Novel Tripartite Motif Containing 22 Interactions in the Context of Very Early Onset Inflammatory Bowel Disease

Khalid Hossain

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto

Khalid Hossain

Master of Science

Department of Biochemistry University of Toronto

2018 Abstract

Genetics predominate in very early onset inflammatory bowel disease (VEOIBD). With poor outcomes and therapeutic response, monogenic causes revealed by whole exome sequencing has potential for personalized therapies. Our recently published E3 ubiquitin ligase TRIM22 variants revealed novel roles in regulating NOD2. Apart from anti-viral pathways, the extent of

TRIM22’s influence remains unclear. We interpreted TRIM22 BioID performed by collaborators. On the list of potential binding partners is the Nucleosome Remodeling and

Deacetylase (NuRD) complex. Functional analysis shows TRIM22 binding HDAC1, the catalytic core of the NuRD complex, but may not ubiquitinate HDAC1 or alter its stability.

VEOIBD variant TRIM22 R442C significantly reduces binding, and is the only variant to not form nuclear bodies. HDAC1 levels may be reduced in TRIM22 R321K patient pre-treatment colon biopsies. Given recent insights into TRIM22 mediated monocyte apoptosis, we suggest interaction with the NuRD complex plays a role in cell fate and inflammation.

Acknowledgments

First and foremost I would like to thank Qi for her early mentorship and guidance. Qi helped build me up from my limited research experience, and developed my skills to a level of self- sufficiency before leaving to grow her career in China. I couldn’t have asked for a more fun, experienced, and accommodating lab manager than Hui. Nor could I have asked for a more “fun”, “experienced”, and “accommodating” tech than Ryan (who really was very helpful). I greatly value the lengthy scientific (and non-scientific) discussions with Jessica, Frozan, Vritika, Zuhra, and anyone else who may have been in the vicinity. I felt safe knowing that anytime I decided to stay for a late night or weekend I would be in the company of one of them, or Neel, Gaby, Lin, or anyone else. I will cherish my food tours with Gaby and Karoline in Boston, which would eventually give rise to the Muise Lab Adventures. Our little band of foodies would later invite Sasha and Emily to indulge in our sins. I thank Abdul, Alessia, and Maggie for the short, but insightful time I’ve spent with them in the lab. There has been an abundance of mentorship; with clinical expertise from Takashi and Eileen, Jie’s expertise in all things imaging, and Neil’s mastery of all things WES and molecular genetics. Neda has helped me during my time at UofT, and has helped greatly during my transition to McGill. I will carry with me the invaluable education bestowed upon me by Gaby and Sasha, on matters inside and outside of the lab. I will not take anything I have been given for granted.

I owe my health and energy to my partner, Monissa, for her undying support at home and her helping me maintain a sustainable life outside of the lab. There is no doubt in my mind that I have only been able to put in as many hours, weekends, and holidays due to her perseverance. I owe my time at UofT to Monissa’s parents, UofT alumni Dr. Avi Chakrabartty and Dr. Nancy Ng, for their guidance during my pursuit of a Master’s in Biochemistry.

I would like to thank my committee members, Dr. Rotin and Dr. Brumell, for shaking up my project early on and setting my project onto a clearly defined path with fresh ideas. Lastly, I would like to thank Aleixo for understanding my motivations from the moment I set foot in his office. Aleixo has never failed to be supportive of my endeavours. Aleixo provided a project which would accomplish my goals, supported me through each step of my academic progress, and played a key role in my scholarships, awards, and presentations at conferences. Most importantly, I’m thankful for a lab Aleixo has filled with brilliantly talented minds.

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Table of Contents

Acknowledgments...... iii

Table of Contents ...... iv

List of Tables ...... vi

List of Figures ...... vii

Abbreviations ...... viii

List of Appendices ...... xiii

Chapter 1 Introduction ...... 1

Introduction ...... 1

1.1 Inflammatory Bowel Disease ...... 1

1.1.1 The Gastrointestinal Tract...... 1

1.1.2 The Diseased State ...... 1

1.1.3 Disease Management ...... 2

1.1.4 Epidemiology and the Environment ...... 3

1.1.5 Very Early Onset Inflammatory Bowel Disease ...... 3

1.2 TRIM22 Controls NOD2 Signalling in VEOIBD ...... 7

1.2.1 Ubiquitin-Proteasome System ...... 7

1.2.2 TRIM22: A Tripartite Motif-containing Protein...... 7

1.2.3 TRIM22-dependent Restriction of HIV-1 ...... 8

1.2.4 TRIM22 Localization...... 9

1.2.5 TRIM22 Regulation of NOD2 Is Associated with VEOIBD ...... 9

1.3 Rationale ...... 13

Chapter 2 Materials and Methods ...... 14

Materials and Methods ...... 14

2.1 BioID and WES Analyses ...... 14

2.2 Plasmid Constructs...... 14 iv

2.3 Co-immunoprecipitation ...... 15

2.4 Western Blotting ...... 20

2.5 Ubiquitination Assay ...... 20

2.6 Cycloheximide Pulse Chase ...... 20

2.7 Immunofluorescence ...... 21

2.7.1 Cell Culture ...... 21

2.7.2 Colon Tissue ...... 22

Chapter 3 Results ...... 24

Results ...... 24

3.1 BioID List and Interpretation ...... 24

3.2 TRIM22 interacts with HDAC1...... 34

3.3 TRIM22 VEOIBD variant shows reduced HDAC1 binding ...... 38

3.4 TRIM22 R442C does not form nuclear bodies ...... 42

3.5 TRIM22 does not ubiquitinate HDAC1 in unstimulated cells ...... 47

3.6 TRIM22 does not alter HDAC1 stability in unstimulated cells ...... 50

3.7 HDAC1 may show reduced levels in TRIM22 patient colon ...... 54

Chapter 4 Discussion and Future Directions ...... 58

Discussion and Future Directions ...... 58

Chapter 5 Conclusion ...... 68

Conclusion ...... 68

References ...... 69

Appendix ...... 76

Contributions...... 78

List of Tables

Table 1: List of Antibodies

Table 2: Mass spectrometry identification of TRIM22 BioID interactors in HEK293T cells.

List of Figures

Figure 1: Factors influencing IBD based on age of onset.

Figure 2: Protein domain schematic of tripartite-containing 22 (TRIM22).

Figure 3: Potential TRIM22 binding partners revealed by BioID.

Figure 4: HDAC1 co-immunoprecipitates with TRIM22.

Figure 5: VEOIBD associated variant TRIM22 R442C shows significantly reduced binding to HDAC1.

Figure 6: TRIM22 R442C does not form nuclear bodies in HEK293T cells.

Figure 7: TRIM22 does not ubiquitinate HDAC1 in unstimulated HEK293T cells.

Figure 8: Cycloheximide chase of endogenous HDAC1 shows unchanged HDAC1 half-life in HEK293T cells transfected with Flag-TRIM22.

Figure 9: Colon tissue immunofluorescence of HDAC1 and TRIM22.

Figure 10: TRIM22 interaction with HDAC1 may be involved in monocyte apoptosis.

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Abbreviations

5-ASA 5-aminosalicylic acid

AP-MS affinity purification coupled to mass spectrometry

ATP adenosine triphosphate

Bak Bcl-2 homologous antagonist/killer

BirA* bifunctional ligase/repressor BirA R118G

BSA bovine serum albumin

CD Crohn’s disease

CDK9 cyclin-dependent kinase 9

CHD(3/4) chromodomain-helicase-DNA-binding protein (3/4)

CHX cycloheximide

Co-IP co-immunoprecipitation

CRAPome contaminant repository for affinity purification

DAPI 2-(4-amidinophenyl)-1H-indole-6-carboxamidine

DIC differential interference contrast

DMEM Dulbecco's modified Eagle's medium

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

E1 ubiquitin activating enzyme

E2 ubiquitin conjugating enzyme

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E3 ubiquitin ligase

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

EOIBD early onset IBD

FBS fetal bovine serum

FFPE formalin fixed paraffin embedded

FITC fluorescein isothiocyanate

Flag DYKDDDDK epitope

Gag group-specific antigen

GALT gut-associated lymphoid tissue

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GATAD2(A/B) GATA zinc finger domain containing 2(A/B)

GI gastrointestinal

GWAS genome-wide association study

HA hemagglutinin tag YPYDVPDYA

HDAC(1/2) histone deacetylase (1/2)

HECT homologous to the E6-AP carboxyl terminus

HEK293T human embryonic kidney 293T

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HIV-1 human immunodeficiency virus-1

HRP horseradish peroxidase

IBD inflammatory bowel disease

IBD-U IBD unclassified

IF immunofluorescence

IFN interferon

IP immunoprecipitation

ISG interferon-stimulated gene

JAK Janus kinase

LPS lipopolysaccharides

MBD(2/3) methyl-CpG-binding domain protein (2/3)

MDM2 mouse double minute 2 homolog

MDP muramyl dipeptide

MG132 benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan- 2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate mRNA messenger ribonucleic acid

MTA(1/2/3) metastasis-associated 1

Myc EQKLISEEDL epitope

NB nuclear body

NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells

NLS nuclear localization signal

NOD2 nucleotide-binding oligomerization domain-containing protein 2

NuRD nucleosome remodeling and deacetylase

PAGE polyacrylamide gel electrophoresis

PBMC peripheral blood mononuclear cell

PBS phosphate-buffered saline

PFA paraformaldehyde

PML promyelocytic leukemia protein

PMSF phenylmethanesulfonyl fluoride p-TEFb positive transcription elongation factor

PVDF polyvinylidene fluoride

RBCC RING–B-box–coiled-coil

RING really interesting new gene

SDS sodium dodecyl sulfate

SPRY named from spla and the ryanodine receptor

STAT signal transducer and activator of transcription

Tat trans-activator of transcription

TNF tumor necrosis factor

TRIM tripartite motif

TRIM22 tripartite motif-containing 22

TRIM5α tripartite motif-containing protein 5 alpha

Tris 2-Amino-2-(hydroxymethyl)propane-1,3-diol

Ub ubiquitin

UC ulcerative colitis

V5 GKPIPNPLLGLDST epitope

VEOIBD very early onset IBD

Vpr viral protein R

WES whole exome sequencing

Y2H yeast two-hybrid

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List of Appendices

Figure A: TRIM22 may bi-directionally co-immunoprecipitate with HDAC1.

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Chapter 1 Introduction Introduction 1.1 Inflammatory Bowel Disease 1.1.1 The Gastrointestinal Tract

The digestive system, responsible for nutrient uptake and waste elimination, is primarily composed of the gastrointestinal (GI) tract and accessory organs. The GI tract extends from the mouth to the anus, processing food through mechanical (mastication, peristalsis, and defecation) and chemical (enzymes within the mouth, stomach, and small intestine) means.1 As a bolus passes through the stomach, into the small intestine, through the colon and finally the rectum, the processing food and waste encounters a diverse microbiome considered by many as effectively forming its own organ not unlike the immune system.2 Significant changes to the host-microbe mutualism in the gut can result in inflammation of the GI tract which, particularly in genetically susceptible hosts, can manifest into a group of chronic disorders referred to as inflammatory bowel disease (IBD).2,3

1.1.2 The Diseased State

IBD is traditionally classified into two types of GI disorders: ulcerative colitis (UC) and Crohn’s disease (CD).3 UC involves inflammation of the intestinal mucosa, the luminal side of the GI tract composed of the epithelium, lamina propria, and muscularis mucosa.1,4 Epithelial differentiation along the GI tract is based on specialized function of the organs, such as secreting a protective mucous barrier, enzymes, and products required for digestion. The lamina propria consists of connective tissue permeated by blood and lymphatic vessels to deliver nutrients and hormones while absorbing products of digestion. The gut-associated lymphoid tissue (GALT) is comprised of lymphocytes and lymph nodules, infiltrating throughout the lamina propria to maintain mucosal homeostasis.1 During IBD, a breakdown of the mucosal barrier resulting in microfloral infiltration into the lamina propria leads to a sustained inflammatory response from

1 2

the immune system. Mucosal injury is associated with dysbiosis of the gut microbiome, possibly aggravating the persistent inflammation further.4,5

Although UC is restricted to the colorectal mucosa, CD is transmural with inflammation involving all layers of the affected GI organs, which in CD are not restricted to the colon. Symptoms of CD depend on location of disease presentation and, coupled with discontinuous localization of inflammation throughout the GI tract, the symptoms can be heterogeneous.5 Since UC is restricted to the colon, it commonly presents with blood in the stool. Most patients present with proctitis (rectal bleeding, urgency) with disease eventually progressing continuously from the rectum to involve the sigmoid and descending colon (proctitis plus bloody diarrhea, weight loss, cramping), to involvement of the colon up to the cecum in extensive colitis, or including the cecum in pancolitis.4 Rectal bleeding and bloody diarrhea also presents in CD when the colon is involved, with roughly a third of patients presenting with perianal disease.5 Whereas a third of UC patients present with extraintestinal manifestations, these symptoms are present in half of CD patients.4,5 Extraintestinal manifestations can be associated with, or independent of, disease activity, exemplifying autoimmune factors of the diseases.5

1.1.3 Disease Management

The goal of treating IBD is to induce and maintain a state of remission with the aim of preventing disability, bowel damage, colectomy in UC, colorectal cancer, and complications.4,5 For mild to moderate UC, the anti-inflammatory drug 5-aminosalicylic acid (5-ASA) can be administered orally, or rectally via enemas or suppositories.4 There is weak evidence to support the use of 5-ASAs in CD, but 5-ASAs are administered for those diagnosed with IBD unclassified (IBD-U).5 Instead, mild CD is first treated with steroids with a combination of immunosuppressants in moderate disease, with biologics like anti-TNF monoclonal antibodies playing a role in more severe disease.5 IBD remains a chronic, incurable disease, with intervention involving treatment of acute symptom flares.

1.1.4 Epidemiology and the Environment

Northern Europe, the United Kingdom, and North America have been historically associated with IBD, with the highest reported incidence and prevalence of both types of the disease. With the exception of Australia, Israel, and South Africa, IBD is relatively rare as reported everywhere else in the world. However, incidence of IBD has since been observed to be dynamic. Incidence of UC has been increasing in previously “low risk” countries in Asia and Latin America.6 Developing countries tend to have low incidence of IBD, but as countries develop through industrialization with changes in diet and lifestyle, incidence of UC increases and incidence of CD begins to emerge later.6,7 This trend has been observed throughout the world, but it’s unclear if incidence is low in developing countries due to low diagnostic awareness.6 The increasing incidence of IBD has been at its highest these past several decades, but the rate of increase may have recently slowed.7 Despite a slowing in the increasing incidence, Canada remains one of the most affected by IBD with one of the highest incidence and prevalence in the world, costing an estimated $2.8 billion in 2012.8 Unlike other parts of the world, rates of CD in Canada are higher than that of UC.8,9

An interesting example of the effect of the environment in disease pathogenesis is seen through immigration studies. The incidence of UC for second-generation Indians in the UK is similar to the country, and is higher than in India. In fact, the incidence of paediatric IBD is higher among South Asian immigrants in British Columbia, Canada than the native population. However, the opposite was seen for East Asian immigrants in Ontario, Canada. Older age at immigration was associated with a decreased risk in IBD.7

1.1.5 Very Early Onset Inflammatory Bowel Disease

Rates of pediatric IBD, particularly CD, has been on the rise globally, with up to 25% of patients developing IBD before the age of 18.10 Paediatric IBD differs from adult-onset disease, such as with an increased disease severity, and impairments to growth and nutrition.10 The Montreal classification of IBD defined onset under age 17 as a distinct group, and the Pediatric Paris

modification of the Montreal classification further classified onset under age 10 as a subdivision of pediatric IBD, sometimes referred to as early onset IBD (EOIBD).11

Despite progress in the understanding of IBD over the past few decades, the etiology of the disease remains unknown.7 Although environmental factors such as vitamin D levels, antibiotic exposure, and diet likely play important roles in IBD, the disease is more likely the result of a combination of complex interactions of many of these factors with the host’s immune system. As such, genetics are believed to play an equally important role in IBD, with heritable inappropriate immune response to the gut microbiota. 2–14% of CD patients and 8–14% of UC patients report a family history of IBD, with the children of two affected parents having a 30% chance of developing IBD. Most striking are twin studies, which show 20–50% concordance rate for monozygotic twins with CD compared to 10% in dizygotic twins.7 Genetics are believed to play a more important role in younger patients due to their increased prevalence of family history, and due to less time having elapsed for environmental factors to influence the disease relative to late onset IBD (Fig. 1).12

Some suggest that diagnosis under age 6 appropriately describes another subdivision within EOIBD, referred to as very early onset IBD (VEOIBD).11 This subdivision of IBD is noted for differences in disease phenotype in relation to older pediatric patients, such as low occurrence of ileal inflammation, high occurrence of pancolitis, higher prevalence of Crohn’s colitis, and an increased risk of surgery and biologic therapy. Since VEOIBD typically responds poorly to conventional therapies, and since the incidence of pediatric IBD is rising in developed and developing countries, the pathogenesis of VEOIBD has become an important area of investigation.13 Onset under age 6 was especially suggested as the upper range of VEOIBD due to an increase in conventional polygenic IBD starting from the age of 7.11 VEOIBD’s relative enrichment of monogenic IBD makes the disease group a fitter candidate for Mendelian genetic studies. Whole exome sequencing (WES) has provided a cost-effective strategy in identifying harmful mutations in the coding regions of the genome.14

Figure 1: Factors influencing IBD based on age of onset. Early onset disease has a higher prevalence of family history and is enriched in monogenic causes, particularly in VEOIBD. As age of onset increases, more time has elapsed for the IBD patient before the culmination of their disease pathogenesis. As such, monogenic causes become less likely, and instead polygenic diseases play a larger role in the presence of environmental triggers. The effect of the environment increases in late onset IBD.12 Adapted from Ruel et al., 2014.

1.2 TRIM22 Controls NOD2 Signalling in VEOIBD

Our recent WES of VEOIBD patients and their parents identified autosomal recessive variants predicted to be damaging in the antiviral E3 ubiquitin ligase TRIM22 previously implicated in cell innate immunity.15–18

1.2.1 Ubiquitin-Proteasome System

TRIM22 is a member of a class of proteins involved in the ubiquitin-proteasome system, the most utilized regulated protein degradation system in eukaryotes.19 In this system, the highly conserved ubiquitin (Ub) protein acts as a signaling molecule, first undergoing ATP-dependent activation by an E1 Ub-activating enzyme. Activated Ub is then transferred to an E2 Ub- conjugating enzyme, which catalyzes the transfer of Ub to the substrate with the help of an E3 Ub-ligase. E3 ligases determine substrate specificity and can be divided into two major classes based on their ligase domains. HECT domains first receive the activated Ub from an E2 enzyme to its own conserved Cys residue before transferring the Ub to the substrate. RING domain E3 ligases, like TRIM22, catalyze E2 enzymes’ direct transfer of activated Ub to substrates.19 Ub is first conjugated by its C-terminus to a specific lysine on the target protein as an isopeptide bond, which could act as a signal referred to as mono-ubiquitination. Poly-ubiquitination signals occur when additional Ub molecules are conjugated by their C-termini to specific lysine residues, or the N-terminal methionine, on the first conjugated Ub, forming complex branches and chains of Ub. The most widely known homotypic Ub conjugate is K48-linked, forming chains which act as a signal for proteasomal degradation. Another known homotypic Ub conjugate is K63-linked, resulting in chains that promote the formation of key signaling complexes with pathways involved in signal transduction.20

1.2.2 TRIM22: A Tripartite Motif-containing Protein

Tripartite motif-containing (TRIM) proteins are a superfamily of RING E3 ligases which contain a tripartite (or RBCC) motif. This motif includes a RING domain, one or two B-box domains involved in protein-protein interactions, and a coiled-coil motif used for self-association (Fig. 2).

TRIM22, along with other TRIM proteins, form homotrimers.21 TRIM22 is known for undergoing self-ubiquitination, targeting itself for degradation by the proteasome.22

The expressions of many TRIM proteins are induced by interferons (IFNs) in association with their roles in innate immunity and countering intracellular pathogens.23 IFNs are small secreted proteins produced in response to the detection of pathogen markers. IFNs then bind to receptors on surrounding cells, bringing receptors into proximity and causing a conformational change resulting in activation of associated JAK proteins in the cytoplasm through transphosphorylation. JAKs then phosphorylate highly conserved tyrosine residues on the receptors, resulting in binding and phosphorylation of STAT proteins which translocate to the nucleus to promote the expression of interferon-stimulated genes (ISGs).24

1.2.3 TRIM22-dependent Restriction of HIV-1

TRIM22 was first discovered as an ISG induced in various cell lines which was able to downregulate transcription directed by the human immunodeficiency virus-1 (HIV-1) promoter.25 TRIM22’s role in HIV-1 pathogenicity and T cell activation was further investigated through DNA microarray analyses; HIV-1 infection, or expression of its Tat transactivator, in immature dendritic cells induced ISG expression, including TRIM22. This results in increased chemokine production which attracts activated T cells and macrophages, the target hosts of HIV- 1.26 Another study also reported TRIM22 expression increasing in monocyte-derived macrophages stimulated with IFNα, LPS, or HIV-1 infection, while TRIM22-transduced macrophages reduced HIV-1 infection by 50–90%.27 Later studies corroborated these findings, with type 1 interferons strongly increasing TRIM22 levels and restricting HIV-1 replication, while also demonstrating that knockdown of TRIM22 increases HIV-1 particle release.

TRIM22 likely disrupts HIV Gag trafficking as TRIM22 overexpression resulted in Gag diffusely distributed throughout the cytoplasm instead of accumulating at the plasma membrane for viral release. The TRIM22-dependent restriction of HIV particle release requires the catalytic residues of its RING domain, C15A and C18A.15 However, TRIM22 has also been shown to inhibit HIV-1 transcription independently of the RING domain catalytic activity. As the only

protein constitutively expressed in U937 cells nonpermissive to HIV-1 when compared to permissive cells, TRIM22 was suggested as interfering with the Tat HIV-1 protein from enhancing viral transcription independently of TRIM22 catalytic activity.28 TRIM22-dependent restriction of HIV-1 was supported by evidence from patient-derived peripheral blood mononuclear cells (PBMCs) of high risk HIV-1-positive and -negative participants. TRIM22, but not its close paralog TRIM5α, was associated with type-1 IFN response and had a negative correlation with plasma viral load.29

1.2.4 TRIM22 Localization

Endogenous TRIM22 cellular localization is dynamic and dependent on specific cellular signals. TRIM22 was shown to form nuclear bodies, and more dramatic nucleolar bodies, following its induced expression by progesterone. These bodies did not form upon induction of expression by IFNγ. TRIM22 localization is also dependent on cell-cycle, with nuclear bodies forming in early G0/G1 and dispersing in the S phase.30 Nuclear body formation was dependent on the RING domain, but not the catalytic cysteines required for ubiquitination. TRIM22 contains a nuclear localization signal (NLS) that is necessary, but not sufficient, for its nuclear localization. More important is the B30.2/SPRY domain, with residues 491–494 being critical for localization and nuclear body formation.31 Species specific localization, when comparing human and rhesus TRIM22, is due to two variable loops in the positively selected B30.2/SPRY domain.32 Endogenous TRIM22 was also found to colocalize with centrosomes independently of cell cycle phase, suggesting possible viral replication and protein degradation roles since some viral assembly occurs in the centrosome.33

1.2.5 TRIM22 Regulation of NOD2 Is Associated with VEOIBD

In 2011, it was discovered that TRIM22 activates NF-κB in a dose-dependent manner and its overexpression significantly induces pro-inflammatory cytokine secretion by U-937 cells.34

Recent investigation of TRIM22 mutations identified in our VEOIBD patients led to the functional characterization of TRIM22’s novel role in regulating NOD2, a cytoplasmic sensor of molecular patterns which itself regulates the anti-bacterial/viral response and inflammatory homeostasis. Defects in NOD2 typically lead to intestinal barrier dysfunction and is a major genetic risk factor in developing CD.35 Immunofluorescence (IF) revealed colocalization of TRIM22 and NOD2, with altered localization and expression in patient colonic biopsies. Co- immunoprecipitation (co-IP) confirmed TRIM22 interaction with NOD2, while reporter assays revealed TRIM22’s enhancement of NOD2-dependent IFNβ and NF-κB activation. Our lab then demonstrated TRIM22’s mediation of NOD2 K63-linked polyubiquitination, regulating NOD2’s localization. The TRIM22 mutations include compound heterozygous variants R150T and S244L (coiled-coil motif) in one patient, homozygous R321K (B30.2/SPRY domain) in another patient, and homozygous R442C (B30.2/SPRY domain) in the final patient (Fig. 2). These VEOIBD associated mutations disrupted NOD2 binding, polyubiquitination, and induction of IFNβ and NF-κB, ultimately reducing antiviral interferons and pro-inflammatory cytokines as confirmed by mRNA knockdown. Correlations of the TRIM22-NOD2 pathway with inflammatory regulators have been computationally shown, but the complete range of pathways influenced by TRIM22 remains a mystery.

Figure 2: Protein domain schematic of tripartite-containing 22 (TRIM22). TRIM22 belongs to the TRIM family of E3 ligases, owing to its TRIM (RBCC) motif that includes a ubiquitinating RING domain, a protein interaction-mediating B-box domain, a coiled-coil motif for homotrimerization, and a B30.2/SPRY domain which is critical for nuclear localization and some protein-protein interactions. VEOIBD associated variants lie within the coiled-coil motif and B30.2/SPRY domain.18 Adapted from Li et al., 2016.

1.3 Rationale

The discovery of TRIM22 as a NOD2 regulator is an example of the potentially undiscovered biological roles TRIM22 plays within the cell. I hypothesize that TRIM22 lies at a crossroad of multiple cellular pathways related to inflammatory disease phenotypes. To uncover unknown roles of TRIM22 in the context of VEOIBD, I investigated:

1. Candidate TRIM22 binding partners and their relation to VEOIBD.

2. The biochemical association of TRIM22 and HDAC1.

3. The possibility of TRIM22 VEOIBD variants impairing its role with HDAC1.

Chapter 2 Materials and Methods Materials and Methods 2.1 BioID and WES Analyses

TRIM22 BioID was performed by the Brumell lab as a collaboration, based on previously described protocols.36 In short, a Flag-BirA*-TRIM22 construct was stably expressed in HEK293T cells, with a Flag-BirA* construct acting as a negative control. Cells were incubated for 24 h with 50 µM biotin and lysed in denaturing buffer. Biotinylated proteins in the lysate were captured with streptavidin beads and analyzed by mass spectrometry. Identified proteins with peptide hits less than 4X the control, and proteins identified by the contaminant repository for affinity purification (CRAPome), were excluded from the final BioID list.37 The remaining list was then ordered by relative peptide hits.

The TRIM22 BioID list was then cross-referenced with a WES (Illumina) dataset involving a SickKids cohort of 1,183 probands with pediatric onset IBD (ages 0-18.5 years), including their affected and unaffected parents and siblings where available (total samples = 2,704). WES was performed in collaboration with the Regeneron Genetics Center. We performed trio-based analysis of 492 complete trios using a proband-based analytical pipeline (Gemini) to identify recessive (compound heterozygous and homozygous) and de novo variants of potential interest in the affected probands.

2.2 Plasmid Constructs

pcDNA3.1 nFlag-tagged TRIM22 for wildtype, R150T, and R442C were previously constructed by site-directed mutagenesis. Site-directed mutagenesis was performed on wildtype nFlag- TRIM22 plasmid with primers generated by ACGT Co. for S244L (5’- cagcatctctactgacagtcccctcaacctccgc-3’ for forward, 5’-gcggaggttgaggggactgtcagtagagatgctg-3’ for reverse) and R321K (5’-cagttttcacttgtttctgatccacagaaatagcaacattcg-3’ for forward, 5’-

14 15

cgaatgttgctatttctgtggatcagaaacaagtgaaaactg-3’ for reverse). pcDNA3.1 nMyc-tagged TRIM22 was generated by Gateway cloning (Invitrogen) with wildtype nFlag-TRIM22. cFlag-HDAC1 was purchased from Addgene, and was a gift from Eric Verdin (#13820).38 cV5-HDAC1 was generated by the SPARC BioCentre. nHA-ubiquitin was purchased from Addgene, and was a gift from Ted Dawson (#17608).39 All constructs were verified by sequencing conducted by ACGT Co.

2.3 Co-immunoprecipitation

HEK293T cells were maintained in DMEM containing 10% heat-inactivated FBS, penicillin, and streptomycin in 5% CO2 at 37°C. Sub-confluent cells were transferred to 10 cm dishes and grown for ~16 h to obtain ~90% confluency. 0.2 µg of HDAC1 plasmid, 0.3 µg of TRIM22 plasmid, and pcDNA3.1 empty vector (where applicable) were co-transfected with PolyJetTM In Vitro DNA Transfection Reagent from SignaGen® Laboratories according to the manufacturer’s protocol. 24 h post-transfection, cells were collected with PBS and the pellet was resuspended with 800 µL lysis buffer (150 mM NaCl, 50 mM HEPES, 1% Triton X-100, 10% glycerol, 1.5

mM MgCl2 and 1.0 mM EGTA) supplemented with protease inhibitors (1 mM PMSF, P2714 inhibitor cocktail [Sigma-Aldrich Co.], and 1 mM sodium orthovanadate). Following a 20 min incubation on ice, samples were centrifuged at 14k rpm for 15 mins and the supernatant was collected. The pellet was homogenized with three 5-second pulses using a pellet pestle on ice with 200 µL of lysis buffer. The homogenization step was repeated twice with 5 min rests. The samples were centrifuged at 14k for 10 mins and the supernatant was combined with the 800 µL of lysate previously collected.

Following a Bradford assay, 1 mg of lysate was diluted in lysis buffer for a total volume of 1 mL for each IP sample. Pre-clearing of samples involved a 30 min incubation on a rotator at 4°C with 20 µL (1:1 slurry) of protein G agarose beads (PRA286, BioShop Canada Inc.) and 1 µg of ChromPure Mouse IgG (015-000-003, Jackson ImmunoResearch Inc.). Beads were pelleted at 2k rpm at 4°C for 10 s, and supernatant was transferred to a new tube for another 30 min pre-clear with 20 µL of beads. Supernatant was transferred to a new tube for a final time and incubated for

2 h with the appropriate antibody (anti-V5, anti-Flag beads, anti-myc), with 20 µL of beads if the antibody is not already conjugated to beads. Beads were then centrifuged at 2k rpm at 4°C for 10 s, washed three times 5 mins each with lysis buffer, and resuspended in 30 µL 1x SDS protein sample buffer (40% glycerol, 240mM Tris/HCl, 8% SDS, 0.04% bromophenol blue, 5% beta- mercaptoethanol). Samples were split into separate SDS-PAGE gels for separate Western blot analyses of TRIM22 and HDAC1 since both proteins are close in band size. Western blot was performed as described in 2.4. Unpaired parametric t test was performed with GraphPad Prism software.

Table 1: List of Antibodies

Primary Antibody Provider of 1° 1° Antibody 2° Antibody 2° Antibody Dilution or Antibody amount Dilution

Immunoprecipitation

Flag beads A2220, Sigma- 20 µL 1:1 N/A N/A Aldrich Co. slurry

Anti-V5 MA5-15253, 1:1000 N/A N/A Thermo Fisher Scientific

Anti-MYC 05-724, EMD 1:1000 N/A N/A Millipore Co.

Anti-HA 901501, 1:1000 N/A N/A BioLegend, Inc.

Western Blot Analysis

Anti-V5 13202S, Cell 1:1000 Peroxidase 1:3000 Signaling AffiniPure Goat Technology, Inc. Anti-Rabbit IgG (H+L), Jackson ImmunoResearch Inc., 111-035-144

Anti-Flag 2368S, Cell 1:1000 Peroxidase 1:3000 Signaling AffiniPure Goat Technology, Inc. Anti-Rabbit IgG (H+L), Jackson

ImmunoResearch Inc., 111-035-144

Anti-GAPDH AM1020b, Abgent 1:2000 Peroxidase 1:3000 AffiniPure Goat Anti-Mouse IgG (H+L), Jackson ImmunoResearch Inc., 115-035-146

Anti-MYC 06-549, EMD 1:1000 Peroxidase 1:3000 Millipore Co. AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch Inc., 111-035-144

Anti-TRIM22 HPA003575, 1:1000 Peroxidase 1:3000 Sigma-Aldrich Co. AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch Inc., 111-035-144

Anti-HA 901501, 1:1000 Peroxidase 1:3000 BioLegend, Inc. AffiniPure Goat

Anti-Mouse IgG, Light Chain Specific for Western blotting after IP, Jackson ImmunoResearch Inc., 115-035-174

Immunofluorescence

Anti-Flag OTI4C5, OriGene 1:1000 Fluorescein (FITC) 1:125

Technologies, Inc. AffiniPure F(ab’)2 (provided by the Fragment Donkey Rotin Lab) Anti-Mouse IgG (H+L), Jackson ImmunoResearch Inc., 715-096-151

Anti-V5 D3H8Q, Cell 1:1000 Alexa Fluor® 594 1:125 Signaling AffiniPure Fab Technology, Inc. Fragment Donkey Anti-Rabbit IgG (H+L)

Anti-TRIM22 HPA003575, 1:200 Rhodamine 1:100 Sigma-Aldrich Co. AffiniPure Donkey Anti-Rabbit IgG (H+L), Jackson ImmunoResearch Inc., 711-025-152

Anti-HDAC1 ab31263, Abcam 1:200 Fluorescein (FITC) 1:100 plc. AffiniPure F(ab’)2 Fragment Donkey Anti-Mouse IgG (H+L), Jackson ImmunoResearch Inc., 715-096-151

2.4 Western Blotting

Equal quantities of protein were loaded based on Bradford assay results and SDS-PAGE was performed. Protein was transferred to either nitrocellulose or PVDF membranes via the Bio-Rad Trans-Blot® TurboTM Transfer system according to the manufacturer’s protocol. Skim milk (5% w/v in PBST) was used for blocking following transfer and for antibody dilution. Primary antibodies with 0.02% sodium azide were either incubated for 1 hour at room temperature or overnight at 4°C. Secondary antibodies were incubated for 1 hour at room temperature. Membranes were washed with PBST for 5 mins three times following each antibody incubation. Western blots were coated with Luminata Crescendo Western HRP substrate (WBLUR0100, EMD Millipore Co.) immediately prior to image capture by the LI-COR Odyssey® Fc imaging system. Image acquisition, processing, and quantification was performed with Image StudioTM Software. Membrane stripping was performed using the RestoreTM Western Blot Stripping Buffer by Thermo Fischer Scientific according to the manufacturer’s protocol. Statistics were performed using GraphPad Prism

2.5 Ubiquitination Assay

HEK293T cells were maintained, seeded, and transfected as described in 2.3 with 0.2 µg of HDAC1 plasmid, 0.3 µg of TRIM22 plasmid, 1.5 µg of HA-Ub, and pcDNA3.1 empty vector where applicable. 24 h post-transfection, media was replaced with warm media supplemented with 10 µM MG132 (M7449, Sigma-Aldrich Co.) and 100 µM chloroquine (C6628, Sigma- Aldrich Co.) for 2 h. Cells were collected and lysed as described in 2.3 with lysis buffer additionally supplemented with MG132 and chloroquine. Co-IP was performed with anti-HA as described in 2.3 without pre-clearing the lysates. The entire sample was analyzed at once by Western Blot as described in 2.4.

2.6 Cycloheximide Pulse Chase

HEK293T cells were maintained, seeded, and transfected as described in 2.3 with 0.3 µg of Flag- TRIM22 or Flag-TRIM22 R442C plasmid. 16 h post-transfection, cells were split into 6-well

plates. The media of each well was replaced with warm media supplemented with 100 µM cycloheximide (ab102480, Abcam plc.) either 12 h, 24 h, 36 h, or 48 h post-seeding. Cells were collected and lysed, as described in 2.3, immediately after the 48 h post-seeding treatment. 60 µg of each sample was loaded for SDS-PAGE and Western Blot analysis as described in 2.4. One phase decay nonlinear regression was performed with the following constraints: K>0, plateau=0.

2.7 Immunofluorescence 2.7.1 Cell Culture

12 mm glass coverslips were soaked in ethanol and flamed prior to placement into 24-well plates. Coverslips were then coated with poly-D-lysine for 5 mins, washed once with PBS, and left to dry for 2 h or overnight. HEK293T cells were maintained, seeded, and transfected as described in 2.3 on the coverslips. Transfections were performed ~5 h post-seeding and cells were incubated overnight. Cells were washed once with PBS, fixed with 4% PFA freshly diluted in PBS for 15 mins at room temperature, and washed with PBS three times. Cells were then permeabilized with 0.1% Triton X-100 freshly diluted in PBS for 15 mins at room temperature, and washed with PBS three times.

Cells were blocked for one hour in blocking buffer (4% BSA, 20% normal donkey serum in PBS) at room temperature. All antibodies used in the following steps are outlined in Table 1. Cells were incubated with primary antibodies diluted in blocking buffer for one hour at room temperature, and washed three times with washing buffer (0.05% Tween in PBS). Secondary antibodies diluted in blocking buffer were incubated on the cells for one hour at room temperature, which was followed by four washes. DAPI (1 µg/mL in PBS) was incubated on the cells for 5 mins, followed by one wash, and coverslips were then mounted with DAKO mounting medium.

Cells were visualized with a Leica DMi8 confocal microscope with a Zeiss objective lens set to 40x (oil immersion), numerical aperture 1.3, and a 0.1 µM step size. Confocal images were

captured with a Hamamatsu C9100-13 EM-CCD and analyzed with Perkin Elmer Volocity. Image acquisition equipment was provided by The Imaging Facility at The Hospital for Sick Children. One experiment was conducted in triplicate. Extended focus of green (Flag-TRIM22 variant) channel captures were quantified with ImageJ by isolating the nuclear area using the DAPI channel in extended focus. The DAPI channel was processed to create a threshold, and the green channel was subtracted. Multi-point tool was used to count the number of nuclear bodies, and averages were used for statistical analysis. Unpaired parametric t test was performed with GraphPad Prism software.

2.7.2 Colon Tissue

Colon biopsy specimens collected from normal, non-monogenic IBD, and the TRIM22 R321K patient were obtained before drug treatment or other clinical therapy. Formalin fixed paraffin embedded (FFPE) sections (5 µm) were cut and dewaxed by heating at 60°C and xylene for 1 h, followed by rehydration with decreasing concentrations of ethanol. Antigen retrieval was performed with high pressure in ethylenediaminetetraacetic acid (EDTA)-borate buffer (1 mM EDTA, 10mM borax [sodium tetraborate, Sigma-Aldrich Co.], 10 mM boric acid [Sigma- Aldrich Co.] with 0.001% Proclin 300 [Supelco, Inc.]) at pH 8.5.

Slides were incubated for 1 h at room temperature in 4% BSA in PBS, containing 20% normal donkey serum. Antibodies and dilutions used are outlined in Table 1. Incubation with mixed primary antibodies was performed overnight at 4°C. Slides were then washed three times with PBS for 10 mins each. A secondary antibody mix was applied and incubated at room temperature in darkness for 2 h. Slides were then washed three times for 10 mins each in darkness. RedDot2 (Biotium, Hayward, CA, USA) was used at 1:200 for nuclear counter staining. Sections were mounted overnight with VECTASHIELD antifade mounting medium (Vector Labs).

Fluorescent confocal images were obtained using a Leica confocal laser scanning microscope (TCS-SPE) and LAS-AF software (Leica Microsystems GmbH). The variable excitation

wavelengths of the krypton/argon laser were 488 nm for FITC conjugate, 568 nm for rhodamine conjugate, and 680 nm for RedDot2 with a Leica 63X/1.4-0.6 oil Flu objective. Images were 3 µM thick, captured at a 0.1 µM step size. Image processing, including resolution, color separation and merging of colors/fields, was carried out with Adobe Photoshop CS3 software (Adobe Systems Inc.).

Chapter 3 Results Results 3.1 BioID List and Interpretation

Our previous association of TRIM22 and VEOIBD identified TRIM22 as a novel regulator of NOD2, a pathogen recognition receptor which mediates pro-inflammatory and host defence pathways in response to intracellular bacteria.18,40 NOD2 was the first gene to be associated with CD and remains one of the greatest genetic risk factors for the disease.35,41,42 Since its discovery as an IFN-induced gene, TRIM22 has mainly been characterized by its roles as an anti-viral protein. However, some limited studies have associated TRIM22 with myeloid cell differentiation and diseases related to cell proliferation and autoimmunity.43 The first step towards revealing the possibility of other undiscovered biological roles of TRIM22 involves identifying potential interacting proteins. Collaborators from the Brumell Lab performed BioID, a method by which TRIM22 is fused with a promiscuous biotin ligase, BirA* (BirA R118G).36 BirA* is derived from the E. coli biotin ligase BirA, with the mutation resulting in the premature release of activated biotin, which then biotinylates amide groups within an approximate 10 nm radius or is quickly hydrolyzed.44 When the Flag-BirA*-TRIM22 construct is expressed in HEK293T cells with biotin, the proximal endogenous proteins are identified following biotin- affinity capture and mass spectrometry.36

Table 2: Mass spectrometry identification of TRIM22 BioID interactors in HEK293T cells. Flag-BirA*-TRIM22 was expressed in HEK293T cells with biotin. Proximal endogenous proteins biotinylated by the construct were captured with streptavidin beads and identified by mass spectrometry. Results were filtered based on a negative control involving BioID with Flag- BirA*. Final protein list is ordered based on number of peptide hits and the ratio of sample hits to control hits. The TRIM22 BioID list was then cross-referenced with a WES dataset involving a SickKids cohort of 1,183 probands with paediatric onset IBD, and their parents and siblings where available (total samples = 2,704). The number of probands from the WES dataset with variants of interest are shown for recessive (homozygous and compound heterozygous) and de novo variants.

Gene Sample Control Sample/ Autosomal Compound De Novo Total Peptide Peptide Control Recessive Heterozygous Variants Variants # # Ratio Variants Variants

TRIM22 2200 0

WRNIP1 147 0 3 4 7

NUP153 103 0

RAB4B 51.5 0

AHCTF1 24 0 2 2

N4BP1 23.5 0 1 1

CHD8 22.5 0 2 2

FIG4 21 0 1 1

RPRD2 19 0

MTA3 18.5 0

PML 18 0

NACC1 17 0

WDR70 16.5 0

ARID3B 15 0 2 2

SUGP2 13.5 0 3 2 5

ZNF318 12.5 0 1 1

MASTL 12 0

NEK10 11.5 0 3 3

RPRD1B 11.5 0 1 1

CBX1 11 0

ZHX3 10.5 0

CWC15 10 0

MED1 9 0

GATAD2A 8.5 0

CFDP1 8.5 0

PRCC 8 0

HAT1 8 0 1 1

GCFC1 7.5 0

KIAA0391 7.5 0

BCOR 7 0 2 2

FAT3 7 0 1 8 1 10

KPNA3 7 0

DGCR14 6.5 0

STUB1 6.5 0

KPNA4 6.5 0

DIDO1 6.5 0 1 14 15

CASC5 6 0

GTF2E2 5.5 0

PPIL4 5.5 0

PPIL3 5 0

TEX10 5 0

CPSF4 5 0

RBM14 5 0

MLLT1 5 0 1 1

XRN2 5 0 1 1

KIF4A 4.5 0 2 2

IK 4.5 0 1 1

CDC5L 4.5 0

MBD3 37 1 37.0

KDM3B 54 1.5 36.0

MTA1 91 3 30.3 26 26

SUGP1 23.5 1 23.5

MTA2 68 3 22.7

JMJD1C 39 2 19.5

CCNT1 26.5 1.5 17.7 1 8 29 38

NUP50 53.5 3.5 15.3

FIP1L1 22.5 1.5 15.0

WAPAL 14 1 14.0

HDAC1 53.5 4 13.4

ZNF281 12.5 1 12.5

DDX42 35 3 11.7

USP7 11.5 1 11.5

SMEK1 26 2.5 10.4

GATAD2B 31 3 10.3

TFIP11 20.5 2 10.3

CBX3 20 2 10.0

RPA1 10 1 10.0

TPX2 45.5 5 9.1

ZCCHC8 27 3 9.0

SCML2 102 12 8.5

U2SURP 54 6.5 8.3

SF3B5 7.5 1 7.5

CDK9 14.5 2 7.3

GNL2 7 1 7.0 2 2

VDAC1 7 1 7.0

CHD4 150 22.5 6.7 2 2

CHERP 26 4 6.5

PNISR 6.5 1 6.5

RBM27 16 2.5 6.4 1 1

XAB2 9.5 1.5 6.3 1 1

PRPF19 27.5 4.5 6.1

MDC1 12 2 6.0 6 6

SCAF4 6 1 6.0

ORC2 20.5 3.5 5.9

RBM17 17.5 3 5.8

RPL18A 14 2.5 5.6

BRD4 33.5 6 5.6

MAPRE2 46 8.5 5.4

MKI67 70 14 5.0 4 4 8

WDR82 21 4.5 4.7

TPGS1 27.5 6 4.6 2 2

LRRC49 27 6 4.5 1 1

SNRPB2 9 2 4.5

PRPF4 4.5 1 4.5

YKT6 26 6 4.3

SF3A2 6.5 1.5 4.3 6 6

SART3 17 4 4.3

TOP2A 8.5 2 4.3

SKIV2L2 19 4.5 4.2

SMARCA5 31.5 7.5 4.2

PPP1R10 61.5 15 4.1

SF1 12 3 4.0 2 2

PSIP1 10 2.5 4.0 1 1

LENG8 6 1.5 4.0

Figure 3: Potential TRIM22 binding partners revealed by BioID. The TRIM22 BioID list is graphically represented with genes ordered vertically based on number of relative peptide hits. Genes are highlighted in the upper panel based on whether they are known interactors of TRIM22, lie within known IBD loci based on genome-wide association studies (GWAS), are linked to primary immune deficiency, regulate NOD2 signaling, or are points of interaction with viral proteins. Also highlighted are components of the Mi-2/NuRD complex (red). Genes with variants of interest in our SickKids WES dataset are highlighted in gray (lower panel).

3.2 TRIM22 interacts with HDAC1

Multiple proteins highlighted by BioID are functionally associated with each other, the most striking of which involves the Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex (Fig. 3). The large macromolecular Mi-2/NuRD complex is the first to couple the independent functions of a chromatin remodeling ATPase and a deacetylase. Together, the ATPase and deacetylase act as the core catalytic proteins of the complex, deacetylating histone tails and mobilizing nucleosomes to increase chromatin compaction and gene repression.45 The combinatorial assembly of subunits allow for diverse specificity in a broad range of tissues with high conservation in plants and animals.46 Mi-2/NuRD subunits of similar function are mainly mutually exclusive, with the deacetylase functions carried out by either HDAC1 or HDAC2, and the ATPase function by CHD3 or CHD4.45 Also in the complex is the methyl-CpG-binding domain protein 2 (MBD2) which can recognize and bind methylated DNA, while the mutually exclusive MBD3 has lost this function. However, knockout models show that knockout of MBD2 are viable, while MBD3 knockout is embryonic lethal.47 MBD3 may act in protein- protein interactions, conferring some specificity of the NuRD complex to its target gene loci via binding with transcription factors. Like the MBD proteins, metastasis-associated 1 (MTA1), and its mutually exclusive counterparts MTA2 and MTA3, are also thought to add a dimension of specificity of the NuRD complex to its target loci through their association with transcription factors. The NuRD complex highlighted by TRIM22 BioID includes CHD4 as the Mi-2 protein, HDAC1 as the deacetylase, MBD3, structural and regulatory subunits GATAD2A/GATAD2B, and MTA1/MTA2/MTA3.

HDACs have been suggested as drug targets for IBD due to pro-inflammatory roles of the proteins. Although many HDAC isoforms exist with a diversity of functions, therapy could involve already established broad spectrum or specific inhibitors.48 HDAC1 has been implicated in colitis, particularly in murine studies where knockout in intestinal epithelial cells resulted in chronic colitis.49 Interestingly, HDAC1 has also been shown to be required for the IFNy induction of TRIM22, controlling TRIM22 at the transcription level.50 Since HDAC1 appears on the TRIM22 BioID list, their potential interaction was investigated.

Figure 4: HDAC1 co-immunoprecipitates with TRIM22. (A) HEK293T cells were transfected with nFlag-tagged TRIM22 and cV5-tagged HDAC1. Immunoprecipitation (IP) with lysates from transfected cells was conducted with anti-V5 (HDAC1) antibody (upper panels). Western blot was conducted with anti-V5 to detect precipitated V5-HDAC1, or anti-Flag (TRIM22). Upper panels also show IP with mouse IgG, acting as a negative control for non- specific IP of V5-HDAC1 and Flag-TRIM22. IP with anti-Flag (TRIM22) beads on lysates from the same transfected cells were blotted with the same antibodies, with V5-HDAC1 showing co- IP (middle panels). Expression of transfected constructs in the lysates was verified using the same antibodies, along with blotting of GAPDH acting as a loading control (lower panels). (B) A similar protocol was performed with nMyc-tagged TRIM22 and cFlag-tagged HDAC1. IPs were conducted with anti-Flag (HDAC1) beads (upper panels) or anti-Myc (TRIM22) antibody. Western blot was conducted with anti-Flag (HDAC1) or anti-Myc (TRIM22). Upper panels also show IP with mouse IgG. Lysates blotted with the same antibodies are shown with GAPDH as a loading control (lower panels).

3.3 TRIM22 VEOIBD variant shows reduced HDAC1 binding

In our recent publication associating four variants of TRIM22 with VEOIBD, the residue changes lie within the coiled-coil and B30.2/SPRY regions.18 These regions are not only important for homotrimerization and nuclear localization, respectively, but have also been implicated in protein-protein interactions.43 We showed that all variants disrupt the binding with NOD2 required for downstream pathways in the inflammatory response.18 Since HDAC1 co- immunoprecipitates with TRIM22 when the latter protein is pulled down (Fig. 4), it is important to investigate whether the four variants associated with VEOIBD affect this binding to test a potential link of this interaction with VEOIBD.

Figure 5: VEOIBD associated variant TRIM22 R442C shows significantly reduced binding to HDAC1. (A) HEK293T cells were transfected with cV5-tagged HDAC1 and nFlag-tagged VEOIBD associated variants of TRIM22 (wildtype, R150T, S244L, R321K, R442C). IP with lysates from transfected cells was conducted with anti-Flag (TRIM22) beads (upper panels). Western blot was conducted with anti-Flag to detect precipitated Flag-TRIM22 constructs, or anti-V5 to detect co-IP of V5-HDAC1. Upper panels also show IP with mouse IgG and equal parts lysates from each set of transfected cells, acting as a negative control for non-specific IP of V5-HDAC1 and Flag-TRIM22. Expression of transfected constructs in the lysates was verified using the same antibodies, along with blotting of GAPDH acting as a loading control (lower panels). (B) Western blot intensity of co-immunopreciptated V5-HDAC1 was normalized to co- IP with wildtype TRIM22. Error bars indicate SEM. ***p=0.0003, ****p<0.0001 (Student’s t test, n=4 independent experiments).

*** 1.5 B ns ns **** ns

1.0

0.5

0.0 Relative HDAC1Relative Band Intensity

R150T S244L R321K R442C W ildtype No TRIM22 Transfected TRIM22 Construct

3.4 TRIM22 R442C does not form nuclear bodies

TRIM22 can localize inside and outside the nucleus depending on a variety of factors, such as cell type, source of protein, stimulation, and stage of cell cycle. The B30.2/SPRY domain is critical for nuclear localization and differentiates the localization of various TRIM proteins.32,43 Two variants lie within this domain, but R422C lies within a particularly important loop in the domain.18,43 Since HDAC1 is able to co-immunoprecipitate with all variants of TRIM22, but shows significantly reduced binding to TRIM22 R442C (Fig. 5), it is important to determine whether this apparent decrease in co-IP is due to a loss in nuclear TRIM22 where HDAC1 normally acts in the NuRD complex. To investigate this, similar conditions and transfections as in the co-IP experiments involving variant TRIM22 and HDAC1 were conducted on coverslips for IF.

Figure 6: TRIM22 R442C does not form nuclear bodies in HEK293T cells. (A) HEK293T cells grown on a coverslip were transfected overnight with V5-HDAC1 and Flag-TRIM22 constructs (wildtype, R150T, S244L, R321K, R442C). Cells were then fixed with PFA, permeabilized with Triton X-100, and labelled with anti-V5 (red) and anti-Flag (green) antibodies. Images were acquired by confocal microscopy, with 0.1 µM Z-stacks. Images are shown in extended focus to show the entire acquisition of the HEK293T samples. (B) Merged images of Flag-TRIM22 wildtype and R442C with V5-HDAC1 from (A) is enlarged to show loss in nuclear bodies. (C) Extended focus of the TRIM22 channel captures were quantified to determine the average number of nuclear bodies (NBs) per nucleus. Error bars indicate SEM. ***p=0.0009 (Student’s t test, n=3 replicates).

3.5 TRIM22 does not ubiquitinate HDAC1 in unstimulated cells

TRIM22 belongs to the TRIM family of proteins which contain a RING domain E3 ligase, important for the ubiquitination of its substrates.22 This ubiquitination has been shown in our recent study to regulate NOD2 through K63-linked ubiquitin chains, allowing for correct localization and function.18 Since TRIM22 interacts with HDAC1, it is important to determine whether HDAC1 may be a substrate of TRIM22 through a ubiquitination assay. This can be conducted by co-transfecting TRIM22, HDAC1, and HA-Ub, followed by the IP of HA-Ub. Since ubiquitination involves covalent linkage of Ub to the tagged protein, IP of HA-Ub will pull down all proteins tagged with the exogenous ubiquitin.20 Should HDAC1 be a substrate of TRIM22, the overexpression of exogenous TRIM22 should increase levels of ubiquitinated HDAC1.

Figure 7: TRIM22 does not ubiquitinate HDAC1 in unstimulated HEK293T cells. HEK293T cells were transfected with V5-HDAC1, HA-Ub, and Flag-TRIM22 constructs (wildtype or R442C). Expression of transfected constructs in the lysates was verified using anti- V5, anti-TRIM22, and anti-HA antibodies, with blotting of endogenous GAPDH acting as a loading control (upper panels). IP with lysates from transfected cells was conducted with anti- HA antibody to precipitate all protein from the lysates ubiquitinated with the HA-Ub. The subsequent Western blot analysis was conducted with anti-V5 to detect V5-HDAC1 ubiquitinated with HA-Ub (lower panel). Lanes with only V5-HDAC1 qualitatively measure non-specific interaction during IP in absence of HA-Ub. Levels of ubiquitinated V5-HDAC1 are shown in the lane with HA-Ub, and this compares with levels of ubiquitinated V5-HDAC1 when Flag-TRIM22 constructs are also co-transfected.

3.6 TRIM22 does not alter HDAC1 stability in unstimulated cells

Ubiquitination of protein through K48-linked chains results in degradation, while other chains have potential for prolonging the half-life of target protein. Although TRIM22 may not alter HDAC1 ubiquitination status (Fig. 7), it’s possible for TRIM22 to alter its stability through its binding. TRIM22 has been previously shown to conduct functions independent of its RING domain or catalytic activity.28 TRIM22 could act as a scaffold, stabilize HDAC1, or ubiquitinate other proteins in the NuRD complex which could ultimately affect HDAC1 stability. To investigate this, a pulse chase assay was performed in which cells transfected with TRIM22 were treated with the protein synthesis inhibitor cycloheximide (CHX) at different time points. This allows the observation of HDAC1 protein levels decaying over time, at its previously described half-life of 24 h, and whether the presence of overexpressed TRIM22 alters this half-life.51

Figure 8: Cycloheximide chase of endogenous HDAC1 shows unchanged HDAC1 half-life in HEK293T cells transfected with Flag-TRIM22. (A) HEK293T cells were transfected with Flag-TRIM22 constructs (wildtype or R442C) for 16 h and subsequently split into 6-well plates. Cell culture media was replaced with media supplemented with either cycloheximide (CHX) or DMSO at 12 h time points, resulting in inhibition of protein synthesis for four different lengths of time prior to cell lysis (0, 12, 24, or 36 h pre-lysis). Expression of transfected constructs in the lysates was verified using anti-TRIM22 antibody, with blotting of endogenous GAPDH acting as a loading control. The expected effect of R442C on TRIM22 stability is visible, with a quick loss in Flag-TRIM22 R442C levels relative to wildtype Flag-TRIM22 following CHX treatment. Detection of remaining HDAC1 levels was performed by immunoblotting lysates with anti- HDAC1 antibody. (B) Western blot intensities of endogenous HDAC1 was normalized to 0 h of their respective treatment and condition. Nonlinear regression curves are shown.

B 1.5

1.0

0.5

0.0

Relative HDAC1Relative Band Intensity 0 10 20 30 40 Hours

M ock M ock + wt Mock + R442C CHX CHX + wt CHX + R442C

3.7 HDAC1 may show reduced levels in TRIM22 patient colon

Although TRIM22 did not alter HDAC1 stability in unstimulated cells (Fig. 8), there is still the possibility of defective TRIM22 within the context of IBD resulting in a decrease in HDAC1. One way to observe HDAC1 levels within the context of TRIM22 associated IBD is by staining colon tissue obtained from one of the VEOIBD patients. IF was conducted with colon biopsies obtained pre-treatment from a normal patient, a patient with severe non-monogenic IBD, and the TRIM22 R321K patient. IF included staining for TRIM22 and HDAC1.

Figure 9: Colon tissue immunofluorescence of HDAC1 and TRIM22. Tissues derived from colon biopsies were obtained pre-treatment from a normal control, a patient with severe IBD without known monogenic defects, and the TRIM22 R321K patient. FFPE (5 µM) sections were cut, dewaxed with heat and xylene, and rehydrated with decreasing concentrations of ethanol. Following high pressure basic antigen retrieval, sections were labelled for endogenous protein using anti-TRIM22 (red) and anti-HDAC1 (green) antibodies. Images were acquired by confocal microscopy, with 0.5 µM Z-stacks. Images are shown in extended focus to show the entire acquisition of the HEK293T samples.

As observed before, TRIM22 R321K shows reduced expression in the colon when compared to normal and non-monogenic IBD colon samples (Fig. 9). Although HDAC1 expression may appear to be lower in the R321K patient, these results should be verified by using sections with a similar cross-sectional angle as the controls. Sections should also be attained where more lamina propria is visible, since localization of HDAC1 appears to be different between the intestinal epithelial cells and cells in the lamina propria. Cell type and environment can play an important role in TRIM22 localization, so further investigation of TRIM22 and HDAC1 in colon samples will prove valuable. In particular, tissue samples from the TRIM22 R442C patient should be obtained and investigated since R442C results in a change in TRIM22 localization and alters co- IP with HDAC1 (Figs. 5 and 6)

Chapter 4 Discussion and Future Directions Discussion and Future Directions

We previously reported novel VEOIBD associated variants of TRIM22 which result in disrupted regulation of NOD2-coordinated pro-inflammatory and anti-viral responses. These patients exhibited similar phenotypes, particularly early onset disease, diarrhea, recurrent infections, severe fistulizing perianal disease, and granulomatous colitis. However, slight differences in phenotypes exist amongst these patients, with the R150T/S244L compound heterozygous patient exhibiting polyarthritis, petechiae, and hepatomegaly, while the R442C patient exhibited a bacterial clearance defect and is recently showing signs of liver defects. Although variants in other regions of the genome may contribute to the slight differences in TRIM22 patient phenotypes, this is less likely as the patients were previously screened for known VEOIBD associated defects and other potential disease causing variants. Differences in environmental exposure may also be possible, although the environment is thought to play a smaller role in VEOIBD.12 Recent studies have highlighted potentially understudied roles of TRIM22, showing potential for unidentified biological functions.43,52,53

To identify candidate TRIM22 binding partners, BioID was performed by the Brumell lab. Traditional protein-protein screening methods involve techniques with significant drawbacks. Two-hybrid screening methods are usually performed with organisms that are cheap, easy, and robust.54 Yeast two-hybrid (Y2H) screens have predominated in interaction screens, resulting in studies affected by the organism’s differences in post-translational modifications and translational system, while restricting the assay to identifying binary interactions without the complementary proteins required to stabilize the complexes. False positives are also possible due to the expression of two proteins at once that may not normally be co-expressed.36 Affinity purification coupled to mass spectrometry (AP-MS) is another common technique, which involves either the IP of the bait protein or purification by affinity columns.54 Although AP-MS has an advantage over Y2H with its identification of larger complexes and indirectly bound protein, AP-MS selects for high affinity interactions and is limited in its ability to capture insoluble protein.36 BioID aims to overcome these challenges by providing a system which labels

proteins in live relevant cells. Since BioID is a proximity-based method of labelling associating proteins, it not only labels direct and indirect interactors, but labels proteins within the 10 nm environment, giving context to the protein’s location.55 An example of this with regards to TRIM22 are the nucleopore proteins Nup153 and Nup50 which are highlighted by BioID, likely labelled due to transit of TRIM22 through the nucleopore when entering the nucleus (Table 2 and Fig. 3).

The ability of BioID to capture transient interactions in living cells also allows for the development of multiple interaction networks of the same protein, differing based on cell type and conditions used during the experiment. For future studies, TRIM22 BioID can be performed in monocytic or intestinal epithelial cell lines to better understand where TRIM22 fits within the interactomes of cells likely playing influential roles in disease. More importantly, the overexpressed TRIM22 construct in the BioID performed in this study occurs in unstimulated cells. Normally, TRIM22 is overexpressed during times of stress brought on by pathogens and cytokines.56 For a BioID list more representative of the in vivo state of the cell when TRIM22 expression is induced, BioID can be conducted following stimulation with factors which modulate TRIM22 expression such as type I/II interferons, viral antigens and lipopolysaccharide.27,57–59 This network can be compared to BioID with unstimulated cells to identify changes in identities and relative abundances of proximal proteins. Endogenous interactions with TRIM22 and their protein levels could be dependent on stimulation as well, as shown with the MDP-stimulation of NOD2 for increased abundance of the binding partner. Therefore, BioID of stimulated cells may result in candidates with more robust interactions with TRIM22, which can be more easily validated by co-IP.

TRIM22 BioID would also likely be sensitive to variations within the protein construct. The VEOIBD associated variants in TRIM22 are located in different functional regions of the protein, all of which resulted in disrupted NOD2 binding and regulation.18 However, interaction with HDAC1 was only significantly impaired by R442C. This variant also lies within the localization domain of TRIM22, resulting in impaired nuclear localization and nuclear body (NB) formation. Impairments caused by the variants, and subtle differences among the TRIM22

variants, can result in differences in BioID candidates. These lists can be used to identify binding partners found within the wildtype list which are lost in the variant TRIM22 lists. These lost interactions could have a significant impact on cellular function in relation to VEOIBD. Subtle variations within the lists could potentially explain the slight variations in patient phenotypes, such as liver and bacterial clearance defects, while highlighting potential druggable targets to modulate the disease.

Interaction of TRIM22 with HDAC1 was the primary focus of the current biochemical investigation due to implications of HDAC1 in colitis, IFN induction of TRIM22, and its potential as a druggable target in IBD.48–50 Although the TRIM22/HDAC1 interaction was conducted with bi-directional co-IP with the pulldown of either protein resulting precipitation of the partner (Appendix Fig. A), replicates of these results were hindered by non-specific interactions (not shown). Following more stringent pre-clearing methods, the non-specific interactions were reduced with consistent co-IP of HDAC1 when TRIM22 is pulled down (Fig. 4). Although it may be possible to perform successful bi-directional co-IP of the two proteins, HDAC1 may be more easily concentrated during TRIM22 pulldown due to the IF results (Fig. 6). Although TRIM22 can localize throughout the cell, HDAC1 is predominantly expressed in the nucleus where the interaction is likely taking place. When in the nucleus, the TRIM22 constructs form NBs. However, the majority of HDAC1 is spread across the nucleus, presumably acting as a histone deacetylase throughout the chromatin, likely making concentrating TRIM22 by HDAC1 pulldown more difficult.

TRIM22 localization patterns have been shown to be dependent on the source of the protein (endogenous vs. exogenous), treatment of cells, and cell types themselves.43 Although studies of endogenous TRIM22 expression have shown the presence of NBs in various cell lines, usually some form of treatment was required.30,43 Studies on localization of the NuRD complex, and how it may change in tandem with changing TRIM22 localization patterns, should be conducted in a variety of relevant cell lines (primarily monocytic and intestinal epithelial) and treatments (cytokines, viral particles, and possibly inducers of apoptosis). Likewise, bi-directional co-IP of TRIM22 and HDAC1 may be improved with the use of these cell lines and treatments.

Since the BioID reveals an entire NuRD complex, other subunits may be required to stabilize the TRIM22/HDAC1 interaction. Preliminary co-IPs were conducted with combinations of myc- TRIM22, V5-HDAC1, and Flag-MTA3 since MTA3 was the subunit of the NuRD complex with the highest number of peptide hits on the BioID list (Table 2 and Fig. 3; co-IP not shown). Although co-IP with V5-HDAC1 and Flag-MTA3 was successful in each combination involving the two proteins, myc-TRIM22 did not pull down in the same complex. It is possible that other subunits mediate the interaction with TRIM22, or the entire complex is required for consistent co-IP.

Since TRIM22 binds to HDAC1, and both proteins localize in the nucleus, co-IP and IF with the VEOIBD associated variants of TRIM22 would shed light on potential mechanisms of disease. Of the four TRIM22 variants, R442C showed significantly reduced binding and a loss in NBs (Figs. 5 and 6). However, it is unclear whether the decreased binding with HDAC1 is due to dysfunction in the protein itself, or is a product of the loss in nuclear TRIM22. In vitro co-IP with purified protein can test for direct binding of TRIM22 and HDAC1, and whether R442C directly impairs binding. The loss in NBs with TRIM22 R442C is not surprising since the residue is highly conserved and lies within the B30.2/SPRY domain previously shown to be required for nuclear localization and NB formation.31 R442C lies near the variable loop 3 thought to be responsible for localization, with the mutation disrupting the putative structure of the domain.32,60 However, R442C may still disrupt direct protein binding since the B30.2/SPRY domain is thought to mediate protein-protein interactions as well, with the variable loops acting as potential contact points for viral recognition.43

TRIM22 NBs have previously been observed as dynamic structures dependent on cell type, cell cycle, and cellular signals.30,43 These structures co-localize partially with Cajal bodies, multifunctional NBs consisting of at least 30 proteins. TRIM22 NBs did not co-localize with PML bodies, the most well recognized form of NBs which may play roles in nuclear storage, post-translational modification, and transcriptional regulation.30,61 However, these studies were limited in the cell types and conditions tested. PML appears on the BioID list, which suggests that PML could be recruited to TRIM22 NBs, or PML NBs partially co-localize with TRIM22

NBs. On the other hand, overexpressed proteins have been known to accumulate in PML bodies, likely to clear foreign or overexpressed nuclear protein.61 However, this does not explain why endogenous TRIM22 forms NBs in other studies.43 A recent study has also observed TRIM22 NBs partially co-localizing with PML bodies and implicated CyclinT1 in the structures.53 CyclinT1 also appears on the TRIM22 BioID list (as CCNT1 in Table 2 and Fig. 3), and is known as a major subunit of the positive transcription elongation factor (p-TEFb) involved in promoting transcription by RNA polymerase II.62 TRIM22 NBs formed independent of its RING domain, recruited CyclinT1, and recruited endogenous PML upon IFNγ stimulation. Since CyclinT1 and PML have been previously associated with HIV-1 restriction, TRIM22 was suggested as recruiting antiviral restriction factors in NBs.53 Unfortunately, the study did not include CDK9, the other major subunit in the p-TEFb, which also appears on the TRIM22 BioID list. Since both components of the p-TEFb are highlighted by TRIM22 BioID, it will be important to follow up these TRIM22 NB studies with binding and localization studies, coupled with an investigation of how the p-TEFb, TRIM22, and PML work together to restrict HIV-1. It is also likely that these newly discovered functions of TRIM22 are disrupted by the R442C variant which loses the ability to form NBs. Preliminary co-IPs of TRIM22, CyclinT1, and CDK9 were attempted, but still need to be optimized (not shown).

As a member of the TRIM family of RING E3 ligases, the catalytic activity of TRIM22 mediates ubiquitination of its substrates. Recently, TRIM22 was shown to ubiquitinate NOD2 through K63 linkage to mediate downstream pro-inflammatory and anti-viral pathways with implications in VEOIBD.18 Although the ubiquitination assays in the current study do not show modulation of HDAC1 ubiquitination status, the cells were unstimulated (Fig. 7). As shown in the co-IP and IF data, most HDAC1 was localized throughout the nucleus and did not precipitate when TRIM22 was pulled down in stringent pre-clearing conditions (Figs. 4 and 6). It is currently unknown whether TRIM22 will colocalize with the rest of HDAC1 during stimulation, but there have been reports of TRIM22 localizing diffusely throughout the nucleus in certain cell types and conditions.43 Not only is there potential for enhancement of co-IP and colocalization following stimulation of specific cell types, this may promote greater ubiquitination activity of TRIM22 on HDAC1. This would suggest that TRIM22 mainly interacts with and alters HDAC1 during times of stress. TRIM22 ubiquitination of HDAC1 could also be better observed by separating

fractions of soluble and chromatin-bound protein. A study on the effect of HIV-1 Vpr protein on class I HDACs showed that Vpr induced the ubiquitination of chromatin-bound HDAC1 in order to overcome latent infection of macrophages.63 Likewise, TRIM22 may be ubiquitinating chromatin-bound HDAC1, or function to protect chromatin-bound HDAC1 from ubiquitination by Vpr during HIV-1 infection. An interesting hypothesis would be that TRIM22 functions to protect HDAC1 from HIV-1 proteins, since it has been suggested that TRIM22 contributes to HIV-1 latency.43,64 To overcome latency, HIV-1 would then bypass TRIM22 to ubiquitinate chromatin-bound HDAC1.

If TRIM22 does not ubiquitinate HDAC1, there is still the possibility of TRIM22 acting on other components of the NuRD complex highlighted by BioID such as CHD4, the Mi-2 protein housing the ATP-dependent histone remodelling activity (Fig. 3).45 Other possible targets of TRIM22 are the MTA proteins, which could modulate NuRD complex specificity, or the GATAD and MBD proteins to modulate scaffolding within the complex and interactions with other proteins.45 As mentioned earlier, a systematic co-IP of TRIM22 and other NuRD complex subunits could reveal new interactions with TRIM22 which can be followed up with ubiquitination assays. However, this may be complicated by possible requirements of specific cell types and stimulation. TRIM22 has also been shown to carry out functions within the cell independent of its RING domain catalytic activity; catalytic residues C15 and C18 are required for restriction of HIV-1 particle release (cytoplasmic function), but not required for restriction of HIV-1 transcription (nuclear function).28,64 It is possible that TRIM22 could be acting on HDAC1 and the NuRD complex through its physical interaction of the complex, modulating protein-protein interactions between the NuRD complex and other proteins, independent of its catalytic activity as in the case of HIV-1 transcription.

Regulation of a protein, particularly by ubiquitination, can involve changes in protein stability. To observe possible changes in HDAC1 stability, pulse chase assays with the protein synthesis inhibitor, CHX, was performed in the presence and absence of overexpressed Flag-TRIM22 or Flag-TRIM22 R442C (Fig. 8). The results in this study suggest that TRIM22 may not affect HDAC1 stability, which is reasonable considering the long 24 h half-life of HDAC1 in the cell

and the multitude of functions carried out by the deacetylase.51,65,66 A more rapid and targeted regulation of HDAC1 at the protein level would involve its deacetylase activity, quickly altering HDAC1 function in response to TRIM22 regulators such as cytokines and antigens. HDAC1 activity can be tested with specific deacetylase assays, involving concentrating HDAC1 and testing its ability to deacetylate a fluorophore-releasing compound.67 HDAC1 activity can be tested in TRIM22 HAP1 knockout cell lines with and without stimulation, while attempting to rescue HDAC1 activity by transfecting with wildtype or VEOIBD variant TRIM22. However, there is still the possibility that TRIM22 could affect HDAC1 stability under certain conditions, such as in an inflammatory environment. Preliminary IF of HDAC1 shows potentially reduced levels in the TRIM22 R321K patient (Fig. 9). However, these results must be repeated with sections of similar orientations as the controls. It is also unclear which cell types show potentially reduced levels in the tissue since it is possible that TRIM22 affects HDAC1 levels in a cell-type dependent manner. Alternatively, TRIM22 may not directly affect HDAC1 levels, but dysfunctional TRIM22 or its decreased levels combined with a chronic inflammatory environment could eventually lead to decreases in HDAC1 levels in the long term.

In 2004, TRIM22 was identified as a p53 target gene.68 The tumor suppressor p53 has been well- studied as an apoptosis inducer through its transcriptional activation of pro-apoptotic factors and direct interaction with apoptosis regulators in the cytoplasm.69 p53 increased TRIM22 expression, and TRIM22 overexpression reduced clonogenic growth of monocytic U937 cells.68 Apoptosis has recently been highlighted as a major role of TRIM22 in monocytes.52 Monocytes recruited to sites of infection trigger immune response and pathogen clearance, but the prolonged survival of monocytes results in overproduced pro-inflammatory cytokines. Monocyte apoptosis attenuates this response, preventing the organ damage that can occur during sepsis. High levels of TRIM22 sensitized monocytes to apoptotic stimuli, was associated with the expression and oligomerization of pro-apopotic Bak, and associated with caspase-9 and caspase-3 activation. These observations were dependent on the TRIM22 RING domain. TRIM22 levels were low in monocytes from septic patients, and positively correlated with Bak levels.52 Since TRIM22 plays a role in monocyte apoptosis and is a p53 target gene, p53 regulation likely plays an important role in this pathway. During times of stress, levels of p53 rise rapidly due to changes in its post- translational modification. In the absence of stress, p53 is ubiquitinated by MDM2 at residues

65 which overlap with acetylation sites. In this MDM2 complex, HDAC1 deacetylates p53 at the overlapping acetylation residues, allowing for MDM2-mediated ubiquitination. During stress, acetylation of HDAC1 reduces its activity, preventing ubiquitination of p53 and increasing p53 stability.70 Reports of the NuRD complex have also shown its promotion of p53 deacetylation.45 As a feedback mechanism, p53 target gene TRIM22 may control monocyte apoptosis through its association of the NuRD complex by participating in p53 regulation. Since TRIM22 may bind the NuRD complex through its association with HDAC1, possibly mainly during times of stress, this may lead to a reduction in HDAC1 activity and decreased p53 deacetylation. Increasing levels of acetyl-p53 would result in increased susceptibility to apoptosis, which agrees with the observation of TRIM22 increasing apoptosis susceptibility in monocytes.52 TRIM22 variants could therefore lose the ability to reduce HDAC1 activity, resulting in persisting monocytes during times of inflammation. TRIM22 mediated inhibition of HDAC1 could also result in a pro- inflammatory response more directly, since HDAC1 has been shown to inhibit inflammation.49,71 This would agree with the recently discovered role of TRIM22 in regulating NOD2 to promote the pro-inflammatory response, the disruption of which leads to VEOIBD.18

However, TRIM22 may also be regulated by HDAC1 during their association, with HDAC1 possibly deacetylating TRIM22 during times of stress and inflammation. HDAC1 would therefore control TRIM22 at the protein level, which would supplement findings of HDAC1 controlling the IFNγ induction of TRIM22 at the transcription level.50 To determine whether TRIM22 or HDAC1 regulate each other at the protein level, HDAC1 activity and the acetylation status of TRIM22, HDAC1, and p53 can be studied with methods similar to a ubiquitination assay, but with the involvement of acetyl-lysine instead of ubiquitinated residues. Since these pathways may play key roles in monocyte apoptosis, apoptosis assays with TRIM22 knockout models can be tested with HDAC1 inhibitors and various apoptotic or inflammatory stimuli.

Figure 10: TRIM22 interaction with HDAC1 may be involved in monocyte apoptosis. TRIM22 was recently implicated in mediating monocyte apoptosis, a process which requires extensive regulation by p53.52 Since TRIM22 has been previously identified as a p53 target gene, and HDAC1 has been shown to regulate p53 at the protein level and TRIM22 at the transcription level, the interaction highlighted in this current study may be related to TRIM22 mediated apoptosis in monocytes.68,70 In this case, p53 would promote monocyte apoptosis through TRIM22, and HDAC1 would suppress this effect. With defective or low levels of TRIM22, as seen in sepsis patients, persisting pro-inflammatory monocytes would result in extensive inflammation.52 Should this be a factor in TRIM22 VEOIBD patients, HDAC1 inhibitors may have therapeutic potential, promoting apoptosis of monocytes to attenuate inflammation.

Chapter 5 Conclusion Conclusion

Due to the recently discovered role of TRIM22 in regulating NOD2, with variants resulting in VEOIBD, bone marrow transplant would not likely be curative. However, alternative methods of therapies may exist for these patients with severe fistulizing perianal disease and granulomatous colitis. BioID reveals multiple pathways of interest, such as ones involving PML and CDK9/CCNT1, which was partially investigated recently.53 Because these pathways involve TRIM22 nuclear bodies, they are likely impaired by VEOIBD variant R442C. In our study, we focus on HDAC1 and the NuRD complex. Although TRIM22’s effect on HDAC1 remains unclear, it is possible that HDAC1 regulates TRIM22 since it is known that HDAC1 is involved in the transcriptional regulation of TRIM22.50 These pathways are likely impaired by R442C as well, with the significantly decreased HDAC1 binding. It is becoming clear that the role TRIM22 plays in inflammation and VEOIBD may be multifactorial, with some pathways containing potential drug targets such as HDAC inhibitors. Further investigation of these pathways highlighted by BioID could not only reveal therapies for difficult to treat patients, but would also provide insight into how TRIM22 controls cell fate and stress response.

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Appendix

Figure A: TRIM22 may bi-directionally co-immunoprecipitate with HDAC1. HEK293T cells were transfected with nFlag-tagged TRIM22 and cV5-tagged HDAC1. IP with lysates from transfected cells was conducted with anti-V5 (HDAC1) antibody (centre panels). Western blot was conducted with anti-V5 to detect precipitated V5-HDAC1, or anti-Flag (TRIM22). IP with anti-Flag (TRIM22) beads on lysates from the same transfected cells were blotted with the same antibodies (right panels). IgG lane shows IP with mouse IgG, acting as a negative control for non-specific IP of V5-HDAC1 and Flag-TRIM22. Expression of transfected constructs in the lysates was verified using the same antibodies (left panels).

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Contributions

Most work presented in this thesis was done by me. Overall direction of the project was led by my supervisor Dr. Aleixo Muise, and further direction was provided by my committee members Dr. Daniela Rotin and Dr. John Brumell. BioID on TRIM22 was conducted by the Brumell lab. Dr. Neil Warner aided in the interpretation of BioID data, and instructed me in the interpretation of the SickKids WES dataset for variants of interest. Dr. Conghui Guo and Dr. Qi Li assisted in in vitro experiments, and Dr. Jie Pan assisted with immunofluorescence experiments.