be preventing this immunostimulant gene from contributing to the over-activation associated with people suffering from depression.

In addition to CCL2, IL2RA was also affected by treatment with Fluoxetine at the

12 and 24 hour time points. IL2RA codes for proteins that act as major mediators of

30 immune response, in addition to regulating different cell functions such as proliferation, differentiation, and apoptosis. IL2RA is activated by several pathophysiological processes such as viral infection, autoimmune diseases and depression. A region that was altered by treatment at both time points was the SRF transcription factor binding site, which lies about 700 bases upstream of the promoter. In a recent study, treatment with Fluoxetine caused the inhibition of signal transduction of this gene in glioma cells suggesting Fluoxetine may attenuate production of this gene (Kovaru et al., 2011). This again is an example of Fluoxetine working to tone down the immune responses associated with depression.

Fluoxetine also had similar effects on STAT1, another immunostimulant gene. At both time points, STAT1 had substantial changes in the positioning of its nucleosomes.

STAT1 is a gene that once activated, acts as a transcription activator that mediates signaling by interferons and drives the cell into an antiviral state. The region that showed change in nucleosomal occupancy near the transcription start site is the NF

Kappa-B binding site, in addition to many other transcription factor binding sites. A recent study (Tsao et al., 2012) showed that Fluoxetine attenuated STAT1 activation and translocation, which may be a mechanism in place to prevent immune system over activation.

In addition to affecting the nucleosomal distribution of immunostimulant genes,

Fluoxetine also altered the positioning of nucleosomes of an immunosuppressant gene,

IL-10. IL-10 inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, and IL-3, which are produced by activated macrophages and by helper T-cells. When treated with Fluoxetine, IL10 experienced more substantial changes to its nucleosomal

31 distribution at the 24 hour time points than at the 12 hour time point. In those regions of change lies Maf K transcription binding site. A study revealed that instead of downregulating the activity of this immunosuppressant gene, treatment with Fluoxetine stimulates the production of the negative immunoregulator, interleukin-10 (Kubera et al.,

2009).

Overall, there is a link between the effects Fluoxetine has on immune regulating genes and Fluoxetine’s ability to modify the nucleosomal distribution of these genes.

Fluoxetine is targeting genes in order to produce an overall immune system suppression to counteract the effects caused by depression. By the further understanding of this mechanism, we can potentially determine a new use for this antidepressant.

Fluoxetine saves millions of people each year from depression and has contributed to the further understanding of depression and its cures. But being a systemic drug that affects other organ system and pathways, it is very important to gain a deeper understanding of the secondary effects associated with this drug.

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Appendix A

Gene Name Chromosome Start Stop ABCE1 chr4 146018156 146020156 ABCF1 chr6 30538170 30540170 ACVR1 chr2 158730623 158732623 ACVR1B chr12 52344486 52346486 ACVR2A chr2 148601570 148603570 ACVR2B chr3 38494790 38496790 ADORA1 chr1 203095836 203097836 ADORA2A chr22 24822530 24824530 AGFG1 chr2 228335888 228337888 AHSG chr3 186329850 186331850 AIF1 chr6 31582033 31584033 ALOX5 chr10 45868629 45870629 AMH chr19 2248113 2250113 AMHR2 chr15 45002685 45004685 AMHR2 chr12 53816639 53818639 ANXA1 chr9 75765781 75767781 APCS chr1 159556616 159558616 APEX1 chr14 20922290 20924290 APOA2 chr1 161192418 161194418 APOBEC3F chr22 39435673 39437673 APOBEC3G chr22 39472025 39474025 APOL2 chr22 36634689 36636689 APOL3 chr22 3652637 3654637 AREG chr4 75479629 75481629 ARHGDIB chr12 15113562 15115562 ASB1 chr2 239334626 239336626 ATRN chr20 3450665 3452665 AZU1 chr19 826831 828831 BAD chr11 64051176 64053176 BAG4 chr8 38033106 38035106 BANF1 chr11 65768550 65770550 BATF chr14 75987784 75989784 BAX chr19 49457117 49459117 BCL11B chr14 99736822 99738822 BCL2 chr18 60985613 60987613 BCL6 chr3 187462475 187464475 BDKRB1 chr14 96721559 96723559 BLNK chr10 98030333 98032333 BMP1 chr8 22021675 22023675 BMP2 chr20 6747745 6749745

BMP3 chr4 81951119 81953119 BMP7 chr20 55840707 55842707 BMPR1A chr10 88515396 88517396 BMPR1B chr4 95678128 95680128 BMPR2 chr2 203240050 203242050 BPI chr20 36931552 36933552 BTK chrX 100640212 100642212 BTRC chr10 103112825 103114825 C1R chr12 7244043 7246043 C2 chr6 31894266 31896266 C3 chr19 6719662 6721662 C3 chr19 6719662 6721662 C3AR1 chr12 8217955 8219955 C4A chr6 31948834 31950834 C5 chr9 123811554 123813554 C8A chr1 57319443 57321443 CAD chr2 27439258 27441258 CAMP chr3 48263862 48265862 CARD18 chr11 105008806 105010806 CASP1 chr11 104971158 104973158 CASP2 chr7 142984402 142986402 CASP3 chr4 185569629 185571629 CASP4 chr11 104838325 104840325 CASP8 chr2 202097166 202099166 CAST chr5 95996941 95998941 CBX5 chr12 54672915 54674915 CCL1 chr17 32689252 32691252 CCL11 chr17 32611687 32613687 CCL13 chr17 32682471 32684471 CCL15 chr17 34328100 34330100 CCL16 chr17 34307523 34309523 CCL17 chr16 57437679 57439679 CCL18 chr17 34390643 34392643 CCL19 chr9 34690274 34692274 CCL2 chr17 32581296 32583296 CCL20 chr2 228677558 228679558 CCL21 chr9 34709147 34711147 CCL22 chr16 57391718 57393718 CCL23 chr17 34344005 34346005 CCL24 chr7 75442033 75444033 CCL25 chr19 8116934 8118934 CCL26 chr7 75418064 75420064 CCL27 chr9 34661689 34663689

CCL28 chr5 43411488 43413488 CCL3 chr17 34416506 34418506 CCL3L1 chr17 34624730 34626730 CCL4 chr17 34430220 34432220 CCL4L1 chr17 34537468 34539468 CCL5 chr17 34206377 34208377 CCL7 chr17 32596240 32598240 CCL8 chr17 32645066 32647066 CCNT1 chr12 49109781 49111781 CCR1 chr3 46248832 46250832 CCR10 chr17 40832845 40834845 CCR2 chr3 46394235 46396235 CCR3 chr3 46282872 46284872 CCR4 chr3 32992066 32994066 CCR5 chr3 46410633 46412633 CCR6 chr6 167524295 167526295 CCR7 chr17 38720724 38722724 CCR8 chr3 39370197 39372197 CCR9 chr3 45927051 45929051 CCRL1 chr3 132315094 132317094 CCRL2 chr3 46447721 46449721 CD14 chr5 140012010 140014010 CD180 chr5 66491617 66493617 CD1B chr1 158300321 158302321 CD1C chr1 158258563 158260563 CD1D chr1 158148737 158150737 CD209 chr19 7811464 7813464 CD22 chr19 35819079 35821079 CD247 chr1 167486847 167488847 CD27 chr12 6553051 6555051 CD276 chr15 73975622 73977622 CD28 chr2 204570198 204572198 CD4 chr12 6897651 6899651 CD40 chr20 44745906 44747906 CD40LG chrX 135729336 135731336 CD44 chr11 35159417 35161417 CD47 chr3 107808935 107810935 CD53 chr1 111414722 111416722 CD55 chr1 207493817 207495817 CD58 chr1 117112715 117114715 CD69 chr12 9912497 9914497 CD70 chr19 6590163 6592163 CD74 chr5 149791323 149793323

CD80 chr3 119242142 119244142 CD86 chr3 121795750 121797750 CD97 chr19 14518533 14520533 CDK7 chr5 68529622 68531622 CDK9 chr9 130547305 130549305 CDKN1A chr6 36645459 36647459 CEBPB chr20 48806376 48808376 CER1 chr9 14721715 14723715 CFB chr6 31912721 31914721 CFP chrX 47488704 47490704 CHIT1 chr1 203197860 203199860 CHST1 chr11 45686172 45688172 CHUK chr10 101988344 101990344 CIITA chr16 10970055 10972055 CKLF chr16 66585472 66587472 CLC chr19 40227668 40229668 CLEC4E chr12 8692558 8694558 CLEC7A chr12 10281868 10283868 CMTM1 chr16 66599294 66601294 CMTM2 chr16 66612351 66614351 CNTFR chr9 34588722 34590722 COLEC12 chr18 499729 501729 COPS6 chr7 99685583 99687583 CR1 chr1 207668473 207670473 CR2 chr1 207626645 207628645 CRADD chr12 94070151 94072151 CREBBP chr16 3929121 3931121 CRISP3 chr6 49711056 49713056 CRLF1 chr19 18716660 18718660 CRLF2 chrY 1280527 1282527 CRP chr1 159683379 159685379 CSF1 chr1 110452233 110454233 CSF1R chr5 149491935 149493935 CSF2 chr5 131408485 131410485 CSF2RA chrX 1386693 1388693 CSF2RB chr22 37308675 37310675 CSF3 chr17 38170688 38172688 CSF3R chr1 36947509 36949509 CTF1 chr16 30906928 30908928 CX3CL1 chr16 57405414 57407414 CX3CR1 chr3 39320527 39322527 CXCL1 chr4 74734109 74736109 CXCL10 chr4 76941273 76943273

CXCL11 chr4 76953842 76955842 CXCL12 chr10 44879542 44881542 CXCL13 chr4 78431907 78433907 CXCL14 chr5 134913969 134915969 CXCL16 chr17 4642223 4644223 CXCL2 chr4 74963997 74965997 CXCL3 chr4 74903490 74905490 CXCL5 chr4 74863416 74865416 CXCL6 chr4 74701273 74703273 CXCL9 chr4 76927641 76929641 CXCR3 chrX 70837367 70839367 CXCR4 chr2 136874725 136876725 CXCR5 chr11 118753541 118755541 CXCR6 chr3 45983973 45985973 CYBB chrX 37638270 37640270 CYP26B1 chr2 72373963 72375963 DCD chr12 55041149 55043149 DEFA4 chr8 6794786 6796786 DEFA5 chr8 6913259 6915259 DEFA6 chr8 6782598 6784598 DEFB1 chr8 6734529 6736529 DEFB118 chr20 29955428 29957428 DEFB127 chr20 137186 139186 DEFB4 chr8 7751199 7753199 DFFA chr1 10531613 10533613 DMBT1 chr10 124319181 124321181 DOCK2 chr5 169063251 169065251 DUSP1 chr5 172197203 172199203 EBI3 chr19 4228540 4230540 ECSIT chr19 11638987 11640987 EDA chrX 68834911 68836911 EDA2R chrX 65834873 65836873 EIF2AK2 chr2 37383190 37385190 ELA2 chr19 851291 853291 ELK1 chrX 47509003 47511003 EP300 chr22 41487614 41489614 EPHX2 chr8 27347645 27349645 EPO chr7 100317423 100319423 EPOR chr19 11494019 11496019 ERAP1 chr5 96142892 96144892 ERBB2 chr17 37855254 37857254 ERBB2IP chr5 65221384 65223384 F11R chr1 160964002 160966002

F2 chr11 46739743 46741743 F3 chr1 95006371 95008371 F8 chrX 154249998 154251998 FADD chr11 70048269 70050269 FAM3B chr21 42687661 42689661 FAS chr10 90749288 90751288 FASLG chr1 172627185 172629185 FCAR chr19 55384549 55386549 FGF10 chr5 44387784 44389784 FGF12 chr3 192125838 192127838 FIGF chrX 15401577 15403577 FLT3LG chr19 49976486 49978486 FN1 chr2 216299791 216301791 FOS chr14 75744481 75746481 GABBR1 chr6 29599962 29601962 GADD45A chr1 68149883 68151883 GAPDH chr12 6642657 6644657 GLI2 chr2 121553867 121555867 GNLY chr2 85920414 85922414 GPC1 chr2 241374115 241376115 GPR44 chr11 60622444 60624444 HAMP chr19 35772410 35774410 HCK chr20 30639057 30641057 HLA-A chr6 29909331 29911331 HLA-C chr6 31238855 31240855 HLA-E chr6 30456271 30458271 HMGA1 chr6 34206070 34208070 HMGB1 chr13 31039081 31041081 HMOX1 chr22 35776087 35778087 HPRT1 chrX 133593175 133595175 HRAS chr11 534550 536550 HSPA1A chr6 31782291 31784291 HSPA4 chr5 132386662 132388662 HSPA6 chr1 161493036 161495036 HSPD1 chr2 198363640 198365640 HTATIP2 chr11 20384231 20386231 HTATSF1 chrX 135578671 135580671 IFNA1 chr9 21439440 21441440 IFNA2 chr9 21384396 21386396 IFNB1 chr9 21076943 21078943 IFNG chr12 68552521 68554521 IFNGR1 chr6 137539567 137541567 IFNGR2 chr21 34774202 34776202

IFNK chr9 27525494 27527494 IKBKAP chr9 111695608 111697608 IKBKB chr8 42127829 42129829 IKBKG chrX 153775062 153777062 IL10 chr1 206944839 206946839 IL10RA chr11 117856106 117858106 IL10RB chr21 34637672 34639672 IL11 chr19 55880814 55882814 IL11RA chr9 34652932 34654932 IL12A chr3 159705629 159707629 IL12B chr5 158756481 158758481 IL12RB1 chr19 18196697 18198697 IL12RB2 chr1 67772047 67774047 IL13 chr5 131992865 131994865 IL13RA1 chrX 117860559 117862559 IL13RA2 chrX 114251207 114253207 IL15 chr4 142556754 142558754 IL15RA chr10 6018537 6020537 IL16 chr15 81586467 81588467 IL17A chr6 52050185 52052185 IL17C chr16 88704001 88706001 IL17RA chr22 17564849 17566849 IL18 chr11 112033840 112035840 IL18R1 chr2 102978097 102980097 IL1A chr2 113541971 113543971 IL1B chr2 113593356 113595356 IL1F10 chr2 113824547 113826547 IL1F5 chr2 113815685 113817685 IL1F6 chr2 113762449 113764449 IL1F7 chr2 113671770 113673770 IL1F8 chr2 113809440 113811440 IL1F9 chr2 113734606 113736606 IL1R1 chr2 102769402 102771402 IL1R2 chr2 102607306 102609306 IL1RAP chr3 190230891 190232891 IL1RAPL2 chrX 103809996 103811996 IL1RL2 chr2 102802433 102804433 IL1RN chr2 113874470 113876470 IL2 chr4 123376650 123378650 IL20 chr1 207038154 207040154 IL21 chr4 123541211 123543211 IL22 chr12 68646281 68648281 IL2RA chr10 6103272 6105272

IL2RB chr22 37544962 37546962 IL2RG chrX 70330403 70332403 IL3 chr5 131395347 131397347 IL4 chr5 132008373 132010373 IL5 chr5 131878214 131880214 IL5RA chr3 3151058 3153058 IL6 chr7 22765766 22767766 IL6R chr1 154376669 154378669 IL6ST chr5 55289763 55291763 IL7 chr8 79716758 79718758 IL8 chr4 74605275 74607275 IL8RA chr2 219030716 219032716 IL8RB chr2 218989746 218991746 IL9 chr5 135230516 135232516 IL9R chrY 59329252 59331252 IRAK1 chrX 153284342 153286342 IRAK2 chr3 10205563 10207563 IRAK3 chr12 66581978 66583978 IRAK4 chr12 44151747 44153747 IRF1 chr5 131825465 131827465 IRF2 chr4 185394726 185396726 IRF3 chr19 50168132 50170132 IRF7 chr11 614999 616999 ITGAX chr16 31365509 31367509 JUN chr1 59248785 59250785 KIR3DL1 chr19 55326893 55328893 KLRD1 chr12 10459417 10461417 LALBA chr12 48962829 48964829 LBP chr20 36973885 36975885 LEAP2 chr5 132208358 132210358 LIF chr22 30641796 30643796 LMNA chr1 156083461 156085461 LMNB1 chr5 126111833 126113833 LMNB2 chr19 2455958 2457958 LTA chr6 31539093 31541093 LTB chr6 31547337 31549337 LTB4R chr14 24781313 24783313 LTBR chr12 6492357 6494357 LTF chr3 46505395 46507395 LY86 chr6 6587934 6589934 LY96 chr8 74902587 74904587 LYZ chr12 69741134 69743134 MADD chr11 47290199 47292199

MAL chr2 95690479 95692479 MAP2K3 chr17 21193781 21195781 MAP2K4 chr17 11923135 11925135 MAP2K6 chr17 67409838 67411838 MAP3K1 chr5 56109900 56111900 MAP3K14 chr17 43393414 43395414 MAP3K5 chr6 137112656 137114656 MAP3K7 chr6 91295907 91297907 MAP3K7IP1 chr22 39794759 39796759 MAP3K7IP2 chr6 149638063 149640063 MAP4K4 chr2 102313488 102315488 MAPK10 chr4 87373283 87375283 MAPK11 chr22 50707779 50709779 MAPK12 chr22 50699089 50701089 MAPK13 chr6 36097262 36099262 MAPK14 chr6 35994454 35996454 MAPK8 chr10 49608687 49610687 MAPK8IP3 chr16 1755221 1757221 MAPK9 chr5 179718071 179720071 MBL2 chr10 54530460 54532460 MIF chr22 24235565 24237565 MX2 chr21 42732950 42734950 MYD88 chr3 38178969 38180969 NCF4 chr22 37256030 37258030 NFATC1 chr18 77154772 77156772 NFIB chr9 14312945 14314945 NFKB1 chr4 103421486 103423486 NFKB2 chr10 104154432 104156432 NFKBIA chr14 35872960 35874960 NFKBIB chr19 39389615 39391615 NFKBIE chr6 44232525 44234525 NFKBIL1 chr6 31514353 31516353 NFKBIL2 chr8 145668812 145670812 NFRKB chr11 129761904 129763904 NGFR chr17 47571655 47573655 NLRC4 chr2 32489801 32491801 NLRP3 chr1 247580351 247582351 NOS2 chr17 26126555 26128555 NR2C2 chr3 14988236 14990236 PAK1 chr11 77184108 77186108 PAK2 chr3 196465728 196467728 PARP1 chr1 226594801 226596801 PELI1 chr2 64370605 64372605

PELI2 chr14 56584093 56586093 PF4 chr4 74846715 74848715 PGLYRP1 chr19 46525323 46527323 PGLYRP2 chr19 15589315 15591315 PGLYRP3 chr1 153282194 153284194 PGLYRP4 chr1 153320022 153322022 PIK3C2B chr1 204458474 204460474 PLUNC chr20 31822802 31824802 PPARA chr22 46545499 46547499 PPBP chr4 74852900 74854900 PPIA chr7 44835241 44837241 PRDX1 chr1 45986609 45988609 PRDX2 chr19 12911694 12913694 PRG2 chr11 57157130 57159130 PRKDC chr8 48871743 48873743 PRKRA chr2 179314958 179316958 PROC chr2 128175017 128177017 PSMG2 chr18 12702064 12704064 PTAFR chr1 28502455 28504455 PTGES chr9 132514344 132516344 PTGS2 chr1 186648559 186650559 PTK2B chr8 27167999 27169999 RB1 chr13 48876883 48878883 RBL2 chr16 53467351 53469351 REL chr2 61107752 61109752 RELA chr11 65429443 65431443 RELB chr19 45503712 45505712 RELT chr11 73086713 73088713 RIPK2 chr8 90768975 90770975 RNASE3 chr14 21358562 21360562 RNASE7 chr14 21509385 21511385 RPL13A chr19 49989865 49991865 RPS27A chr2 55458635 55460635 RPS27A chr2 55458635 55460635 S100A12 chr1 153347075 153349075 S1PR3 chr9 91605362 91607362 SARM1 chr17 26697987 26699987 SCYE1 chr4 107235853 107237853 SELL chr1 169679843 169681843 SERPINA1 chr14 94854153 94856153 SERPINC1 chr1 173885516 173887516 SERPINE1 chr7 100769379 100771379 SFTPD chr10 81707861 81709861

SIGIRR chr11 416397 418397 SLC11A1 chr2 219245752 219247752 SLPI chr20 43882206 43884206 SMARCB1 chr22 24128150 24130150 SPP1 chr4 88895802 88897802 SPTAN1 chr9 131313866 131315866 STAB1 chr3 52528356 52530356 STAT1 chr2 191877976 191879976 STAT3 chr17 40539513 40541513 TBK1 chr12 64844937 64846937 TFCP2 chr12 51565664 51567664 TGFB1 chr19 41858816 41860816 TICAM1 chr19 4830737 4832737 TICAM2 chr5 114937109 114939109 TIRAP chr11 126151982 126153982 TLR1 chr4 38805412 38807412 TLR10 chr4 38783589 38785589 TLR2 chr4 154604441 154606441 TLR3 chr4 186989309 186991309 TLR4 chr9 120465460 120467460 TLR5 chr1 223315624 223317624 TLR6 chr4 38830160 38832160 TLR7 chrX 12884202 12886202 TLR8 chrX 12923739 12925739 TLR9 chr3 52259179 52261179 TMPRSS11D chr4 68748716 68750716 TNF chr6 31542350 31544350 TNFAIP3 chr6 138187581 138189581 TNFRSF10A chr8 23081680 23083680 TNFRSF10B chr8 22925700 22927700 TNFRSF10C chr8 22959434 22961434 TNFRSF10D chr8 23020540 23022540 TNFRSF11A chr18 59991548 59993548 TNFRSF11B chr8 119963383 119965383 TNFRSF12A chr16 3069313 3071313 TNFRSF13B chr17 16874402 16876402 TNFRSF13C chr22 42321821 42323821 TNFRSF14 chr1 2486805 2488805 TNFRSF17 chr16 12057964 12059964 TNFRSF18 chr1 1141089 1143089 TNFRSF19 chr13 24152639 24154639 TNFRSF1A chr12 6450261 6452261 TNFRSF1B chr1 12226060 12228060

TNFRSF21 chr6 47276680 47278680 TNFRSF25 chr1 6525255 6527255 TNFRSF4 chr1 1148512 1150512 TNFRSF6B chr20 62327021 62329021 TNFRSF8 chr1 12122434 12124434 TNFRSF9 chr1 7999887 8001887 TNFSF10 chr3 172240269 172242269 TNFSF11 chr13 43147291 43149291 TNFSF12 chr17 7451375 7453375 TNFSF13 chr17 7460609 7462609 TNFSF13B chr13 108920977 108922977 TNFSF14 chr19 6669599 6671599 TNFSF15 chr9 117567408 117569408 TNFSF18 chr1 173019103 173021103 TNFSF4 chr1 173175471 173177471 TNFSF8 chr9 117691770 117693770 TNFSF9 chr19 6530010 6532010 TOLLIP chr11 1329839 1331839 TRADD chr16 67192812 67194812 TRAF1 chr9 123688173 123690173 TRAF2 chr9 139779965 139781965 TRAF3 chr14 103242816 103244816 TRAF6 chr11 36530822 36532822 TREM1 chr6 41253457 41255457 TRIM5 chr11 5705293 5707293 TSG101 chr11 18547489 18549489 TYMP chr22 50967514 50969514 UBE2N chr12 93835026 93837026 UBE2V1 chr20 48731494 48733494 VPS4A chr16 69344287 69346287 XCL1 chr1 168544856 168546856 XCR1 chr3 46067979 46069979 XPO1 chr2 61764418 61766418 YY1 chr14 100704102 100706102 ZBTB7A chr19 4065816 4067816