Wild-Derived Allele of Tmem173 Potentiates an Alternate Signaling Response to Cytosolic DNA

Wild-derived allele of Tmem173 potentiates an alternate signaling response to cytosolic DNA.

A dissertation

submitted by

Guy Surpris

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Immunology

TUFTS UNIVERSITY

Sackler School of Graduate Biomedical Sciences

May 2016

Adviser: Alexander Poltorak

ABSTRACT

The cellular recognition of cytosolic DNA is critical for maintaining homeostasis and to signal warnings to prevent the spread of pathogens such as

HSV1 or Listeria. Inborn mutations in the human population determine the susceptibility or ability to clear infection. Mouse models of infectious disease are an invaluable resource for the study of these mechanisms of disease progression.

However, classical laboratory mouse strains do not always recapitulate the diversity in immune responses found in the human population. Wild derived mice are an excellent source of genomic and phenotype diversity in the lab. Herein, we report and characterize phenotype variations in the wild-derived mouse strain MOLF/Ei and classical lab mouse strain in interferon stimulated gene induction to cytosolic

DNA species. Using forward genetic analysis, we identified multiple loci that confer attenuated IFNβ production in MOLF/Ei macrophages to pathogen derived cytosolic di-nucleotides. Fine mapping of a major locus of linkage revealed a novel polymorphic allele of Tmem173 (STING). The MOLF allele of Tmem173 produces a protein with multiple amino acid changes, and an internal 6 amino acid deletion.

Most of these amino acid changes are confined to the understudied N-terminus.

These polymorphisms in MOLF STING altogether confer a lack of induction of the

IFNβ promoter in an overexpression assay that seems to be attributed to the most

N-terminal proximal mutations. The loss-of-function of MOLF STING is inherited in macrophages of C57Bl/6 mice congenic for the MOLF Tmem173 allele. C57Bl/6

2 macrophages congenic for MOLF STING display a shifted interferon signal profile in response to different c-di-nucleotides when compared to wild-type C57Bl/6.

Furthermore, expression of MOLF STING in STING-/- MEFs show attenuated trafficking of MOLF STING after c-di-nucleotide activation compared to B6 STING.

Several, human alleles of STING confer a loss-of-function or gain-of-function phenotype that may dramatically affect immunity. The study of this novel allele in mice will aid in the mechanistic study the STING pathway that has recently garnered much attention as being central to the response to cytosolic DNA.

To know, is to know that you know nothing. That is the meaning of true knowledge. Socrates

4 Acknowledgements

I would like to thank my family for loving and nurturing my curiosity and love for science.

Thank you Sasha for bringing me into your lab. Thanks for sharing excitement for the work that we have done together. It was a blessing to have a mentor such as you who always encouraged, and believed in me. I have learned how to think original scientific ideas, and many life lessons.

Thank you Irina for your friendship, and for your dependable support. I will not forget all the molecular biology techniques that I have learned.

DEDICATION

I would like to dedicate this work to my family, and to the people whom I intend to

serve.

TABLE OF CONTENTS

ABSTRACT ...... 2

Acknowledgements ...... 5

DEDICATION ...... 6

TABLE OF CONTENTS ...... 7

LIST OF TABLES ...... 11

LIST OF FIGURES ...... 12

LIST OF ABBREVIATIONS ...... 15

CHAPTER 1 - Introduction ...... 17

Type I IFNs in Physiology and Pathogenesis ...... 17

DNA Damage sensing and Type I Interferons ...... 19

PRRs ...... 22

DNA Sensors ...... 26

STING ...... 29

SELF-DNA and the overwhelmed DDR leads to STING mediated autoimmunity ..... 38

Forward genetics is an unbiased approach of explaining gene’s function ...... 38

MOLF mice variability ...... 41

CHAPTER 2 - Methods ...... 44

Reagents ...... 44

Mice ...... 44

DNA Sequencing ...... 44

SNP analysis ...... 45

Species alignment ...... 45

Isolation of Primary Peritoneal Macrophages ...... 46

Genotyping and Quantitative Trait Locus Mapping (QTL) ...... 46

Plasmids ...... 47

Luciferase assay ...... 47

Overexpression assay ...... 47

CDN stimulus dependent Luciferase assay ...... 48

Western Blotting ...... 48

Listeria monocytogenes, HSV1, and Sendai infection ...... 49

ELISA ...... 49

Quantitative PCR RNA analysis ...... 50

RNASeq workflow ...... 50

Lentiviral transduction of STING -/- MEFs ...... 51

Confocal imaging of MEFs ...... 51

CHAPTER 3 - MOLF macrophages lack interferon responses to DNA viruses, Listeria, and cytosolic nucleotides...... 53

MOLF macrophages lack IFNβ responses to DNA viruses ...... 55

CHAPTER 4 - Forward genetic screen reveals linkage to Tmem173 as candidate gene.

...... 66 8

N2 Panel responses to cytosolic nucleotides and pathogens ...... 67

CHAPTER 5 - Identification of polymorphisms in MOLF Tmem173...... 79

CHAPTER 6 - MOLF STING is deficient in activating the IFNβ and NF-kB promoter elements in Luciferase assay...... 92

CHAPTER 7 - Characterization of Interferon responses in Congenic mice reveals further defect of MOLF STING...... 106

CHAPTER 8 - Mapping genes that interact with MOLF STING on the MOLF background to confer responsiveness...... 117

CHAPTER 9 - Localization of STING during signaling...... 126

CHAPTER 10 - RNA-seq and Pathway analysis of STING dependent responses in macrophages ...... 135

DISCUSSION ...... 147

Summary Findings ...... 148

STING dependent IL-6 production ...... 150

Complementation ...... 151

STING signalosomes ...... 154

Pathway analysis...... 156

B6 StingMOLF in vivo studies ...... 158

Conclusion ...... 159

APPENDIX I – Genes significantly differentially regulated between STINGB6 and

STINGMOLF peritoneal macrophages activated with 2’3’cGAMP ...... 160

APPENDIX II – Cell signaling pathway analysis of 2’3’cGAMP stimulated macrophages...... 169

BIBLIOGRAPHY ...... 180

LIST OF TABLES

Table 1: QTL analysis: Linkage of MOLF loci with differential IFNβ production...... 77

Table 2: List of Known Human STING Polymorphisms and alleles ...... 85

LIST OF FIGURES

Figure 1: MOLF macrophages display and attenuated IFNβ production to HSV1 (DNA

virus), and mCMV (DNA virus)...... 57

Figure 2: MOLF peritoneal macrophages respond with attenuated IFNβ production to

DNA agonist yet are proficient in IL-6 production...... 60

Figure 3: MOLF macrophages are attenuated in cytokine and signaling responses leading

to IFNβ production...... 63

Figure 4: Profile of inflammatory transcripts are shifted in MOLF macrophages

compared to B6 macrophages in response to HSV1 and Listeria...... 65

Figure 5: N2 Panel reveals wide range of responses to CDNs and DNA pathogens

indicating that lack of IFNβ production in MOLF is a genetic trait ...... 68

Figure 6: IFNβ production of individual N2 mice to Listeria and HSV, and Listeria and

c-di-AMP are correlate in intensity, while N2 mice responses to LPS and HSV

responses are discordant...... 70

Figure 7: Marker regression analysis reveals that several loci confer differential IFNβ

between B6 and MOLF macrophages...... 72

Figure 8: F2 Panel reveals wider ranges of responses to CDNs and DNA pathogens

indicating that lack of IFNβ production in MOLF is a genetic trait ...... 76

Figure 9: QTL analysis links MOLF loci with low interferon responses...... 78

Figure 10: MOLF Tmem173 (Sting) contains multiple polymorphisms and a 18 base pair

deletion...... 82

Figure 11: Alignment of amino acid sequence of MOLF, B6, Human STING, and select

mammals display unique amino acid substitutions in MOLF STING ...... 83

Figure 12: MOLF STING and Spretus STING contain unique divergent alleles of STING

compared to common lab strains...... 84

Figure 13: MOLF amino acid changes are N-terminally located...... 88

Figure 14: MOLF amino acid changes disrupt evolutionary conserved sequences in

STING N-terminus ...... 91

Figure 15: MOLF STING promotes reduced induction of the IFNβ promoter...... 95

Figure 16: L47V-A48G, and S53L mutations confer the strongest individual effect on the

defective regulation interferon induction downstream of the STING pathway...... 98

Figure 17: MOLF N-terminal mutations to B6 partially restores function of STING. ... 100

Figure 18: S53A (structurally similar to Serine) mutation causes hyper-activation of the

IFNβ promoter...... 103

Figure 19: Recapitulation of MOLF mutations on human STING provides diverse effects

on human STING induction of interferon promoter...... 105

Figure 20: B6-StingMOLF IP Macs lack IFNβ to all STING dependent agonists...... 109

Figure 21: B6 StingMOLF congenics exhibit differential responses to STING agonists. . 111

Figure 22: p65 and IRF3 phosphorylation is reduced in B6 Sting MOLF IP macrophages

(PECS)...... 113

Figure 23: Dominant negative inheritance of STING in B6 StingB6/MOLF can be explained

by haploinsufficiency...... 116

Figure 24: Analysis of DNA responses in C57BL/6, B6-StingMOLF, and MOLF/Ei

macrophages suggests the existence of a 2nd genetic component on the MOLF

background which permits 2’3’cGAMP IFNβ responses in MOLF macrophages. 120

Figure 25: Comparison of IFNβ production in MOLF, B6-StingMOLF, and F1-StingMOLF

reveals importance of genetic background for MOLF STING responsiveness...... 122

Figure 26: Responses of F2-STINGMOLF/MOLF mice to CDNs reveals interaction of genes

to confer 2’3’cGAMP dependent IFNβ production by MOLF STING...... 125

Figure 27: MOLF N-terminal mutations in STING disrupt membrane topology model of

B6 STING...... 130

Figure 28: MOLF STING show aberrant localization compared to B6 STING during

activation...... 132

Figure 29: Localization of L47V-A48G, and S53L mCherry-STING in stably transduced

MEFs stimulated with CDNs ...... 134

Figure 30: MOLF STING inheritance confers distinct ISG signature...... 137

Figure 31: MOLF and B6 StingMOLF congenic vary in upregulation of ISGs in a ligand

specific manner...... 141

Figure 32: Comparison of B6 and MOLF STING dependent signaling in response to

2’3’cGAMP reveals significant decrease in IRF, NF-kB, and AP-1 family

transcription factor activation...... 143

Figure 33: Cell signaling pathway analysis of StingB6, StingMOLF, and MOLF/Ei

macrophages stimulated 4hrs with 2’3’cGAMP...... 146

LIST OF ABBREVIATIONS

Abbreviation Full name AGS aicardi goutieres syndrome AIM2 absent in melanoma 2 AMFR Autocrine Motility Factor Receptor AP-1 Activator protein 1 AP-3 Activator protein 3 ATM Ataxia telangiectasia mutated c-di-AMP Cyclic diadenylate monophosphate c-di-GMP Cyclic diguanylate monophosphate CARD Caspase activation and recruitment domains CD14 cluster of differentiation 14 CDN c-di-nucleotide cGAMP Cyclic guanosine monophosphate–adenosine monophosphate cGAS Cyclic GMP-AMP synthase CMV Cytomegalovirus CoV Coronavirus CTT c-terminal tail DAI (aka ZBP) Z-DNA-binding protein 1 DDX41 DEAD (Asp-Glu-Ala-Asp) Box Polypeptide 41 E1a Adenovirus early region 1A E7 HPV E7 oncoprotein ERC endocytic recycling compartment F1 1st Filial (generation) F2 2nd Filial (generation) HIV human immunodeficiency virus HSV herpes simplex virus IFI16 Interferon, Gamma-Inducible Protein 16 IFN interferon IFNAR interferon receptor IKK IkB kinase INSIG1 Insulin Induced Gene 1 IRF Interferon regulator factor ISGF3 Interferon-stimulated gene factor 3 ISRE Interferon-sensitive response element JAK Janus kinase KSHV Kaposi's sarcoma-associated herpesvirus LANA latency-associated nuclear antigen LC3 light chain 3A

LPS Lipopolysaccharide LRR leucine-rich repeat LTA lipoteichoic acid MAM mitochondria-associated membranes MAVS Mitochondrial antiviral-signaling protein MDA5 Melanoma Differentiation-Associated protein 5 Mre11 Meiotic recombination 11 homolog 1 MyD88 Myeloid differentiation primary response gene 88 N2 2nd F1 backcross NBS1 Nibrin NFkB nuclear factor kappa-light-chain-enhancer of activated B cells NL63 NL63 species of coronavirus PAMP Pathogen-associated molecular patterns PLCγ Phospholipase C gamma PLP2 Papain-like protease 2 from SARS-CoV PLpro The papain-like protease domain, of coronovirus protein RAD50 double strand break repair protein RING (Really Interesting New Gene) finger domain containing RNF protein RSV Respiratory syncytial virus severe acute respiratory syndrome, caused by SARS SARS coronavirus. SCAP SREBP cleavage-activating protein SeV Sendai Virus SLE Systemic lupus erythematosus SNP single nucleotide polymorphism SOCS1 Suppressor Of Cytokine Signaling 1 SOCS3 Suppressor Of Cytokine Signaling 3 STAT signal transducer and activator of transcription STING Stimulator of interferon genes Syk Spleen tyrosine kinase TBK1 TANK-Binding Kinase 1 TIR Toll/interleukin-1 receptor homology domain TLR Toll-like receptors TRAF TNF receptor associated factors TRAM TRIF-related adaptor molecule TRIF TIR-domain-containing adapter-inducing interferon-β TYK tyrosine kinase ULK1 Unc-51 Like Autophagy Activating Kinase 1 vIRF viral interferon regulatory factor VSV Vesicular stomatitis virus 16

CHAPTER 1 - Introduction

Type I IFNs in Physiology and Pathogenesis

Type I Interferons (IFN) are a family of cytokines, that activate cellular antimicrobial defense genes to promote local cellular defense responses, and to activate innate and adaptive responses to the incoming pathogen (Schindler and

Brutsaert 1999). Type I IFN is produced after activation of particular microbial pattern-recognition receptors (PRRs) (Kawai and Akira 2010). These cytokines are necessary for the control of the spread of microbial infection, especially that of viruses. IFN cytokines serve as an early warning sign to neighboring cells to mount defenses against a possible impending infection (Isaacs and Lindenmann 1957).

These cytokines act by activating cellular immunity, innate effector cells, such as

NK-cells, and the adaptive immune response.

Type I interferons are comprised of IFNβ, and 14 IFNα family members.

Almost all cells produce IFNβ, but mainly only cells of hematopoietic origin can produce IFNα. IFNβ is produced by a single gene, which is comprised of a single exon. Meanwhile, IFNα isoforms are each encoded by distinct genes. IFNβ and IFNα bind the heterodimeric IFNAR receptor that is comprised by IFNAR1 and IFNAR 2.

IFNAR ligation activates tyrosine kinases Janus kinase 1 (JAK1) and tyrosine kinase

2 (TYK2). JAK1 and TYK2 phosphorylate transcription factors, signal transducer and activator of transcription 1 and 2 (STAT1) and (STAT2) (Li et al. 1996) (Li et al. 17

1997). STAT1 and STAT2 dimerize after phosphorylation. The STAT1-STAT2 heterodimer translocates to the nucleus where it binds interferon regulatory factor

(IRF9) to form a complex known as IFN-stimulated gene factor 3 (ISGF3) (McComb et al. 2014). ISGF3 then binds to DNA consensus sequences called ISREs (Marié,

Durbin, and Levy 1998; Li et al. 1996). ISREs are located upstream of hundreds of genes termed Interferon stimulated genes (ISGs) (Zitvogel et al. 2015) (De Veer et al. 2001). In addition to the canonical ISGF3 activation, IFNAR can activate other

STAT dimers in certain types of cells (Schindler and Brutsaert 1999). Overall, these

ISGs encode proteins that inhibit or subvert infection and proliferation of an invading pathogen.

When particular PRRs sense patterns-associated molecular patterns

(PAMPs) Type I interferon is rapidly induced. Downstream of these PRRs, transcription factors IRF3 or IRF7, and NF-kB and AP-1 are activated and translocate to the nucleus to associate with p300/CREB-binding protein

(p300/CBP) (Merika et al. 1998). p300/CBP serves as a chromatin remodeler, as

IRF3/IRF7, AP-1, and NF-kB induce transcription of genes containing IFN promoter sequences (Merika et al. 1998). This complex is known as the IFN-enhanceosome and serves to produce the first wave of IFNβ or IFNα cytokine dependent on cell type.

Downstream of Type I IFN receptor ligation ISGs USP18, SOCS1, and SOCS3 serve as negative regulators to limit IFNAR signaling (Song and Shuai 1998)

(Sarasin-Filipowicz et al. 2009). USP18 competes for JAK1 binding to IFNAR2

(François-Newton et al. 2011) (Francois-Newton et al. 2012). SOCS1 and SOCS3 competitively inhibit STAT1 and STAT2 binding to IFNAR, thus blocking STAT1/2 activation by JAKs(Song and Shuai 1998) (Sarasin-Filipowicz et al. 2009. Other cytokines and pro-inflammatory signals, such as IL-6 or TLR activation, activate

STATs family members to induce SOCSs members. Thus, a preceding signal such as

IL-6 IL-1, or a PAMP can attenuate IFNAR activation via induced negative regulators.

{Hu, 2007 #2447) Other cytokines and pro-inflammatory signals, such as IL-6 or

TLR activation, activate STATs family members to induce SOCSs members. Thus, a preceding signal such as IL-6 IL-1, or a PAMP can attenuate IFNAR activation via induced negative regulators. {Hu, 2007 #2447} . Other cytokines and pro- inflammatory signals, such as IL-6 or TLR activation, activate STATs family members to induce SOCSs members. Thus, a preceding signal such as IL-6, IL-1, or a

PAMP can attenuate IFNAR activation via induced negative regulators. (Hu et al.

2007; Shuai and Liu 2003)

DNA Damage sensing and Type I Interferons

The immune response downstream of nucleic acid receptors is crucial for protection against viruses or other infections. However, it must be carefully

controlled to prevent recognition of self-DNA and RNA. Studies have shown that consecutive or chronic activation of many pattern recognition biosensors cause autoimmune diseases by chronic cytokine overproduction (Sharma et al. 2015). It has also been recently shown that levels of expression or location of such sensors are also crucial to prevent recognition of self-RNA and DNA. In addition, multiple nucleic acid sensors avoid sensing self DNA and RNA by recognizing particular motifs specific to viral or bacterial DNA or RNA, such as poly A-T rich sequences in

DNA or lack of 7-methylguanosine-cap in mRNA(Kariko et al. 2005). However, some

DNA sensors, such as cGAS in the STING pathway, will recognize the host DNA if it presents itself in the cytosol(Hartlova et al. 2015).

Aicardi-Goutierers Syndrome (AGS) is an autoimmune disease caused by loss of function mutations in human TREX1 gene(Crow and Rehwinkel 2009). AGS patients have elevated type I interferon responses and severe inflammation as a result of build up of cytosolic DNA. TREX1 is the DNA metabolizing digesting enzyme that is found in the cytoplasm. Its function is to digest ssDNA and dsDNA that naturally gets out of the nucleus during homeostasis. The source of this DNA can be from extra genetic material from the DNA damage response or from extra

DNA material after division. The function of TREX1 is to get rid of this extra DNA before it is sensed by cytosolic sensors (Lee-Kirsch et al. 2007), where DNA will trigger IFNβ mainly via the cGAS-STING pathway (Cai, Chiu, and Chen 2014; Wang et al. 2015; Gray et al. 2015).

Trex1 deficient mice share the severe phenotype of AGS and SLE patients.

These mice were completely rescued from the phenotype by the lack of IRF3,

IFNAR1, and STING, and partially by RAG2, TCRa, and Ighm. This shows that this is dependent on sting pathway detection of DNA and disease is mediated by IFN responses effect on the hematopoietic compartment(Stetson et al. 2008; Hartlova et al. 2015; Lee-Kirsch et al. 2007; Gray et al. 2015)..

In another study, STING also rescued DNAseII-/- lethality in mice. DNAseII-/- is a lysosomal endonuclease enzyme that digests phagocytosis DNA mainly sourced from apoptopic cells(Lan et al. 2014; Evans and Aguilera 2003). During embryogenesis in DNAseII-/- mice, DNA builds up in phagosomes and triggers DNA sensors. IFNAR-/- partially rescues the phenotype, because these mice present with polyarthritis; STING-/- fully rescued disease in mice. This revealed that the STING pathway was the major pathway to detect phagocytozed DNA, and that cytokines downstream of STING other than interferons are important mediators of disease, as

IFNAR-/- did not fully rescue DNAseII-/- dependent disease. (Ahn et al. 2012).

Therefore, unregulated activation of antiviral sensors by self nucleic acids can lead to autoimmune disease showing further promise for studying the STING pathway to discover promising modes of intervention.

PRRs

Cellular detection of pathogen-derived molecular patterns PAMPs are crucial for rapid mounting of defenses. Cells express a dedicated set of proteins to sense

PAMPs. These Pattern Recognition Receptors (PRR) are confined to spaces in the cells that signal presence of specific PAMPs, in a location specific manner. (Kawai and Akira 2010)

Nucleic acids are an important component to survey, as they are necessary for life, and its presence in the wrong location or form can signify an infection in the cell. For instance, the presence of DNA in the cytosol is a Danger signal, hence DNA sensors, such as DDX41, and STING are situated in the cytosol to mount a warning to the presence of a pathogen or DNA damage. RNA in the cytoplasm that lack host features, such as a 7-methylguanosine cap typical of viral RNA, will be detected by

RIG-I(Loo et al. 2008).

Toll like receptors (TLRs), are transmembrane proteins whose pattern recognition receptors (PRR) are directed toward the extracellular and endosome spaces. The extracellular or lumenal domain of TLRs contain leucine rich repeats

(LRRs), through which they detect specific PAMPs. The binding of PAMPs by TLRs induces dimerization of LRR, which in turns dimerizes the TIR domains at the cytoplasmic tails of TLRs. This initiates the formation of a supramolecular signaling

complex known as the myddosome. TIR domains of TLRs associate with

TIRAP(Yamamoto et al. 2002) and MyD88, with the exception of TLR3. MyD88 activates IRAK4 to phosphorylate IRAK1 which in turn activates TRAF6 to mediate downstream NF-kB and MAPK signaling. TLR3 and TLR4 associate with TRIF and

TRAM to recruit RIP1(Meylan et al. 2004) and TRAF6 to activate TAK1 and TBK1 (in a TRAF3 dependent manner), (Tseng et al. 2010) also leading to the activation of pro-inflammatory cytokine and Type 1 IFNs(Tseng et al. 2010). TLR7(Lund et al.

2004) and TLR9(Barton, Kagan, and Medzhitov 2006) are endosomal, dependent on location and state of the endosome, and can signal through MyD88 to activate transcription factors IRF7 and NF-kB. (Kawai and Akira 2010)

TLRs that detect nucleic acids are contained in the endosomal pathway.

These TLRs are TLR3, TLR7, TLR8, and TLR9. As viral or bacterial products are endocytosed, nucleic acids can be exposed during digestion of pathogens via the lysosomal pathway and presented to TLRs. TLR3 senses double stranded RNA, which is usually viral. TLR7 and TLR8 senses single-stranded RNA(Diebold et al.

2004; Larange et al. 2009). TLR9 senses DNA that contain particular patterns of

CpG sequences(Sharma et al. 2015; Kawai and Akira 2007; Takeda and Akira 2004).

(Chow, Franz, and Kagan 2015)

Endosomal populations and trafficking can specify TLR activity. Nucleic acid sensing TLRs are restricted spatiotemporally to avoid aberrant activation to host

nucleic acid. Pools of endosomal TLR7, 3, 8, and 9 are regulated by

UNC93B1(Brinkmann et al. 2007; Sasai and Iwasaki 2011; Fukui et al. 2011).

UNC93B1 traffics TLRs from the ER to the endocytic pathway via the Golgi Body complex(Brinkmann et al. 2007; Sasai and Iwasaki 2011; Fukui et al. 2011). An additional form of regulation comes in the form of proteolytic priming of the endosomal TLRs that needs to precede signaling. These TLRs need to be cleaved by proteases such as cathepsins that are active in maturing acidic endolysosomal compartments(Chiang et al. 2012). This all ensures that these nucleic acid sensing

TLRs are activated in the right time and place, so that improper responses to host nucleic acids are avoided(Moretti and Blander 2014). Yet another form of regulation occurs in that TLR9 can activate differential cytokine signaling dependent on endosomal compartment. Recruitment of TLR9 phagosomes or endosomes by

LC3 or AP-3 dependent non-canonical autophagy(Henault et al. 2012) bifurcates a pool of TLR9 to vesicles where IRF7 dependent IFN responses are activated(Sasai,

Linehan, and Iwasaki 2010; Moretti and Blander 2014; Engel and Barton 2010).

TLR2 and TLR4 are localized to the plasma membrane, yet can sense viruses.

TLR2 and TLR4 have been originally described as sensors of bacterial LTA or

LPS(Takeuchi et al. 1999), respectively. However, these TLRs have been shown to detect non-nucleic acid components of viruses as well(Barbalat et al. 2009). TLR2 can be activated by HSV(Aravalli et al. 2005; Sato, Linehan, and Iwasaki 2006),

CMV(Kijpittayarit et al. 2007), or vaccinia(Zhu et al. 2007) glycoproteins. RSV and

VSV trigger TLR4 activation and internalization and signaling through the TRIF pathway, and can serve as a mechanism of entry for the viruses(Kurt-Jones et al.

2000; Haynes et al. 2001; Georgel et al. 2007).

As one of the earliest discovered PRRs, much has been uncovered about

TLR4 signaling(Poltorak 1998). Dissection of this pathway can give hints about regulation of other pathways. Notably, TLR4 signaling bifurcates down two major pathways upon receptor ligation, a TIR and MyD88 pro-inflammatory signaling pathway, and a TRIF and TRAM mediated IFN signaling pathway(Tanimura et al.

2008; Fitzgerald et al. 2003). The MyD88 dependent pathway activates NF-kB, while the TRIF/TRAM pathway leads to IRF3 activation. TLR4 location determines which pathway is activated(Fitzgerald et al. 2003). Early TLR4 signaling occurs at the plasma membrane where TIRAP and MyD88 signaling occur minutes after ligation of the receptor by LPS. However, there is a delayed TRIF mediated response that is dependent on CD14 mediated receptor internalization and trafficking to the perinuclear endocytic recycling compartment (ERC) (Zanoni et al. 2011; Jiang et al.

2005). E. coli-induced IFNβ responses mediated by TLR4 requires internalization of the receptor and bacteria to Rab11a enriched compartments where TLR4 and TRAM localize with IRF3. Silencing of Rab11a reduced TLR4 trafficking and IRF3 phosphorylation(Husebye et al. 2010). Another study, discovered that Ca2+ flux was needed to induce LPS mediated IFNβ production(Husebye et al. 2010; Kagan

2010), as treatment with Ca2+ chelators blocked IRF3-phosphorylation and

induction of ISGs. PLCγ2 activity is required Ca2+ flux and translocation of TLR4 to endosomes(CHIANG et al. 2011). Syk and PLCγ2 are activated downstream of

CD14(Zanoni et al. 2011). Altogether, a very specific pathway must be induced to prompt TLR4 to traffic to a perinuclear endosomal signaling complex for optimal induction of IRF3.

While TLRs detect PAMPs in luminal and extracellular spaces, RIG-I and melanoma differentiation-associated protein 5 (MDA5) are cytoplasmic RNA sensors that bind short phosphorylated dsRNA, and long dsRNA respectively(Loo et al. 2008). RIG-I and MDA5 interact and form oligomers with mitochondrial antiviral signaling protein (MAVS) through caspase recruitment domain (CARD) interactions(Seth, Sun, and Chen 2006). A signalosome develops at the mitochondria, mitochondria-associated ER membranes (MAMs), or peroxisomes to induce a type I, or type III interferon response through TBK1 dependent IRF3 activation(Dixit et al. 2010; Kagan 2012).

DNA Sensors

Given the importance of DNA, there are a plethora of cytosolic DNA sensors that differ by cell type distribution and specificity. The variety of DNA sensors permits redundancy of DNA detection. The earliest cytosolic DNA sensor to be

discovered is DNA-dependent activator of IFN-regulatory factors (DAI). (Takaoka et al. 2007) Fibroblasts deficient in DAI were found to be deficient in IFN responses to transfected B-DNA and HSV1. DEAD-Box Polypeptide 41 (DDX41) was discovered to be an important DNA sensor in Dendritic cells(Zhang et al. 2011; Parvatiyar et al.

2012), and B-cells(Lee et al. 2015). DDX41 ablation reduced STING dependent IFN responses to B-DNA, or HSV. Interestingly, DDx41 was also found to be a cooperative sensor and binder of c-di-GMP and c-di-AMP to mediate STING responses(Parvatiyar et al. 2012). RNA polymerase III can mediate cytosolic DNA sensing through the transcription of RNA species that can be then recognized by the

RIG-I pathway. (Ablasser et al. 2009) IFI16 is a DNA sensor(Unterholzner et al.

2010) that binds viral DNA to induce STING dependent interferon or AIM2(Ablasser et al. 2009) (Fernandes-Alnemri et al. 2009) dependent inflammasome activation.

IFI16 is crucial for immunity to many DNA viruses, due to its ability to shuttle between the nucleus and cytoplasm. IFI16 shuttles between the cytosol and nucleus, and is crucial for immunity to DNA viruses HIV(Jakobsen et al. 2013),

HSV(Orzalli, DeLuca, and Knipe 2012), CMV(Gariano et al. 2012), KSHV(Thompson et al. 2011), and viruses that replicate in the nucleus(Thompson et al. 2011). While

IFI16 has been shown to bind HSV-1 DNA in the nucleus and shuttle it to the cytosol(Li et al. 2012). IFI16 facilitates IL-1/IL-18 and IFNβ cytokine responses dependent on AIM2 and STING, respectively. While IFI16 and Aim2 complexes have been shown, It is not yet mechanistically clear how IFI16 instigates STING

activation, but it seems that cGAS stabilizes IFI16:DNA complexes(Storek et al. 2015;

Orzalli et al. 2015) in some cases to fulfill this signal.

Binding of dsDNA(Shu, Li, and Li 2014) or Y-form, single-stranded template

DNA (sstDNA) (Herzner et al. 2015) by cGAS causes a change of confirmation that activates the enzymatic domain of cGAS to produce cyclic-GMP-AMP (2’3’-cGAMP). cGAS produced cGAMP(Chatzinikolaou, Karakasilioti, and Garinis 2014; Xiao and

Fitzgerald 2013) promotes STING dependent signaling in bystander cells by the propagation of 2’3’cGAMP through gap junctions or the incorporation of 2’3’cGAMP into the capsid of HSV-1(Gentili et al. 2015) and maybe other virions. cGAMP thus is an efficient second messenger to bystander cells warning of the infection of nearby cells(Xiao and Fitzgerald 2013; Li et al. 2013), thus to mount rapid induction of ISGs and an antiviral state. This is especially important in nearby cells that would otherwise not have upregulated cGAS or other DNA sensors.

Aim2 binds cytosolic DNA to induce inflammasome assembly, which is a signaling complex that includes caspase1 and ASC. The inflammasome then acts to cleave and activate the IL-1 and IL-18(Hornung et al. 2009). Activation of AIM2 interestingly has an antagonistic effect on the STING pathway, as knockdown or deletion of the

AIM2 pathway increases STING activation and type I interferon responses. (Corrales et al. 2016)

STING

STING is central to the IFN response against viruses. As STING-/- mice have difficultly controlling DNA viruses, such as HSV1(Ishikawa, Ma, and Barber 2009), and some RNA viral infections like VSV(Ishikawa and Barber 2008). Most DNA sensors require STING to signal Type I interferons. Furthermore, STING directly binds and is activated by both pathogenic CDNs, and endogenous cGAMP produced by cGAS activation(Li et al. 2013).

STING is highly expressed in immune effector cells and various other cell types to survey the presence of DNA. DNA in the cytosol is considered a Danger signal, and drives a type I interferon responses. The cytosolic PRR STING is crucial for the induction of cytokine responses to aberrant cytosolic DNA species. The genomes of intracellular pathogens such as Listeria or HSV-1 can be a source of

DNA(Hansen et al. 2014), highlighting the urgency of mounting a proper response.

STING (Stimulator of Interferon Genes) – (aka MITA, MPYS) (Jin et al. 2008)

(Ishikawa and Barber 2008) (Jin et al. 2013) is a 378 or 379 amino acid protein in mice and humans, respectively, that is encoded by the gene Tmem173. At the inactivated state it exists as a dimer. It is localized to the endoplasmic reticulum(Ishikawa and Barber 2008) by tetraspanning, N-terminal,

transmembrane domain. A putative transmembrane domain forms the N-terminus of the protein (1-145), followed by the dimerization domain (145-155). The globular domain (156-378 - in mice) is cytosolic and is the portion of STING that binds CDNs. The C-terminal tail (CTT) (341-378) of STING recruits and activates

TBK1. Cyclic dinucleotides (CDNs) (Yin et al. 2012) (Yin et al. 2012) are produced by bacteria and by cellular cGAS as second messengers, to serve as a warning to signal the presence of aberrant DNA. STING binds CDNs, undergoes an allosteric conformational change that brings together the dimerization domains, and exposes the CTT(Wu et al. 2014) . The CTT then recruits factors (TBK1, IKK) to activate

(IRF3, STAT6, and NF-kB) that results in the activation and induction of host ISGs and cytokines.

Cyclic-dinucleotides are comprised of 2 subunits of either AMP and/or GMP linked by phosphoesters of their sugar groups. Cyclic-di-nucleotides (CDNs) are produced by bacteria as necessary messengers to coordinate quorum sensing based on bacterial species. Bacteria use CDNs as second messengers to activate gene networks to coordinate activity in plaque formation or other chemotactic responses.

(Danilchanka and Mekalanos 2013). Generally, c-di-GMP is produced by gram negative bacteria, c-di-AMP is produced by gram positive bacteria such as Listeria, and 3’3’cGAMP, a unique cyclic GMP-AMP hybrid linked by 3’5 linkages, is produced by vibrio species. 2’3’cGAMP is a relatively novel CDN produced in mammalian cells by the DNA sensor cGMP-cAMP synthase (cGAS).

c-di-AMP was found to be the crucial factor responsible for Type I Interferon production by Listeria infection(Burdette and Vance 2013; Burdette et al. 2011;

Barker et al. 2013). Introduction of c-di-GMP into the cytoplasm was also found to induce IFNβ production(Burdette and Vance 2013; Burdette et al. 2011; Barker et al.

2013). STING was discovered as the responsive element through an ENU mutagenesis genetic screen in mice. The mutant mouse discovered, named

Goldenticket, has a loss-of-function I199N mutation in STING that rendered the mouse deficient in mounting an IFNβ response to intracellular Listeria(Sauer et al.

2011). Further studies by the same group show direct binding of c-di-GMP by

STING(Burdette et al. 2011). Many studies afterwards have shown crystal structures of c-di-GMP bound to the STING globular C-terminal domain(Yin et al.

2012; Ouyang et al. 2012; Huang et al. 2012). The conformation of the N-terminus of STING with or without ligand can only be guessed, as the N-terminus has been omitted from crystallization studies.

The N-terminal structure and function of STING is largely unknown.

Overexpression of STING in 293T induces a ligand independent induction of ISRE promoter element in a luciferase assay. The Russell Vance lab introduced amino- acids changes to charged residues in the N-terminus. They noted a strong reduction of induction of the ISRE elements without affecting c-di-GMP binding. Deletion of the N-terminus of STING in this assay promoted a lack of induction of ISREs, and co-

expression of the N-terminus fraction along with full length STING also reduced signaling(Burdette et al. 2011). This indicates that the N-terminus of STING is crucial for the function of the C-terminus, even though the C-terminus by itself binds

CDNs and activates TBK1(Tanaka and Chen 2012). Recent work is highlighting the significance of the N-terminus, as some novel inhibitory viral proteins and host activators of STING target the transmembrane domains(Dempsey 2015).

During STING signaling there are multiple steps of posttranslational activation. Upon activation, ubiquitin ligases TRIM32(Zhang et al. 2012) and

TRIM56(Tsuchida et al. 2010) bind and catalyze K63 ubiquitination of STING at multiple lysine residues, but mainly at K150. TRIM32 ubiquitination of STING at multiple residues has been shown to be crucial for TBK1 interaction with STING.

TBK1 binds STING, sometimes in a TRAF3(Abe and Barber 2014) mediated fashion, to autophosphorylate itself and phosphorylate STING at residue 365 or 366 in mice or humans, respectively .102 TBK1 phosphorylation of STING at 365-366 precedes

IRF3 recruitment and phosphorlylation.102 Ubiquitination of STING also allows

Traf6 interaction with STING for IKK activation. Activated NF-kB, AP-1, and IRF3 transcription factor complexes translocate to the nucleus to activate the first wave of interferon, IFNβ.10 ULK1 a kinase is activated downstream of IFNAR to activate autophagy that negatively regulates signaling. ULK1 phosphorylated STING at

365/366 serine residue in a manner that is correlated with negative

regulation(Konno, Konno, and Barber 2013). E3 ligase, RING finger protein (RNF) family members RNF5(Zhong et al. 2009) and RNF26(Qin et al. 2014) regulate

STING levels through ubiquitination. RNF5 K48 ubiquitinates STING at K150 to induce proteasome degradation of STING, while RNF26 can competitively induce

K11 ubiquitination at the same K150 site to promote STING stability. The order of these events that leads to STING activation and translocation is still far from fully understood.

Recently, it has been determined that factors normally involved in the ER stress response help mediate the first steps of activation of STING. INSIG1/AMFR is an ER resident complex, that serves as an E3 Ubiquitin-ligase that catalyzes K48- ubiquitination of misfolded proteins at the ER. Recently, autocrine motility factor receptor (AMFR) and insulin-induced gene 1 (INSIG1) complex has been shown to mediate the K27-ubiquitination of STING in response to cytosolic DNA or

CDNs(Wang et al. 2014). This ubiquitination is necessary for STING interaction with

TBK1. Afterwards, TBK1 goes on to phosphorylate IRF3. However, the mechanism by which IRF3 was recruited to the STING signaling complex was still not understood until SREBP cleavage-activating SCAP(Chen et al. 2016), another ER resident protein was found to interact with STING. SCAP was found to translocate to perinuclear microsomes, in a STING dependent manner, to facilitate STING-TBK1-

IRF3 interactions.

Autophagy components have been implicated in both the translocation based activation and degradation of STING(Woo et al. 2014; Konno, Konno, and Barber

2013; Konno and Barber 2014). Upon activation, STING traffics from the ER to vesicles via the Golgi apparatus to induce IFN. The initial trafficking to Golgi is shown to be dependent on vacuolar sorting protein 34 (VPS34) (Woo et al. 2014;

Konno, Konno, and Barber 2013; Konno and Barber 2014), which is a Class III PI3K, and calcium flux from an unknown source (Liu, Zeng, et al. 2012) (Hare et al. 2015).

Blocking either calcium flux (Hare et al. 2015) or VPS34 activation retains STING at the ER (Konno, Konno, and Barber 2013). From the Golgi, STING continues to yet undefined perinuclear vesicles where IRF3 is robustly activated. From there, STING associates with LC3, in an ATG9 dependent manner, to be degraded(Saitoh et al.

2009).

The DNA damage response and IFN responses are more connected than previously realized. It makes sense with the discovery of many cytosolic and endosomal sensors that sense DNA to induce IFN responses. When DSB repair enzymes are overcome by the level of DNA damage, or ATM is nonfunctional as in

Ataxia-telangiectasia mutants, DNA leaks and builds up in the cytoplasm to trigger the STING pathway(Hartlova et al. 2015). The MRN complex (MRE11, RAD50, and

NBS1) is a sensor of DNA Damage activity and activator of ATM(Buis et al. 2012).

Interestingly, MRE11 and RAD50 acts as a sensor for STING recognition of

DNA(Takeshi Kondo and Kenshi Komatsu 2013). This may be one mechanism of delivery of self-DNA to activate STING.

The STING-cGAS pathway supports immunity to viruses. It is no surprise that viruses have also derived mechanisms to subvert the cGAS-STING pathway.

HSV-1 has been shown to activate the STING pathway in vivo and in vitro, and is necessary for an effective immune response to virus, as STING-/- MEFs are attenuated in IFN responses to HSV-1 infection, and STING-/- mice succumb to fulminant HSV-1 infection(Ishikawa and Barber 2008).

The study of how viruses subvert or modify the STING pathway to permit viral persistence can uncover novel STING-dependent pathways. Understanding how viral proteins target the pathway, can lead to more effective therapies to block viral persistence, and can lead to mechanistic understanding of the pathway.

Infection with another herpetic virus, KSHV, activates the cGAS-STING pathway(Jacobs et al. 2015). However, expression of multiple KSHV gene products antagonize pathway activation. Viral interferon regulatory factor 1 (vIRF) inhibits

STING dependent IFNβ induction by an interaction with STING that prevents TBK1 binding to STING(Jacobs et al. 2015). The latency associated nuclear antigen

(LANA) of KSHV blocks cGAS activity to block the pathway induction(Zhang et al.

2016).

The polymerase of HBV suppresses IFN responses by binding STING and reducing K63-linked poly-ubiquitination necessary for STING signaling. HPV E7 protein blocks STING mediated interferon responses to DNA, by binding to STING N- terminus in a manner dependent on the LXCXE motif of E7. Interestingly, this

LXCXE motif is also responsible for STING’s ability to bind retinoblastoma (Rb).

(Laura Lau 2015) E1a protein of adenovirus, not a herpes virus, was also found to inhibit STING activity in a manner dependent of LXCXE binding of the STING N- terminus. Strangely, adenovirus infection did not reduce STING mediated IRF3 and

TBK1 phosphorylation. Thus E1a, and E7 inhibit STING function by acting at the N- terminus suggests an unknown arm of STING signaling mediated by the N-terminus along with a possible, yet to be discovered, accessory protein. (Lam, Stein, and

Falck-Pedersen 2014)

STING has been shown to have a role in the control of RNA viruses, by an unclear mechanism. This is attributed to STING interactions with MAVS at MAMs

(Marchi, Patergnani, and Pinton 2014). RIG-I, a DEAD-box helicase upstream of

MAVS, has been shown to have direct interaction with STING (Biacchesi et al. 2012).

STING -/- cells have weaker interferon responses to RNA viruses SeV and VSV.

(Ishikawa, Ma, and Barber 2009) Consequently, some RNA viruses evolved viral proteins which inhibit STING responses as well (Nitta et al. 2013). Coronovirus suppresses Interferon responses via viral protein transmembrane proteins. Plpro

(Chen et al. 2014), a protein of SARS CoV, and PLP2 (Sun et al. 2012), of NL63 CoV, both interact with STING to disrupt dimerization and activation at the ER., and attenuate K63 poly-ubiquitination. Thus these CoV proteins appear to block protein interactions (probably at the N-terminus) at the early phases of STING activation.

This probably prevents STING translocation to the MAM, where it facilitates MAVS-

RIG-I signaling.

The Human population contains multiple alleles of TMEM173, with variants that change the outcome signaling. The evolutionary basis of most of these polymorphisms and amino acid changes is still not understood. The original human reference allele of STING has an arginine at 232 (Diner et al. 2013) (R232) that is activated by canonical CDNs, bacterial CDNs c-di-AMP, c-di-GMP, and 3’3’cGAMP, and non canonical 2’3’cGAMP produced by the host. STING alleles with the histidine at H232 retain robust responses to 2’3’cGAMP yet respond inefficiently to pathogen derived CDNs. (Yi et al. 2013) R71H-G230A-R293Q (HAQ) allele is activated by all

CDNs though the responses are generally weaker than the WT ref allele (Jin et al.

2011). The R293Q allele only responds well to cGAMP, but not other CDNs. Two anti-tumor synthetic compounds, 10-carboxymethyl-9-acridanone (CMA) and 5,6- dimethylxanthenone-4-acetic acid (DMXAA) (Cavlar et al. 2013; Conlon et al. 2013) produce type I interferon in mice, and have been found to do so by activating the

STING pathway. However, human STING is unresponsive to DMXAA due to G230 amino acid residue that doesn’t allow binding (Gao et al. 2014) (Gao et al. 2013)

(Conlon et al. 2013). Overall, there is much work to be done in studying how the genetic variations in human STING alleles affect the immune response to pathogens and self-DNA.

SELF-DNA and the overwhelmed DDR leads to STING mediated autoimmunity

The DNA damage response is a cellular program to control damage caused by host DNA breaks, which when are exacerbated lead to interferon responses . Ataxia telangiectasia mutated (ATM) is a kinase downstream of the DDR pathway, and is activated in response to double strand DNA breaks. It’s response to DNA damage subdues type I interferon signaling. In the normal homeostatic state of the cell, type

I interferon is basally expressed in effector cells to prime the cell for quick responses. In cases where ATM is defective, and thus so is DDR, DNA fragments build up in the nucleus, and leak into the cytoplasm where they activate the STING pathway. In patients or mice with loss-of-function ATM mutations, this constant flow of DNA fragments in the cytosol lead to chronic IFNβ production and autoimmunity. (Hartlova et al. 2015)

Forward genetics is an unbiased approach of explaining gene’s function

During human history and still today, Infectious diseases has been a major selective pressure and determination of human fitness and health. Furthermore, with the onset of globalization increase of human travel, the increased chances for rapid spread of diseases are evident. Human diversity leads to variations in susceptibility of disease. Most of these variations are conferred by genetically inheritable traits. It is more important than ever to study the genetic factors that confer the resistance to infection in as what seems to be a increasing smaller world.

People groups are increasing coming into contact with pathogens that to which as a population they are naïve. (Patarcic et al. 2015; Kodaman et al. 2014)

ENU mutagenesis is a common genetic screen where an inbred mouse strain is subjected to mutagen. The result of this mutagenesis is phenotypic variation in a panel of mice. STING was discovered in a mutant the via the STING gt/gt mutation in an ENU mutagenesis panel.(Sauer et al. 2011) However, wild-derived inbred strains, and recombinant congenic crosses can also be used in a Forward Genetic screen to find novel interactions. ENU mutagenesis variations are caused on a common background, yet with multistrain intercrosses there may multiple genes in a phenotype that naturally evolved to interact.

Classical lab strains are great models to dissect how the body fights and overcomes infectious disease. Individuals an inbred line are almost genetically identical leading to the reproducibility of laboratory experiments.(Festing and

Altman 2002) It has been noted that susceptibility to disease can differ between

strains. Reproducible and reliable differences responses to various pathogens between lab strains give opportunity for genetics screens to elucidate novel mechanisms of immunity. Various studies reveal that these traits can segregate in a

Mendelian ratio. Our lab, and some others, has had great success exploiting

MOLF/Ei genetic variation to map loci that confer traits. (Surpris et al. 2016; Conner et al. 2010; Conner, Smirnova, and Poltorak 2009, 2008; Khan et al. 2012; Vidal et al.

2008)

Forward genetics is a procedure to identify genes or loci that confer a trait or phenotype by providing a causative link between the phenotype and the genotype.

In mouse genetics, this link is established by comparing a phenotype and the genotype in a second-generation progeny derived from parental lines that are different in the phenotype of interest. This technique usually reveals mode of inheritance (dominant or recessive) of the phenotype thus providing information about interaction of the gene product encoded by the wild type and mutant alleles.

This can be inferred by a quantifiable change in phenotype after crossbreeding. This method has lead to the discovery of a host of genes that modulate host resistance to pathogens, which is how Lps (TLR4) (Poltorak 1998) and Irak2c(Conner, Smirnova, and Poltorak 2009) were discovered. A Quantitative trait locus (QTL) is a genomic region, identified usually by markers and/or sequencing, that correlates with an inheritable phenotype in a crossbred population of individuals. (Smith et al. 2009)

We use J/QTL to run the statistical analysis to find QTLs(Salinger and Justice 2008;

Valdar et al. 2006)

MOLF mice variability

Mus musculus diverged about a million years ago into three major subspecies: mus musculus domesticus, mus musculus castaneus, and mus musculus musculus.

These sub species were segregated by geographical location to Western Europe;

Western and Southeastern Asia; and Eastern Europe, Russia, and Northern China respectively. Mus musculus molossinus originated 10,000 years ago from a hybridization of mus musculus castaneus and mus musculus musculus in Japan.

(Guénet and Bonhomme 2003)

Laboratory mice can be categorized as Classical inbred or Wild derived strain of mice dependent on origins. Classical lab strains originated from the collections of mouse fanciers, whose mice were already subject to inbreeding. According to extensive genome-wide analysis of haplotype of the classical strains, these strains have been derived from a few founder mice. These strains have varying contributions of all the original subspecies. The relative lack of variation of classically inbred strains to those found in the wild, has led to the derivation of new strains from wild-caught mice. These mice represent a more natural hybrid of subspecies. (Morgan et al. 2015; Yang et al. 2011)

The genomic contribution mus musculus species to MOLF mice are 11% domesticus, 74% musculus, and 15% castaneus. The MOLF/Ei wild-derived lab strain is of the m. m. molossinus subspecies and contributes a mere 10% genetic contribution to the classical lab strains. (Morgan et al. 2015; Yang et al. 2011)

(Frazer et al. 2007) On average, MOLF mice contain a differentiating SNP every 100-

200bp compared to classical lab strains such as C57Bl/6. This genetic diversity leads to much phenotypic diversity which can be exploited in a genetic screen.

Interbreeding of the two evolutionarily divergent strains leads to variable gene interaction networks that when mapped and described can lead to deeper understanding of the affected pathway or gene. In the Poltorak lab, we intercross

B6xMOLF to create F1s, and then we intercross F1s to create an F2 panel of mice.

Alternatively, we can backcross F1 to C57Bl/6 to create N2 mice. Due to the meioses and homologous recombinations that occur in between generations each individual F2 or N2 mouse is a genetically distinct and unique combination of the founder parental genes.

To further understand the mechanisms of innate immune responses to cytosolic DNA, we established a genetic screen in which we measured cytokine production in macrophages from a genetically diverse wild- derived mouse strain,

MOLF, in response to HSV1 (Herpes Simplex Virus) and Listeria monocytogenes.

Here we identify a novel loss-of-function allele of Sting that fails to activate IFN- production because of previously uncharacterized mutations in its N-terminal domain (NTD). We also show that amino acid substitutions at positions 47, 48 and

53 of the N-terminus of MOLF STING impair IFN-responses to DNA in MOLF mice.

The identified mutations are unique in that despite low production of IFN both in vitro and in vivo, MOLF mice exhibit normal levels of IL6 - thus showing underappreciated cross-talk between different signaling pathways of innate

MOLF immunity. Using Sting congenic mice, we show that STING is required but not sufficient for IL6-production, as DNA-responses in congenic mice, containing MOLF

STING, do not lead to high levels of IL6. Based on our linkage analysis, imaging data, genetic complementation and in vitro reporter analysis, we propose a model in which the amino terminus of STING plays a central role in controlling the balance between production of IFN and other cytokines, namely IL-6, in the DNA sensing pathway. Our data for the first time uncover important biological functions for the

NTD of STING, made possible by studies of wild-derived mice whose STING alleles may elucidate evolutionary pressures that may have also shaped human STING polymorphisms.

CHAPTER 2 - Methods

Reagents

Poly(I:C) and Poly(A:T) [dAdT] was purchased from Sigma Aldrich. c-di GMP, c-di

AMP, 2’3’ cGAMP, 3’3’ cGAMP, and DMXAA was purchased from Invivogen.

Lipofectamine 2000 (Life Technologies) was used to transfect c-di GMP, c-di AMP,

2’3’ cGAMP, 3’3’ cGAMP, and dAdT. Xtremegene 9 to transfect poly(I:C).

For stimulation experiments macrophages were plated at a density of 106 cells/mL.

C-di-nucleotide stimulations done as indicated at 2ug or 4ug/mL. materials and methods.

Mice

C57BL/6J and MOLF/EiJ were obtained from Jackson Laboratories (Bar Harbor,

ME); Sting -/- obtained from Glen Barber (U. Miami), and described in reference:

Ishikawa and Barber, 2008. Mice were housed in a pathogen-free facility run by

Division of Laboratory Animal Medicine (DLAM) at the Tufts University School of

Medicine. For generation of the B6.MOLF-Tmem173molf (STINGMOLF/MOLF) congenic mice, F1

(B6 x MOLF) hybrids were backcrossed for 9 generation on the B6 background.

DNA Sequencing

Genomic DNA or cDNA was amplified using Phusion high-fidelity DNA polymerase

NEB). PCR products were resolved on a 1.2% agarose gel and extracted using a 44

QIAquick gel extraction kit (Qiagen). DNA was sequenced from primers flanking using an ABI 3130XL DNA sequencer at the Tufts University Core Facility.

Genomic primers: 5’-GAACCCCAGAAGCATAGCTG-3’, and 5’-

GCACACCAGCACTAGCCATA-3’. cDNA cloning primers: 5’-TATCTCGAGGCCACCATGCCATACTCCAACCTGCATC-3’ &

5’-ATGGATTCGATGAGGTCAGTGCGGAGTGGGAGAGG-3’

SNP analysis

Common lab strains SNPs were probed on JAX SNP database. MOLF, MSM, and SPRET polymorphic regions were verified with genomic sequencing of exons 3 -

4 using primers: 5’-GAACCCCAGAAGCATAGCTG-3’, and 5’-

GCACACCAGCACTAGCCATA-3’. Sequence alignment of murine allelic variants compared and Hierarchal Clustering Dendrogram created using ClustalW2

(http://www.ebi.ac.uk)

Species alignment

STING cDNA was cloned from B6, MOLF, MSM, and SPRET macrophages and sequenced. FASTA sequences of various mammalian STING genes were downloaded from NCBI. DNASTAR Structural Biology Suite was used to analyze alignment of

Sting gene variants and effect on secondary structure.

Isolation of Primary Peritoneal Macrophages

Peritoneal macrophages were isolated from mice 8-12 weeks of age. Cells were elicited by injection of 1 ml of 3% thioglycollate. 3-5 days post injection mice were sacrificed by CO2 asphyxiation and cervical dislocation, and peritoneal cavity was immediately lavaged with cold PBS. Cells were pelleted at 1000 rpm @ 4˙C for

10mins, then resuspended in DMEM (GIBCO) with 10% FBS (Atlas Biologicals) and

1% penicillin/streptomycin. Cells were plated and incubated overnight at 37˙C with

5%CO2 before stimulation.

Genotyping and Quantitative Trait Locus Mapping (QTL)

Genomic DNA was isolated from mouse tails using DirectPCR Tail lysis kit

(Viagen) according to Manufacturer protocols. Genome wide genotyping screen was perform with 2-4 polymorphic microsatellite markers per chromosome using MIT primer sequences obtained from The Jackson Laboratory Mouse Genome

Informatics Web site: http://www.informatics.jax.org/marker. ~80-200bp fragments from Microsatellite region were amplified by PCR using Jumpstart

REDtaq (Sigma) and resolved on 3% gels.

Genotyping of MOLF/Ei and C57BL6 allelic variants of STING was done using the following primers and the aforementioned REDtaq protocol. 5’-

TTTCCTCCAAAACACTGC-3’ & 5’-ACCCACCTCTTCTGCTTC-3’.

Phenotyping data from stimulated macrophages (IFNβ and IL-6 cytokine levels measured by ELISA) and genotyping data were used for QTL mapping of loci which conferred traits. QTL mapping was done using J/qtl v.1.3.x and R versions

2.14 – 3.0 (http://churchill.jax.org/software/jqtl.shtml)

Plasmids

Using High-Fidelity Phusion polymerase, B6 and MOLF STING was cloned from macrophage cDNA and subcloned into pEF-BOS-HA or pC1-mCherry. Single

Mutations were created by site directed mutagenesis with primers containing SNPs.

Parental STING and Mutated variants were subcloned into pC1-mCherry or pLex

(OpenBiosystems).

Luciferase assay

Overexpression assay

293T cells were plated at 5x105 cells per well in a 96 well plate and allowed to adhere overnight. 30 ng STING pEF-BOS, 12.5ng Renilla Luciferase - pRL-TK, and

12.5ng Firefly Luciferase plasmids expressing the full IFNβ promoter, or

PRDII(NFkB), or PRDIII(IRF3) promoter regions were transfected per well with

1ul/well Lipofectamine 2000 (Life Technologies).

CDN stimulus dependent Luciferase assay

293T cells were plated at 2.5x105 cells per well in a 96 well plate and allowed to adhere overnight. 0.5 ng STING pEF-BOS, 12.5ng Renilla Luciferase - pRL-TK, and

12.5ng Firefly Luciferase plasmids expressing the full IFNβ promoter transfected per well with 1ul/well Xtremegene9 (Xtremegene9). 24hr later, media was changed and, 293Ts were stimulated with CDNs at 1-4ug/mL with Lipofectamine 2000

(Roche). 16 hrs post stimulation, luciferase activity was determined with Dual-Glo®

Luciferase Assay System on Spectramax M5

Western Blotting

Whole cell lysates were prepared by first washing cells with ice cold PBS, followed by cell lysis with lysis buffer (50 mM Tris [pH 8], 150 mM NaCl, 2 mM

EDTA, 1% Triton-X100, 1 mM sodium vanadate, 10 mM NaF, and protease inhibitor cocktail) on ice 10 mins. Lysates were centrifuged for 10mins at 13,000 rpm at 4˙C, then lysates were transferred into fresh tubes mixed with Laemmili buffer and boiled for 10mins. Protein lysates were run on 4-12% gradient Novex Bis-Tris SDS gel (Life Technologies), transferred to a nitrocellulose membrane, and blocked in

5% BSA, before incubating in 1˙ and then 2˙ antibodies. Western Blots were detected using and ECL chemiluminescent assay (Pierce)

Antibodies: (Cell Signaling): STING #3337; p65 #8242; Phospho-p65 #3033; ERK

#4695; Phospho -ERK #4370; p38 #9212; Phospho -p38 #4511; TBK1 #3013;

Phospho -TBK1 #5483; Phospho -IRF3 #4947; JNK #9258; Phospho -JNK #9251;

HA-Tag #3724.

(ENZO): Grp94, #ADI-SPA-850

Listeria monocytogenes, HSV1, and Sendai infection Cells were infected with

Listeria monocytogenes strain 10403S at multiplicity of infection MOI of 10. Cells were washed at 1h after infection, and gentamicin added to kill bacteria.

Supernatant was collected 18hrs after infection. For viral infection, HSV-1, KOS strain, and Sendai virus, Cantell strain (Charles River Labs), were used at MOI of 20 and 10 respectively.

ELISA

The concentration of IFN-β, and IL-6 in culture supernatants were measured by

ELISA (IL-6, R&D Systems; IFN-β, antibodies from PBL InterferonSource and Santa

Cruz used as per protocol described in reference: Roberts, Goutagny et al. 2007.

RNA analysis by Nanostring.

Macrophages were treated as previously indicated, and RNA was purified using

RNeasy Kit (Qiagen). Total RNA was hybridized to a custom designed gene codeset

(using bar-coded fluorescent probes) according to manufacturer’s instructions

(Nanostring technologies), and quantified on nCounter Analysis System. Log- transformed values were displayed by Heat map.

Quantitative PCR RNA analysis

RNA extracted from macrophages using TriPure, was first subjected to DNAse treatment before Mouse IFNβ mRNA was measured by real-time PCR with the

TaqMan gene expression assay probe: Mm00439552_s1. As a control Mouse GAPD

(GAPDH) Endogenous Control (VIC®/MGB probe) was used.

RNASeq workflow

Total RNA isolated, using TriPure(Roche), from macrophages stimulated with indicated agonists (lipo, 2’3’cGAMP 4ug/mL, dAdT 4ug/mL, or DMXAA

1ug/mL). Total RNA prepared for reading in MiSeq platform (Illumina) with TruSeq

RNA Sample Preparation Kit. Double-stranded cDNA library was generated from mRNA. Samples were sequenced with 75bp pair-end reads on Illumina MiSeq. Raw data sets were analyzed with Tuxedo application suite. TopHat2/Bowtie2 was used to align 75bp reads and simultaneously map them to the annotated NCBI/mm10 genome. Biological replicates were mapped independently. Mapped reads were input into Cufflinks package to assemble transcripts for each replicate, merge transcript expression of replicates, calculate expression levels and significance, and provide a report of differential expression between results.

Lentiviral transduction of STING -/- MEFs

MEFs isolated form 13.5 day old STING -/- embryos. Lentiviral particles were produced by transfecting STING expression constructs (pLex with truncated

CMV) with packaging constructs pMD2.G and psPAX2 into HEK293T cells using

Xtremegene-9 DNA transfection reagent (Roche). Virus containing media was collected 48hrs and 72hrs post-transfection pool and filtered using 45μM filter.

Resulting media was used at 25-30% to infect STING Knockout MEFs for 24hrs.

STING -/- MEFs were puromycin selected with 2 μg/ml Puro for 2 two days, and allowed to recover at least 3 more days before use.

Confocal imaging of MEFs

Transduced MEFs were grown on glass coverslips, stimulated as indicated, fixed in 4% Paraformaldehyde and permeabilized with cold Methanol. Coverslips were blocked in 5% goat serum before incubation in Grp94 ab (ENZO) 1:1000 at room temperature 1 hr in 1% BSA in PBS. anti-Rat IgG-Alexa 488 added at 1:1000 at room temperature. Imaging runs were performed on a spinning-disc confocal microscope using a modified live-cell imaging system (UltraVIEW; PerkinElmer) consisting of a 5-line laser launch (442/488/514/568/647 nm; Prairie

Technologies), a spinning-disc confocal head (CSU-10; Yokogawa Corporation of

America), an Axiovert 200M stand (Carl Zeiss), a 40× Plan-Neofluar oil immersion objective lens (NA 1.3; Carl Zeiss), and an intensified CCD (ICCD) camera (XR MEGA-

10; Stanford Photonics). ImageJ analysis was done using ImageJ software (NIH).

Colocalization analysis was done using Coloc2 plugin.

CHAPTER 3 - MOLF macrophages lack interferon responses to DNA viruses, Listeria, and cytosolic nucleotides.

Problem: The human population contains much genetic diversity that shapes the outcome of disease. Mendelian inherited interferonopathies cause autoimmune diseases, such as SLE, or increase susceptibility to infections, such as Listeria. Mouse models of these variations of disease outcomes can aid in understanding the mechanistic onset of disease due to aberrant induction of Type I Interferons.

(Hartlova et al. 2015; Reder and Feng 2014; Deng et al. 2014; Moraru et al. 2012;

Brzoza-Lewis, Hoth, and Hiltbold 2012; Baker and Antonovics 2012; Woodward,

Iavarone, and Portnoy 2010; Stockinger and Decker 2008; Sancho-Shimizu et al.

2007; Decker, Muller, and Stockinger 2005; O'Connell et al. 2004; Carrero, Calderon, and Unanue 2004; Auerbuch et al. 2004)

Approach: MOLF mice are genetically distinct from common lab strains.

Originating from subspecies that diverged ~ 100,000 to 106 yrs ago, MOLF mice contain polymorphisms that distinguish them from common lab strains.

Macrophages detect viral and/or bacterial components through PRRs. Ligation of

PRRs with PAMPs trigger signal cascades that lead to transcription and translation of immune response genes, including cytokines. Question: How do MOLF macrophage and C57BL/6 macrophage compare when challenged with DNA viral, RNA viral, or Listeria infections?

Chapter Summary: Our studies reveal that MOLF intraperitoneal macrophages are hyporesponsive in IFN responses to DNA virus HSV1 and Listeria, but not the RNA virus Sendai. Further investigation of this defect reveals that MOLF mice also lack responses in IFNβ to cytosolic DNA or ci-di-nucleotides

A portion of this work is originally published in The Journal of Immunology. Guy Surpris, Jennie Chan, Mikayla Thompson, Vladimir Ilyukha, Beiyun C. Liu, Maninjay Atianand, Shruti Sharma, Tatyana Volkova, Irina Smirnova, Katherine A. Fitzgerald, and Alexander Poltorak. “Novel Tmem173 Allele Reveals Importance of STING N Terminus in Trafficking and Type I IFN Production.” 2016 Jan 15; J. Immunol. 196(2):547-52. Copyright © [2016] The American Association of Immunologists, Inc. doi: 10.4049/jimmunol.1501415. Epub 2015 Dec 18.

MOLF macrophages lack IFN responses to DNA viruses

As mentioned previously, MOLF/Ei mice have genomic variability compared to common lab strains. Because of these evolutionary divergences, MOLF have many differences in phenotypes and cellular responses that are conferred by their genomes when compared to common lab strains. These differences in phenotypes better relate to variation seen in the wild. Most importantly dissecting a role, genetic element, or reason for these differences in phenotype leads to increased understanding of how genetic elements and signaling pathways work, or may even lead to the discovery of a novel gene or pathway. Herein, we begin to dissect inter- strain differences in Interferon responses to cytosolic deoxynucleotide detection.

We collaborated with the Kate Fitzgerald Lab to investigate the variation of

MOLF macrophage responses to viral infection. Thioglycollate derived peritoneal macrophages and bone marrow derived macrophages (BMDMs), were infected 16 hrs with HSV or mCMV DNA viruses. IFNβ cytokine levels are a measure of the Type

I Interferon response, and IL-6 is a measure of the pro-inflammatory response in macrophages. IFNβ and IL-6 levels were measured by ELISA. Typically, infection of macrophages by a DNA virus will produce a significant type I interferon response as seen with IFNβ production from both B6 peritoneal and BM derived macrophages.

(Figure 1) However, MOLF/Ei macrophages produce significantly less IFNβ to DNA viruses when compared to B6. Interestingly, MOLF/Ei are responsive to infection by these viruses as IL-6 pro-inflammatory responses in MOLF are similar to (HSV), or 55

slightly higher (mCMV) than B6. MOLF macrophages have a deficiency in type I interferon responses to DNA viruses.

Viral components can trigger multiple components, such as TLR2, while entering the cell to produce pro-inflammatory cytokines. The DNA component of the viruses can be detected by endosomal TLR9s to produce IFNβ. However, the capsids of HSV and mCMV directly traffic the genome to the nucleus without appreciable direction to endosomes, as these viruses disrupt early endosomal trafficking. Thus, it is unlikely that much DNA will be available to the late endosome compartment to allow signaling via TLRs. MOLF have many aforementioned differences cytokine regulation downstream of TLRs, but it is necessary to discount contribution of the TLR9 compartment to viral infection. In Figure 1D, while there is defective IFN production to HSV, CMV, and poly-dAdT in MOLF and MSM macrophages, B6 TLR9-/- are sufficient in IFN production to these agonists. This shows that the endosomal TLR9 pathway is not important in detection of these pathogens.

A caveat is TLR9 KO in MOLF is not available. MOLF have differential mechanisms to phagocytose virus or usurp cytosolic DNA to make DNA available to

TLR9. However, our forward genetic screen should reveal any impact of this pathway if it exists in MOLF. Overall, A genetic component in MOLF macrophages confers IFN production to DNA viruses.

Figure 1 - C57Bl/6 and MOLF/Ei strains show a polymorphic response to DNA agonists A B 12000 1400

10000 1200 1000 8000 800 6000 600 4000 400 2000 200 0 0 B6 peri mφ MOLF peri mφ B6 BMDM MOLF BMDM B6 peri mφ MOLF peri mφ B6 BMDM MOLF BMDM HSV mCMV C IL-6 ELISA 7.5 B6 peri mφ 5.0 MOLF peri mφ B6 BMDM 2.5 MOLF BMDM

0.2

0.0 HSV mCMV D Peritoneal macrophages 1500 B6 PEC MOLF PEC 1000 MSM PEC TLR9 -/- PEC F1 MOLF 500 F1 MSM

Experiment from 10.28.10 APM and Fitzgerald Lab

Figure 1: MOLF macrophages display and attenuated IFNβ production to HSV1 (DNA virus), and mCMV (DNA virus). ELISA IFNβ MOLF/Ei and B6 peritoneal cells and BMDMs in response to overnight infection with (A) HSV or (B) mCMV. IL-6 ELISA of MOLF/Ei and B6 peritoneal cells and BMDMs in response to overnight infection with (C) HSV or mCMV. ELISA of IFNβ responses. (D) IFNβ ELISA of Peritoneal Macrophages from B6, MSM, MOLF, TLR9-/-, F1(MOLFxB6), and F1(MSMxB6) were treated overnight with poly dAdT, Sendai virus (SV), HSV, or mCMV. Preliminary Data from Annie Moseman and Kate Fitzgerald Lab A) representative of one experiment. B) representative of N=2 experiments

To further dissect the pathway, we transfected dAdT or the cyclic dinucleotide, c-di-AMP, directly into the cytosol with liposomes, and compared responses to infection with Listeria or HSV1. Listeria is a gram positive intracellular bacteria. Listeria breaks into the cytosol in a listeriolysin dependent manner after phagocytosis by the macrophage. In the cytosol, Listeria induces a type I interferon dependent on its secretion of c-di-AMP. HSV1 DNA is presented to the cytoplasm via leakage from viral capsid or presentation of DNA in the cytoplasm by DNA binding sensors, such as IFI16, that shuttle between the nucleus and cytoplasm.

As previously shown, MOLF peritoneal macrophages were deficient compared to B6 in interferon responses to HSV1. However, RNA virus Sendai virus produced strong and equivalent IFNβ responses in B6 and MOLF macrophages

(Figure 2A). Sendai virus releases RNA into the cytoplasm to produce a RIG-I dependent type I interferon responses. B6 and MOLF macrophages respond to

Sendai virus with equivalent IFNβ production (Figure 2A). The equivalent responsiveness between the strains, generally demonstrates that Type I Interferon responses are intact in MOLF macrophages. Therefore, the components to induce a

Type I interferon response are functional in MOLF. The defect in MOLF type I interferon responses appears to be specific to DNA species. (Figure 2A)

To evaluate that cytosolic DNA species are the principle component to which

MOLF macrophages lack responsiveness, we transfected dAdT or c-di-AMP into the

cytoplasm of peritoneal macrophages. dAdT and c-di-AMP represent the purported type 1 interferon stimulatory DNA of HSV1 and Listeria respectively. MOLF macrophages were defective in IFNβ production to both dAdT and c-di-AMP when compared to B6. Interestingly, MOLF produced robust IL-6 production to these agonists. These results suggest that MOLF has one or more variations in the cytosolic DNA sensing pathway that confer alternate cytokine production (Figure

2B).

Figure 2: MOLF peritoneal macrophages respond with attenuated IFNβ production to DNA agonist yet are proficient in IL-6 production. A) IFNβ or B)IL-6 production in thioglycollate elicited peritoneal macrophages after 16h of treatment with dAdT, c-di-AMP, HSV, Listeria, or Sendai virus (SeV); ELISA of Intraperitoneal Treatments: 2x105 Macrophages in 200 uL DMEM treated with liposomal poly dAdT (2ug/ml), liposomal c-di-AMP (2 ug/ml), SeV (MOI 10), HSV (MOI 20), or Listeria (MOI 10). *p , 0.05, **p , 0.01 representative of 3+ experiments.

Since MOLF macrophages have shown a defect in IFNβ production to c-di-

AMP, we extended our study to include all known natural CDNs. Peritoneal macrophages were stimulated with c-di-AMP, c-di-GMP, 2’3’cGAMP, 3’3’cGAMP, and the unnatural synthetic analog DMXAA. MOLF peritoneal macophages were significantly weaker in IFNβ production in all cases except in response to 2’3’cGAMP

(Figure 3). Compared to MOLF macrophages IFNβ responses with viruses (Figures

1 and 2), the MOLF responsiveness to 2’3’cGAMP are substantial. This is analogous to the responsiveness seen with STINGH232 allele, where a previously thought to be

CDN unresponsive allele was responsive primarily to 2’3’cGAMP.

To see how the IFNβ induction pathway compares between B6 and MOLF peritoneal cells we probed for activating phosphorylation of key signaling proteins downstream of Type I interferon activation. MOLF macrophages were weaker in activation of signaling components downstream of 2’3’cGAMP and barely activated signaling downstream of DMXAA. We stimulated peritoneal cells with the weakest and strongest agonists in inducing MOLF peritoneal IFNβ production – DMXAA and

2’3’cGAMP. Phosphorylation signals of IRF3 and NF-kB(p65) were both severely attenuated in MOLF PECs stimulated with 2’3’cGAMP or DMXAA(Figure 3C). This signaling data seems to conflict with MOLF 2’3’cGAMP cytokine production data, but it may be that MOLF STING is deficient in inducing the IRF3 signaling pathway while other MOLF genetic components compensate for the weaker induction during the

16hr stimulation for ELISA. There are multiple components that lead to and

enhance IFNβ promoter induction, transcription, and translation. One or more of these pathways may be skewed in the divergent MOLF background, or a novel yet undiscovered pathway may synergize to create this response.

62 LISTERIA HSV-1 A B6 MOLF B C 800 ** P<0.0001 B6 MOLF B6 MOLF 600 viperin ,U/ml

β 400 cxcl10 ** * ** tnfa IFN 200 20 rig-i 0 Il-1a 10 Il1ra lipo SeV IFN, U/ml mda5 media HSV1 Listeria 0 Ifi205 ci-di-AMP C57BL6 MOLF Il6 poly (dAdT) * * mnda 10 * Ifi204 P=0.1169 tlr2 Figure 3: MOLF macrophages are 5 * cox2 2

IL-6 ng/ml Il-1b 0 a20 a4enuated in IFNβ produc;on to cytosolic Irf7 lipo SeV 1 trex1 HSV1 media Listeria cxcl1 nucleo;des, but are hyper-responsive in IL6 ci-di-AMP IL-6 ng/mL stat1 poly (dAdT) 0 zbp1 C57BL6 MOLF casp1 produc;on compared to B6 lgp2 nlrc5 D E nlrp3 socs1 A tlr8 B6 PECs C 80 MOLF PECs ** tlr3 ml

/ tlr7

U ***

, Il12a β 40 DMXAA 2'3'-cGAMP * * Il-18

IFN duba * C57BL6 MOLF C57BL6 MOLF 0 aim2 Hrs 0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4 tlr4 Il-10 lipo DMXAA nos2 c-di-AMPc-di-GMP pTBK1P&TBK1& tlr1 2'3' cGAMP3'3' cGAMP Il12b B 1.5 pIRF3P&IRF3& tlr9 *** Ifnb1 1.0 ** * pERKP&ERK& nlrc4 tlr5 0.5 IL6 ng/ml * ns Ifna4 P&p65&pp65 Il21 0.0 nlrp6 P&p38&pp38 tlr11 lipo nlrp12 DMXAA GAPDHGAPDH& Il23a c-di-AMPc-di-GMP 2'3' cGAMP3'3' cGAMP Log2(FPKM+0.1) © Journal of Immunology 2016 0 2 4 6 8 10

Figure 3: MOLF macrophages are attenuated in cytokine and signaling responses leading to IFNβ production. Peritoneal macrophages C57Bl/6 and MOLF/Ei mice were harvested from mice primed 4 days prior with thioglycollate. Peritoneal macrophages were treated with 4ug/mL CDNS c-di-AMP, c-di-AMP, 2’3’ cGAMP, or 3’3’ cGAMP; or 10ug/mL DMXAA. A) IFNβ and B) IL-6 ELISA were done after 16-18hr stimulations. Stimulated peritoneal macrophage lysates were probed by WB for phosphorylated signaling components. Peritoneal macrophages stimulated 0 – 4 hrs with C) DMXAA or 2’3’ cGAMP. A), B) Representative of more than 3 independent experiments *p , 0.05, **p , 0.01, ***p , 0.001. C) Representative of 2 independent experiments.

IFNβ and IL-6 cytokines were only diagnostic measures of global responses to viruses. To attain deeper understanding of MOLF and B6 peritoneal macrophage responses we measured levels of a profile of inflammatory mRNAs, which will give us a deeper picture of transcriptional responses downstream of Listeria infection or

HSV1 infection (Figure 4A, 4B).

MOLF and B6 macrophages display considerable variation in induction of inflammatory transcripts upregulated downstream of Listeria and HSV1 infection. In

Figure 4, RNA from MOLF peritoneal macrophages and B6 peritoneal macrophages was extracted for hours after infection with Listeria or HSV1. With both Listeria infection and HSV infection MOLF peritoneal macrophages produce robust cytokine transcripts to IL-1 family members such as Il-18, Il-1a, Il-6, Tnf, and IL-1b, and these responses were significantly higher than B6 responses. However, MOLF upregulation of ISG’s such as IFI204, IFI205, Cxcl10, Viperin, and IFNβ remain significantly lower than B6 (Figure 4A, 4B). Comparable to Figure 2, IFNβ and IL-6 transcripts phenocopy cytokine levels (Figure 4C, 4D) seen in response to Listeria and HSV infection. Altogether, the inflammatory mRNA profile data reinforces that

MOLF macrophages respond with different inflammatory responses to Listeria and

HSV.

64 Figure 4: mRNA response to Listeria in Macrophages (Nanostring)

A 400000 HSV-1 200000 100000 B6 MOLF 80000

60000

40000 Relative Expression 20000

0 Il6 tlr2 tlr3 tlr7 tlr8 Irf7 a20 tnfa rig-i Il1ra lgp2 Il-1a Il-1b aim2 cox2 nlrc5 zbp1 stat1 trex1 nlrp3 duba cxcl1 Iﬁ204 Iﬁ205 mda5 mnda socs1 casp1 cxcl10 viperin

B 200000 LISTERIA

100000 60000 B6 MOLF

40000

Relative Expression 20000

0 Il6 tlr2 tlr3 tlr7 tlr8 Irf7 rig-i tnfa a20 lgp2 Il-1a Il-1b Il1ra cox2 aim2 nlrc5 stat1 zbp1 trex1 nlrp3 duba cxcl1 Ifi204 Ifi205 mda5 mnda socs1 casp1 cxcl10 viperin

C IFNβ D IL-6 40000 1200 B6 1100 B6 1000 MOLF MOLF 900 30000 800 700 600 20000 500 400 300 10000 Relative Expression Relative Expression 200 100 0 0

Media HSV-1 Media HSV-1 Listeria T gondii Listeria T gondii

Figure 4: Profile of inflammatory transcripts are shifted in MOLF macrophages compared to B6 macrophages in response to HSV1 and Listeria. Thioglycollate elicited macrophages were infected with HSV or Listeria for 4 hrs. Total RNA was hybridized to a custom designed gene codeset (using bar-coded fluorescent probes) according to manufacturer’s instructions (Nanostring technologies), and quantified on nCounter Analysis System. Transcriptional upregulation of B6 and MOLF genes in response to 4hr (A) HSV1 or (B) Listeria infection. (C) Excerpt of IFNβ and (D) IL-6 mRNA production in response to HSV-1, or Listeria infection from profile.

CHAPTER 4 - Forward genetic screen reveals linkage to Tmem173 as candidate gene.

Problem: In contrast to B6, MOLF/Ei macrophages are defective in IFNβ production and ISG induction to HSV1, Listeria, and CDNs, while producing hyperactive IL-6 responses to the same agonists. Due to the F1 dominant inheritance of the trait, the assumption is that a genetic locus in MOLF confers the trait. What genetic loci confer the attenuated IFN-response and/or IL-6 hyper- response in MOLF progeny.

Approach: Using forward genetics analysis in order to identify loci that confer the trait, we found a high degree of linkage of the trait with IFNβ responses to c-di-GMP, dAdT, or c-di-AMP to chromosome 18. Fine mapping of loci revealed linkage to

Tmem173.

Chapter Summary

Forward genetic analysis is unbiased approach of identifying genes that are responsible for a phenotype of interest. Accordingly, to identify gene(s) that confer the IFN-defect, we generated panels of N2 (F1(B6xMOLF)xB6) then F2 (F1xF1) mice and measured IFNβ production by peritoneal macrophages in response to HSV,

Listeria, and CDNs. Using Quantitative Trait Locus (QTL) analysis we identified

Tmem173 as a gene candidate for differential IFNβ production to cytosolic DNA.

N2 Panel responses to cytosolic nucleotides and pathogens

Since macrophages form F1 mice display IFN-defect, they show that the trait is indeed inheritable (Figure 1). In our first mapping experiment we generated N2 mice, by backcrossing F1 mice to B6. The progeny of this cross can be either heterozygous or homozygous for the B6 strain at any given locus. We phenotyped this panel of 35 genetically unique individual mice for IFNβ or IL-6 production in peritoneal macrophages (Figure 5). This phenotype was measured after infection or stimulation of macrophages with Listeria, HSV1, cyclic di-AMP, and LPS. This breeding strategy is expected to reveal loci responsible for the lack of IFNβ production in MOLF macrophages to cytosolic DNA.

Figure 5: N2 Panel reveals wide range of responses to CDNs and DNA pathogens indicating that lack of IFNβ production in MOLF is a genetic trait A panel of 48 N2 mice were generated. Thioglycollate elicited macrophages from N2 48 mice were stimulated as follows: 2x105 Macrophages in 200 uL DMEM treated with liposomal poly dAdT (1ug/ml), liposomal c-di-AMP (2 ug/ml), SeV (MOI 10), HSV (MOI 20), or Listeria (MOI 10). (A) Distribution of IFNβ production in N2 mice to LPS, HSV1, Listeria, c-di-AMP, and Sendai Virus. (B) Distribution of IL6 production in N2 mice to LPS, HSV1, Listeria, c- di-AMP, and Sendai Virus by ELISA.

To determine the relevance of interferon production to particular stimuli in the N2 panel, we plotted the correlation of the intensity of IFNβ production of LPS to

HSV1, or HSV1 and Listeria. Spearman's rank correlation (rho) is a nonparametric measure of statistical dependence between two variables that measures the rank order between points. Spearman’s rho coefficient of individual mouse responses to

LPS and HSV1 was very weak (Figure 6A). However, the correlation between HSV1 and Listeria IFNβ production in these N2 panel mice was quite strong with the

Spearman r ranking of 0.8025(Figure 6B). This suggests that responses to both

HSV and Listeria are conferred, to large extent, by the same genes. This is significant because unlike LPS, Listeria and HSV purportedly release DNA species into the cytoplasm. The genetic contributions leading to increase IFNβ production may be highlighted by these correlated responses. Thus, it is not surprising in Figure 7C that common loci map to both HSV1 and Listeria dependent IFNβ production.

Figure 6: IFNβ production of individual N2 mice to Listeria and HSV, and Listeria and c-di-AMP are correlate in intensity, while N2 mice responses to LPS and HSV responses are discordant.

Correlation of individual N2 mice (A) IFNβ responses to LPS/HSV show low correlation. Spearman r 0.4303 (MOI 20:1) and 0.3112 (MOI 10:1). (B) IFNβ responses to Listeria/c-di-AMP show high correlation. Spearman r 0.8025. (C) IFNβ responses to Listeria/HSV1 show high correlation. Spearman r = 0.5992 (MOI 10:1), and 0.5527 (MOI 20:1).

Genome wide QTL analysis displayed linkage to many MOLF loci that conferred differential IL-6 or IFNβ production when inherited in a heterozygous fashion. Also many of these loci were common between HSV and Listeria. Loci corresponding to markers D3Mit98, D11Mit4, D16mit189, and D18Mit202 were enriched for both Listeria and HSV1 (Figure 7c). However, none of these linkages were significant, to our criteria for confidence. We consider Log-of-odds (LOD) scores of 3 or more as significant, which corresponds to a Likelihood-Ratio-Score

(LRS) of greater than 13.8 (Figure 7A+B). This criteria for confidence represents a p-value < 0.001, or a less than 1:1000 chance that random assortment of genotypes led to a phenotype output.

HSV and Listeria infections constitute multiple components, such membrane proteins, DNA, etcetera, that can potentially stimulate various macrophage cellular components. Thus, one issue with attaining multiple peaks of linkage for HSV1 and

Listeria is that each of these linkages can be due to different components of the infectious agent.

A Marker Regression Analysis of N2 panel - IFNβ responses 15 LPS dAdT c-di-AMP HSV MOI 20:1 HSV MOI 10:1 Listeria MOI 20:1 10 Listeria MOI 10:1 LRS score

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Chromosome

B Marker Regression Analysis of N2 panel - IL6 responses

15 LPS dAdT c-di-AMP HSV MOI 20:1 HSV MOI 10:1 Listeria MOI 20:1 10 Listeria MOI 10:1 LRS score

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Chromosome

Figure 7: Marker regression analysis reveals that several loci confer differential IFNβ between B6 and MOLF macrophages. QTL analysis: linkages to differential IFNβ production. (A) Likelihood Ratio Score (LRS) for linkage with loci conferring IFNβ defect in N2 macrophage samples treated with LPS, dAdT (1ug/mL), c-di-AMP (2ug/mL), HSV MOI 20:1, HSV MOI 10:1, Listeria MOI 20:1, Listeria MOI 10:1. Calculated using QTXb software. (B) same as A except LRS scores link to IL-6 production. (C) Linkages as in A except highlighting similar loci in HSV and Listeria.

Figure 7c: QTL analysis: Shared linkage to differential IFNβ production between HSV1 and Listeria

C D3Mit98 Marker Regression - IFN-B 10 D16mit189 9 D11Mit4 8 D18mit202

6 HSV MOI 20:1 5 HSV MOI 10:1 LRS SCORE 4 Listeria MOI 20:1

3 Listeria MOI 10:1

D8Mit1(6) D9MIT2(17) D2MIT61(34) D4MIT172(9) D4MIT42(81) D5MIT95(68) D11Mit4(37) D12Nds2(59) D14Mit60(15) D19Mit28(12) D2MIT148(105) D3MIT98(39.7) D6MIT14(71.2) D7MIT350(41) D9MIT273(49) D10MIT279(2) D10MIT103(70) D15Mit115(24) DXMit50(14.1) D6MIT274(20.5) D8Mit163(45.32) D12MIT103(1.0) D13Mit311(32.8) D13Mit66(34.54) D16Mit189(55.2) D17Mit10(24.5) D18Mit64(4.46) D1MIT411(12.62) D1MIT440(44.98) D15MIT246(60.1) D18Mit124(32.15)

Because of the weak linkage obtained in the N2 panel, we switched to mapping using F2 intercross progeny, which is independent of the mode of inheritance. In our F2 panels we retained significant distributions in intensity of IL-

6 and IFNβ responses to agonists. (Figure 8)

As mentioned previously, QTL mapping is a powerful method to determine loci that confer phenotypes. In this panel we found strong linkages in response to c- di-AMP and c-di-GMP, and notable responses to dAdT. C-di-AMP and c-di-GMP displayed significant linkages with LOD scores 4.5 and 7.2 respectively at just one shared locus. (Table 1, Figure 9) This locus peaked over D18mit202. dAdT had dual linkages both with LOD scores of 2.4 loci at D11Mit4 and D18Mit202.

Interestingly, there is similar linkage confer by IFNβ responses to HSV and

Listeria. However, as with the N2 panel these associations remains of lower significance. HSV and Listeria responses give many more peaks than the simple agonists such as c-di-AMP. The increased number of peaks in the case of Listeria and HSV may be due to the multiple components contained within them. Other than

DNA, Listeria and HSV have lipid or protein components that trigger DNA independent immune responses. For example, HSV1 and Listeria can trigger TLR2 upon entry into the cell. The multiple components triggered by the Listeria and HSV can activate multiple pathways, and thus distract from a clear defined prominent linkage to Listeria or HSV.

Going forward, to determine genes that conferred the loss-of-interferon phenotype we continued to employ genome-wide mapping of this phenotype

inheritance in a series of F2 panels of (B6xMOLF) F1 intercrosses. We found that mice that inherited the MOLF locus at chromosome 18 near 19cM lacked interferon production to c-di-AMP. (Figure 9A) Fine mapping with additional markers revealed linkage with loci containing Trim11, Trim17, and Nlrp3 as candidate genes on chromosome 11 (Table 1, Figure 9B-C), Mice that inherited the MOLF locus at chromosome 18 near 19cM lacked interferon production to c-di-AMP. The gene for

STING, Tmem173, is located in this locus, and became a major candidate in this area.

(Table 1, Figure 9A). Genes that could obviously be involved in the interferon pathway were considered as preliminary candidates. Altogether, this suggests that

MOLF derived an alternate network of responses to cytosolic nucleotides.

In addition to IFN-response loci, we mapped a linkage to the MOLF hyper- production of IL-6 in response to both dAdT and c-di-AMP to loci on chromosome

10. Many of the Loci that conferred MOLF macrophage IL-6 hyper-responsiveness to DNA agonists were also linked to IL-6 hyper-responsiveness to non-DNA agonists in previous QTL screens in the lab, such to LTA and LPS. This may be a general enhance of IL-6 production that is generally downstream of proinflammatory activation. Further study of IL-6 hyper production downstream of DNA agonist stimulation can involve the investigation of previously mapped loci that conferred

IL-6 trait in MOLF macrophages, such Why1 and Why2 loci.

Figure 8: F2 Panel reveals wider ranges of responses to CDNs and DNA pathogens indicating that lack of IFNβ production in MOLF is a genetic trait Thioglycollate elicited macrophages from N2 48 mice were stimulated as follows: 2x105 Macrophages in 200 uL DMEM treated with liposomal poly dAdT (1ug/ml), liposomal c-di-AMP (2 ug/ml), SeV (MOI 10), HSV (MOI 20), or Listeria (MOI 10). A) Distribution of IFNβ responses in F2 mice to HSV1, Listeria, c-di-GMP, poly dAdT, and Sendai Virus. B) Distribution of IL6 production in F2 mice to HSV1, Listeria, c-di- GMP, poly dAdT, and Sendai Virus. Data representative of 3+ F2 panels.

Primary linkage to Tmem173 loci where MOLF FIGURE 2 allele has nega;ve eﬀect on IFNβ produc;on A B Linkage analysis of responses to cytosolic DNA

TRAIT Locus 1 LRS Pvalue Effect 600 c-di-GMP - c-di-AMP - 600 c-di-AMP D1Mit411 9.2 0.0021 - **** Tmem173 21.2 0.00001 - 400 400 pdAdT D11Mit4 13.3 0.00076 - ns Tmem173 14.2 0.00032 - IFNß UI/ml IFNß UI/ml 200 Listeria D9Mit151 8.7 0.0034 - 200 Tmem173 11.5 0.0011 - ns DXMit193 9.2 0.0022 - 0 0 HSV Tmem173 13.3 0.0004 - B6/B6 B6/B6 SV D1Mit102 8.3 0.0055 - B6/MOLF B6/MOLF D2Mit61 11.5 0.0043 + MOLF/MOLF MOLF/MOLF D3Mit117 7.8 0.008 - Tmem173 D19Mit90 9.2 0.0051 + Tmem173

Table 1: QTL analysis: Linkage of MOLF loci with differential IFNβ production. Association of IFNβ production to various agonists (Traits) with Loci. Likelihood Ratio Score (LRS) and p-value display power of MOLF loci to positively or negatively produce IFNB (QTL Effect – Analysis-/+). linkage results.

Supplementary Figure 1. QTL analysis of F2 IFNβ production to c-di-AMP reveals low IFNβ conferred by locus on Chromosome 18. AA Figure 9: QTL analysis links MOLF loci with low interferon responses. ELISA IFNβ production data (phenotypic) and genotyping data from multiple F2 panels were entered into J/qtl program to determine QTLs A) Significant linkage of MOLF loci with lack of IFNβ BB production to c-di-AMP, B) Linkage of MOLF loci with increased IFNβ production to dAdT, C) Assortment of responses to c-di-AMP, and D) c-di-GMP at Chromosome 18 by genotype at loci, FIGURE 2 Assortment of responses to A B E) dAdT or F) c-di-AMP at Linkage analysis of responses to cytosolic DNA Chromosome 11. TRAIT Locus 1 LRS Pvalue Effect C 600 c-di-GMP - D c-di-AMP - 600 c-di-AMP D1Mit411 9.2 0.0021 - **** C) and D) represent 2 Tmem173 21.2 0.00001 - 400 400 different F2 Panels. One-way pdAdT D11Mit4 13.3 0.00076 - ns Tmem173 14.2 0.00032 - IFNß UI/ml IFNß UI/ml ANOVA with DUNNs mutilple 200 Listeria D9Mit151 8.7 0.0034 - 200 Tmem173 11.5 0.0011 - ns comparison test done for C, DXMit193 9.2 0.0022 - 0 0 D, E, and F – P-value *p<0.05, HSV Tmem173 13.3 0.0004 - B6/B6 B6/B6 **p<0.01, ***p<0.001, ****p < SV D1Mit102 8.3 0.0055 - B6/MOLF B6/MOLF D2Mit61 11.5 0.0043 + MOLF/MOLF MOLF/MOLF D3Mit117 7.8 0.008 - 0.0001. Tmem173 Tmem173 D19Mit90 9.2 0.0051 +

c-di-AMP E dAdT F 800 *** 1000 ** *** 800 600

600 400

400 IFNß UI/ml IFNß UI/ml 200 200

0 0

B6/B6 B6/B6 B6/MOLF B6/MOLF MOLF/MOLF MOLF/MOLF Genotype @ D11Mit4 Genotype @ D11Mit4

CHAPTER 5 - Identification of polymorphisms in MOLF Tmem173.

Problem: We discovered strong linkage to attenuated CDN induced IFNβ responses to a locus containing; Stimulator of Interferon Genes (STING)

Approach: We hypothesize that there will be a genetic lesion in the region that confers the trait. This was before whole-shotgun-sequencing data of the MOLF genome was available at http://www.sanger.ac.uk/science/data/mouse-genomes- project, so we cloned our candidate genes from mRNA or gDNA, and sequenced them.

Chapter Summary:

Cloning the Tmem173 gene of MOLF and B6 mice revealed multiple polymorphisms and an 18bp - deletion in MOLF allele of Tmem173. Comparison of Tmem173 from mice of various sub-species reveals that molossinus has a unique allele of STING. All polymorphisms in the coding region of molossinus STING lead to amino acid changes in evolutionary conserved regions.

Defects in IFNβ production strongly correlated with inheritance of the locus containing Tmem173. Since this gene encodes STING, a product central to cytosolic

DNA responses, we reasoned this was our major gene candidate. To determine if

MOLF STING had any functional polymorphisms we created primers that flanked the coding regions for Tmem173 and amplified cDNA from macrophages mRNA by

PCR. PCR fragments were sent for Sanger sequencing at Tufts University Genomic

Core Facility.

Sequences of B6 and MOLF STING cDNA were combined using Cap3

Sequence assembly program and aligned using Clustal software. cDNA sequence of

MOLF revealed 8 SNPs and an 18 base pair deletion (Figure 10A+B). All of these

SNPs were in the coding region and conferred amino acid changes compared to the

B6 reference (Figure 11 A).

To dismiss artifacts in sequence of cDNA and to confirm our findings, we sequenced the STING genomic region. We used primers flanking the N-terminal exons of Tmem173 to clone and sequence genomic fragments from high quality gDNA of MOLF, MSM, and B6. Genomic sequencing of MOLF and MSM genomic DNA confirmed the MOLF Sting cDNA data. (Figure 10B) that all the SNPs and the 18 bp deletion were present as lesions in the molossinus genome. MOLF and MSM are closely related mouse strains of the subspecies molossinus, so it was not surprising to see that their genomic sequences for STING were identical.

Multiple alleles of STING have been found in the human population (Jin et al.

2011) (Table 2). Our discovery of the MOLF variant of STING represent the first

murine variant of STING found. We extended our inquiry to determine variability of

Tmem173 in various common lab strains. We sequenced, or determine the sequence from various online SNP databases, the Tmem173 region from genomic

DNA of common lab strains such as 129, PWD, and BALB/c to represent the other sub-species of mice. Many of these mice are less evolutionary divergent from B6 than MOLF/Ei. In these lab strains the Tmem173 sequences were nearly identical to

B6 with a few synonymous SNPs in a few cases. Therefore, molossinus species present a unique novel allele of Tmem173 in the mus musculus species (Figure12).

However, when we sequenced the Tmem173 of the SPRETUS species of mice, we found an equally unique allele of STING. Our collaborators tested IFNβ and Il-6 response of Spretus, B6, MOLF and MSM mice to viral and Listeria infection. Spretus was globally defective in mounting IFNβ responses to SeV, HSV1, Listeria or LPS.

This data is in accordance with a 2006 publication, were Mathieu et al show that macrophages from SPRET mice were deficient in IFNβ and IFNβ-dependent gene induction in response to various agonists, including: Listeria, LPS, or Influenza virus.

The SPRET IFNβ was due to a genetic defect in the IFNβ feedback loop that amplifies

IFNβ-dependent signaling. (Mahieu et al. 2006) For our current purposes SPRETUS is a poor model to study STING pathways due to the lack of general induction of IFN responses and the in ability to intercross with mus musculus to produce fertile offspring

Figure 10: MOLF Tmem173 (Sting) contains multiple polymorphisms and a 18 base pair deletion. Sequence of Mus musculus molossinus (MOLF) Sting aligned to B6. B6 STING and MOLF STING cDNA was amplified from mRNA isolated from B6 or MOLF peritoneal macrophages by PCR. B6 STING and MOLF STING were sequenced from cDNA; coding sequence is aligned and shown in A). B) Partial alignment of genomic sequence of B6, MOLF, and MSM.

Figure 11: Alignment of amino acid sequence of MOLF, B6, Human STING, and select mammals display unique amino acid substitutions in MOLF STING (A) Amino acid sequence of STING aligned using ClustalW2 (default settings). Sequences translated from cDNA: “pubSTING.seq” from ENSEMBL reference ENSMUST00000115728; “B6STING.seq” and “MOLFSTING.seq” from internal sequencing. (B) Multispecies alignment of N- terminus of STING compared to MOLF STING. (using DNASTAR software).

Figure 12: MOLF STING and Spretus STING contain unique divergent alleles of STING compared to common lab strains. Evolutionary tree of STING from different mouse species and subspecies. Nucleotide sequence of Tmem173 of strains shown were downloaded from EBI.AC.UK, and www.informatics.jax.org/snp. Coding sequences were entered into CLC sequence viewer to create alignment and phylogeny tree. Distance represents fraction of genetic changes compared to sequence length. Scale bar = 0.012

Table 2: List of Known Human STING Polymorphisms and alleles Residue Position Name Frequency 71 230 232 293 % R G R R (WT) 57.9 H A R Q (HAQ) 20.4 R G H R (R232H) 13.7 R A R Q (AQ) 5.2 R G R Q (R293Q) 1.5 H A R R

H G R R (R71H) H A R R (G230A) H G R Q (HQ) red is variation from reference allele

other mutants

gain of function SAVI Adapted from: mutants N154S V155M V147L Yi, G; Kao et al 2013

The SNPs found in the coding region of the MOLF allele of Tmem173 all confer amino acid changes. These amino acid changes are mostly N-terminal; they are L47V, A48G, S53L, S103F, I114M, Y115C Y126S, N210D and a 6 amino acid deletion of residues 116-121 (Figure 11). Humans have multiple known alleles of

STING, however none of these polymorphisms confer amino acid substitutions found in MOLF STING. Furthermore, the only N-terminal/TM proximal modification in humans is H71 (Table 2), while MOLF STING has 3 mutations even more N-terminal than H71 at L47V, A48G, and S53L (Figure 11-13). Interestingly,

MOLF L47V, A48G, and S53L and human H71residues are in low complexity region determine by SMART – a online protein domain architecture tool (Figure 13). The significance of this region in STING signaling is unknown. In addition, MOLF has a deletion of six amino acids in an un-described portion of STING. The MOLF STING C- terminus is almost completely identical to B6, except for one amino acid change,

N210D. On the other hand, most of the Human STING variations are on the well- studied C-terminus. Most of the defects conferred by the C-terminal variations have been explained by crystal structure studies or biochemistry. (Tanaka and Chen

2012) (Shaw, Ouyang, and Liu 2013) The N-terminus, which has trans-membrane domains, has been omitted from crystal structures. The N-terminus is not well understood, and much has to be uncovered in its functions in STING signaling.

MOLF STING mutations are therefore unique and can lead to novel characterization of N-terminal interactions to confer differential pathway activation. Ultimately, a

great question to answer would be to understand what fitness advantage do these mutations confer on the m. m. molossinus background.

Loca%ons of MOLF Amino Acid changes.

TBK1 IRF3 binding

L47G, A48G S103F Y126S N210D S53L I114M, Y115C del 116-122

Figure 13: MOLF amino acid changes are N-terminally located. Schematic of STING mutations relative to N-terminus. SMART database schematic depicting MOLF amino acid changes relative to full-length protein. Red, Black, and Blue lines represent intron positions. Red (2) = phase 2 intron; Blue (1) = phase 1 intron; Black (0) = phase 0 intron. Transmembrane region 1: start – 20 end – 37 Low complexity start – 47 end – 71 Transmembrane region 2: start – 85 end – 107 Transmembrane region 3: start – 114 end – 136

Source: smart.embl-heidelberg.de

The signaling function of the N-terminus of STING is unknown. Since, MOLF

N-terminal changes seem to act to perturb STING signaling, we aimed to look for functional significance of the amino acid changes that disrupt evolutionarily conserved sequences of STING. The online database: Eukaryotic Linear Motif resource (ELM.eu.org)23 was used to align the STING protein sequence with other species and screened for murine consensus with protein domains of known function, with the aim of identifying functional sites in STING. Alignment of mutant and wild type STING shows that the L47V+A48G mutations disrupted homology with probable cyclin1 or Atg8 binding sites, and that the S53L mutation disrupted a possible 14-3-3 ligand site. Disruption of the cyclin binding site may abrogate binding of cyclin/CDK–like complex and thus disrupt phosphorylation events. 14-3-

3 ligand domains are found in signaling proteins that dimerize, and stimulate protein-protein interactions24. In this way STING dimers can act as a clamp to enforce the dimerization of a binding partner important in signaling transduction.

This can also explain the dysfunctional IFNβ production in mice heterozygous for

Tmem173, as the interaction with the target protein or protein complex and this region would be abrogated. Recent studies show several viral or host targets that bind/interact to respectively block or facilitate signaling downstream of STING.

Though these functional sites are not proven in the context of STING, these novel site mutations may highlight undiscovered signaling domains in the STING N- terminus.

The C-terminal domain (CTD) of STING is conserved in the MOLF mutant.

STING binds IRF3, TBK1, and STAT6 and activates them at its CTD20. This may be important to retain robust SeV (RNA) induced IRF3, TBK1, and STAT6 activation19 in the context of STING-MAVS-Rig-I mediated interactions21. MOLF mutations are concentrated in proposed binding domains near the N-terminal. These I hypothesize are binding partners in the context of cytosolic DNA or c-di-AMP activation. STING activation is dependent on translocation to perinuclear spaces2 and K150 ubiquitination22 upon c-di-AMP or DNA sensing through binding partners.

MOLF mutations may block translocation and ubiquitination events that are necessary for assembly of signaling complexes.

90 A/J B A:J.pro AKR:J.proAKR BALB:J.proBALB CBA:J.proDBA A DBA.proCBA FVB:J.proFVB A Alpha, Regions - Garnier-Robson LP:J.proLP Alpha, Regions - Garnier-Robson I114M, Y115C, del 116-120 NOD.proNOD SPRETUS.proSPRETUS A S53L Alpha, Regions - Chou-Fasman 0.015 Alpha, Regions - Chou-Fasman SEG.pro 0.003 SEG 0.007 B Beta, Regions - Garnier-Robson MOLF.proMOLF Beta, Regions - Garnier-Robson 0.009 A S103F Alpha, Regions - Garnier-Robson 0.002 MSM.proMSM Alpha, Regions - Garnier-Robson B Beta, Regions - Chou-Fasman PWK.pro Beta, Regions - Chou-Fasman Y126S 0.003 PWK L47G,A A48G Alpha, Regions - Chou-Fasman Alpha, Regions - Chou-Fasman T Turn, Regions - Garnier-Robson WSB.proWSB Turn, Regions - Garnier-Robson 129 B Beta, Regions - Garnier-Robson Beta, Regions - Garnier-Robson 129:J.proC57BL6 T Turn, Regions - Chou-Fasman C57BL6:J.pro Turn, Regions - Chou-Fasman C57BL6/J.proB Beta, Regions - Chou-Fasman Page 1 Beta, Regions - Chou-Fasman Monday, CAugust 4, 2014 4:43 PM Coil, Regions - Garnier-Robson Coil, Regions - Garnier-Robson T Turn, Regions - Garnier-Robson Turn, Regions - Garnier-Robson 4.5 C57BL6 SMART- 0.01 C57BL6/J.proT 10 20 30 40 50 60 70 80 90 100 110 120 130 140MOLF150 160 170 Turn, Regions - Chou-Fasman Page 1 Turn, Regions - Chou-Fasman MOLF.proMonday,A August 4, 2014 4:43 PM motifsAlpha, Regions - Garnier-RobsonPage 1 Alpha, Regions - Garnier-Robson C Coil, Regions - Garnier-Robson Coil, Regions - Garnier-Robson Monday,C57BL6/J.proMOLF.pro 0August 4, 2014 4:44 PM Hydrophobicity Plot - Kyte-DoolittlePagePage 1 1 Hydrophobicity Plot - Kyte-Doolittle A Alpha, Regions - Chou-Fasman Alpha, Regions - Chou-Fasman Monday,Monday,4.5 AugustAugust 4, 102014 4:434:4420 PM 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 MOLF.pro Page 1 BA 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Alpha,Beta, Regions Regions - -Garnier-Robson Garnier-Robson Alpha,Beta, Regions - Garnier-Robson Monday, August 4, 2014 4:44 PM C57BL6 -4.5A 10 2020 3030 4040 5050 60 7070 80 90 100 110110 120 130 140 150 160 170 Alpha-helixAlpha, Regions - Garnier-Robson Alpha, Regions - Garnier-Robson BA Alpha,Beta, Regions Regions - -Chou-Fasman Chou-Fasman Alpha,Beta, Regions - Chou-Fasman 0AA MOLF Alpha,HydrophobicityAlpha, Regions Regions Plot- Garnier-Robson - Garnier-Robson- Kyte-Doolittle Alpha,HydrophobicityAlpha, Regions Regions -Plot Garnier-Robson -- Kyte-Doolittle Garnier-Robson AT 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Alpha,Turn, Regions Regions - Chou-Fasman - Garnier-Robson Alpha,Turn, Regions Regions - Chou-Fasman - Garnier-Robson *B Beta,Alpha, Regions Amphipathic - Garnier-Robson Regions - Eisenberg D Beta,Alpha, Regions Amphipathic - Garnier-Robson Regions - Eisenberg AA Alpha,Alpha,Alpha, Regions Regions Regions - -Garnier-Robson Chou-Fasman - Chou-Fasman Alpha,Alpha, Alpha,Regions Regions Regions - Garnier-Robson- Chou-Fasman - Chou-Fasman BT Beta,Turn, Regions Regions - Garnier-Robson - Chou-Fasman Beta, RegionsTurn, Regions - Garnier-Robson - Chou-Fasman -4.5*B Beta,Beta, Regions Amphipathic - Chou-Fasman Regions - Eisenberg Beta,Beta, Regions Amphipathic - Chou-Fasman Regions - Eisenberg BAB C57BL6 Alpha,Beta,Beta, Regions Regions - Garnier-RobsonChou-Fasman - Garnier-Robson 1000 C57BL6 MOLF SPRETUS Alpha,Beta, Beta,RegionsRegions Regions -- Chou-FasmanGarnier-Robson - Garnier-Robson BC Beta,Coil, Regions Regions - Chou-Fasman - Garnier-Robson Beta, RegionsCoil, Regions - Chou-Fasman - Garnier-Robson FT Beta-sheetTurn,Flexible Regions Regions - Garnier-Robson - Karplus-Schulz Turn,Flexible Regions Regions - Garnier-Robson - Karplus-Schulz BB Beta,Beta,Beta, Regions Regions Regions - -Garnier-Robson Chou-Fasman - Chou-Fasman Beta,Beta, Regions Beta,Regions Regions - Garnier-Robson- Chou-Fasman - Chou-Fasman 4.5T* MOLF Turn,Alpha, Regions Amphipathic - Garnier-Robson Regions - Eisenberg Turn,Alpha, Regions Amphipathic - Garnier-Robson Regions - Eisenberg 1.7T Turn, Regions - Chou-Fasman Turn, Regions - Chou-Fasman 4.5TBT Beta,Turn,Turn, Regions Regions Regions - -Chou-Fasman Garnier-Robson - Garnier-Robson Beta,Turn, Regions Turn,Regions Regions - Chou-Fasman- Garnier-Robson - Garnier-Robson T* Turn,Beta, Regions Amphipathic - Chou-Fasman Regions - Eisenberg 600 Turn,Beta, Regions Amphipathic - Chou-Fasman Regions - Eisenberg C Coil, Regions - Garnier-Robson Coil, Regions - Garnier-Robson TT Turn,Turn,Turn, Regions Regions Regions - -Garnier-Robson Chou-Fasman - Chou-Fasman Turn,Turn, Regions Turn,Regions Regions - Garnier-Robson- Chou-Fasman - Chou-Fasman 0CF Coil,FlexibleHydrophobicity Regions Regions - Garnier-Robson - Plot Karplus-Schulz - Kyte-Doolittle Coil,Flexible RegionsHydrophobicity Regions - Garnier-Robson - Karplus-Schulz Plot - Kyte-Doolittle 4.50 C57BL6 Antigenic Index - Jameson-Wolf Antigenic Index - Jameson-Wolf

CC Coil,Coil, Regions Regions - Garnier-Robson - Garnier-Robson IFN (IU/ml) Coil, RegionsCoil, Regions - Garnier-Robson - Garnier-Robson 4.51.7T HydrophobicityTurn, Regions - Chou-Fasman Turn, Regions - Chou-Fasman -4.54.54.5 C Coil, Regions - Garnier-Robson 200 Coil, Regions - Garnier-Robson -4.50 Hydrophobicity Plot - Kyte-Doolittle Hydrophobicity Plot - Kyte-Doolittle C57BL6/J.pro-1.74.5 Page 2 0 MOLF HydrophobicityAntigenic Index Plot - Jameson-Wolf - Kyte-Doolittle HydrophobicityAntigenic Index Plot - Jameson-Wolf - Kyte-Doolittle Monday, August 4, 2014 4:43 PM 00 * HydrophobicityAlpha,Hydrophobicity Amphipathic Plot Plot - Kyte-Doolittle Regions- Kyte-Doolittle - Eisenberg HydrophobicityAlpha,Hydrophobicity Amphipathic Plot - PlotKyte-Doolittle Regions- Kyte-Doolittle - Eisenberg 6 Lipo HSV SeV -4.5 0* HydrophobicityBeta, Amphipathic Plot - Kyte-Doolittle Regions - Eisenberg HydrophobicityBeta, Amphipathic Plot - Kyte-Doolittle Regions - Eisenberg MOLF.pro-4.5-1.75 Page 2 Poly dA:dT -4.5-4.5 Cyclic-di-AMP Monday, FAugust* 4, 2014 4:44 PM Alpha,Flexible Amphipathic Regions - Karplus-Schulz Regions - Eisenberg 5000 Alpha,Flexible Amphipathic Regions - Karplus-SchulzRegions - Eisenberg 6 1.7* Alpha, Amphipathic Regions - Eisenberg Alpha, Amphipathic Regions - Eisenberg -4.5* Beta, Amphipathic Regions - Eisenberg Beta, Amphipathic Regions - Eisenberg 1** Alpha,Alpha,Surface Amphipathic Amphipathic Probability Regions RegionsPlot - -Emini Eisenberg - Eisenberg Alpha,Alpha, SurfaceAmphipathic Amphipathic Probability Regions Regions Plot - Eisenberg - Emini - Eisenberg * Beta, Amphipathic Regions - Eisenberg Beta, Amphipathic Regions - Eisenberg 00F C57BL6 SurfaceFlexible Regions - Karplus-Schulz Flexible Regions - Karplus-Schulz *** Alpha,Beta,Beta, Amphipathic Amphipathic Regions Regions - Eisenberg - Eisenberg 3000 Alpha,Beta, Beta,AmphipathicAmphipathic Amphipathic RegionsRegions Regions - - Eisenberg Eisenberg - Eisenberg 1.70F FlexibleAntigenic Regions Index - Karplus-Schulz - Jameson-Wolf FlexibleAntigenic Regions Index - Karplus-Schulz - Jameson-Wolf FF FlexibleFlexible Regions Regions - Karplus-Schulz - Karplus-Schulz FlexibleFlexible Regions Regions - Karplus-Schulz - Karplus-Schulz 1.7* 60 70 80 90 100 110 120 130 140 150 160 170 Beta,probability Amphipathic Regions - Eisenberg Beta, Amphipathic Regions - Eisenberg

1.71.71 MOLF Surface Probability Plot - Emini IL-6 (pg/ml) Surface Probability Plot - Emini 0F Flexible Regions - Karplus-Schulz Flexible Regions - Karplus-Schulz -1.70 Antigenic Index - Jameson-Wolf 1000 Antigenic Index - Jameson-Wolf 1.7 0 Antigenic Index - Jameson-Wolf Antigenic Index - Jameson-Wolf 00 60 70 80 90 100 110 120 130 140 150 160 170 60 70 80 90 100 110 120 130 140 150 160 170 AntigenicAntigenic Index Index - Jameson-Wolf - Jameson-Wolf AntigenicAntigenic Index Index - Jameson-Wolf - Jameson-Wolf -1.7 0 Antigenic Index - Jameson-Wolf Lipo HSV SeV Antigenic Index - Jameson-Wolf -1.7 -1.7-1.7 60 70 80 90 100 110 120 130 140 150 160 170 Poly dA:dT Cyclic-di-AMP 60 70 80 90 100 110 120 130 140 150 160 170 -1.7 60 7070 80 90 100 110110 120 130 140 150 160 170 C 60 70 80 90 100 110 120 130 140 150 160 170 LIG_BRCT: LIG_BRCT: A CYCLIN 14-3-3 TRG_END2 Predicted motifs affected BRCA1 BRCA1

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Elephant.pro. s. .. c. .. gvi a. vs . .g . l . g. e. .. .i..hdh. .. s...... q. .ws. . . .vr .s.h.q...... g.mi l.q q.k .a. ... rf.h. .gl .. ls. l.g.ci .. ..v sca .v. ... fg..l .sg .e p.. ..mdi . h. .l qp. w.. .. .v. .s. q.l .a...... rlg .q. .l .q. .a. .v..nm.i g.. i. .s.s.... r.g .h. .g. .. l. il h...... c...... f . .r s. .. .p. . . mm . .. .l . pr .h g. .. l . l l. a. .. c. .g. .f l. sq .apv. nm i. .. l . ps. .. . .g l . a . . g . l q a v n i . . s . . g . Rat.pro ...... Rat.pro. s . . . . . s y . f . l a . f v l . . .. g...... s .. .. m. ts g. e .. .. s. .. .s. yh .. f. .. .l . av .. f. qv ql . . Rat.pro. .. g. .. .. k. . s. .. . m. . t . g. e...... s...... h. ..s...... vs .y. ...vf q.. sql .a. ...cf.fv ..l .. l. .gk...... fs .. ... m. .ct .sg .e e.. .. s. .s. .. .l hr. ... a...... h .l v.. .i. .. .q. .q. .s ...... v.. k.t .s. .d...... c. . .f...... l...... f...... c. s. .. .e. . . .v s. s. .l . rc .f a. .. lh. l. .. .i f...... sc s. .. e. . .. s. .t l . r d. .a . h. l . . i . . . s . . . . . t . d . . . . Guinea pig.pro . . . c . s . . Guinea. s . . pig.pro...... a q q a . . v l . . .g v. c .. vs. .. .. g. ts g. e .. .. d...... r .. a. vq .qya. .. . lv ql . . Guinea. g. v. c.pig.pro.. evv .. .y . .g . t . g. e. .. .i...dc. .. s.. .s. . .r. ..ss. . .v...... y...... al.q q.q .a. ... r..sv .gl .. vg. lve.cv.. ..v yc. .. l.. sg.st .sg .e p.. t.. dis. .g. k. pr. .f. w. sv. ..t y.l .as ...... lg .q. .s .. .a. .v..t ..i e.. v. ...y.... r.p .s. .g. .. v. il ...... cs .. .l ss. s. s. .. .p. t . d. s. .g .k . pr fs wg .. vt l l. a. .. c. .gl .s s s. .apvt td si g. k. p .f .w . tp l . a . . g . s . a v t i . . . . . p . Brown bat.pro . . . h . s . . Brown. s . .bat.pro. . . s . k a . i a . a v l . . .i .. ch .. asa .f .. . ls g. e .s .. k. .. is. .q kc a. l. .i . a. .. a. lv ql . . Brown. i. .. bat.procf .. rav a. fs . v. . l . g. e. .s. ....hkh. .. si. . . . .q. .cs. . r .lr ...... s . .. .k..al.. sqi .a. ... ra..v .gl vf. li. v.r .cv.. ..a sca .f. ..v .. .dl .pg .e s. .. ek . . hgi .l qp. cf. .i .l. ... .l .p. .fr .. rlg .q. .s .e. .a. .. .f. ..i r.i vs. .d.s.... vrg ... .g. v. l. v. h...... c...... r dr p. .. .s . . . e. . sg .l . pr f. gi v. l . v l. p. .f c. .g. .. sd pe .as . . .e i. gi l . pdf .i . .g l . p f . g . s e a . . i i . d . . g . C57BL6:J.pro - M P Y S N L H C57BL6:J.proP A I P R P R G H R S K Y V A L I F-LMV PA SY LSMNI LL HW VPAAKIDPP RP NP HRTGLHK RY SL AK LYHVLAA LS HI EFLLGC57BL6:J.proVL AL LSKLNML IC LC WL AVEAEKL DC-HPMVPPQNYSHSRNTYLQLHGKPSYAYLIWPAKRALPVHRRLGAHACRSLSHGKCEYPVL IAGHLLCI MLFALLMKV IANLSLLLCMS CSI YL WFAYVEAF EK- LD QCP NPHNTVHAQDT SLI KYRYLYLSQAWGML HSF LGYALWSLKHVAEL VLYGKRLSALLCLSLKMNGL LCLCGPCLILQHASECLEMLACMH VI QL SLRSY SQ GY SFYYWFK -A VL RQANCTL AG CDPI I YH LC MS AWMMI FL GL SL SLYVFLY YF K- LSQLN ST MA DL ILYGL LS WQMSFLG L L V L Y K S L S M L L G L Q S L MOLF.pro ...... MOLF.pro...... V. G...... L...... MOLF.pro...... V. . .G...... L...... F...... M. C.. -..V- .G- ..- .- .-. ... L...... S ...... F...... M.C. -. -. .- .- . -. -. .. F...... S...... M.C.- .- -. -. - . - . . . . S ......

Ruler 2 1 Ruler10 2 20 1 30 10 40 2050 Ruler60 230 70 401 80 10 50 90 20 60 10030 7011040 1208050 13060 90 70 100 80 110 90 120100 110130 120 130 4.3 4.3 4.3

S S F F F A 2.2 S K 2.2 E K C AE T C A C T T 2.2 K H E H R V R V T I H V V V T I Y V V R Y V VA Y G A T Y I G R I CQ Y R C P L Y A Y Sequence LogoG I Q Y R C Y I Q P A L A P L K Sequence Logo R N F A K E F G C G G N D F C Q R P S Sequence Logo E F R Q P K G R C G N G D K C Q M P S R R N Y F R Q P N E M K F G C G G D C Q P S R Y M K I R H A I Q P V L LN S I G Y H A V L L S G L P L I R L M V G I L S G L P C I A L R L H M V G D E I R A H A V A L I N R G D I C G L P I L N C L S R LR M V HA F G I E I LR A F D S C I A L S H A Q G F D E I R A L D A L F D C N R I S A L S AQ F L C T D H R A A L A L A G D A L C C T H F R S A A Q L A A L F G Q Q I C T A L A Q M F D H F L I A L Q Q L F I V D Q Q S H T M G P L F Q I T K A A M E S F D H F G L R I K G C T H R T F C G I A F S E H L A A G A L V W H I Q L F I V K Q S I H T M G P S Q L F T F Q Q I C TV S W G A Q GM V I E I T F D S H F L K I F H C I G A R S C K S G G E E Q H G K A S I Q I G E M Q L F V K G E T S S H T M G R K S P G H K S T F S G V I P F K L C H A L C E A G V R L G Y A N I L S L V W I H I S Q M P L H L A E V R L Y N L S L P S G V T L H L K A C A V R L C G Y N M L GQ S K A S I L V D D 0.0 R Q T SV K W S S S S I M W T A H Y K I Q A G S V M D H V L S Y V L M Q V A V L D S R T W V C S M C V V V L S I T N S Y T F P I Y E T T S F P 0.0 Q T S G A L G G E H S S I W P L T R Y K I A G S V M V S L Y M V K A V T S L C W D H T W V YM C V V V S S S T N S M Y P I Y E T T S P A S H G A L G G E H H A V QS VS L V Q F K D Y S R E C S L P I T F L F V Q C 0.0 T R A S H G Y K I Q A A L T G G S V M AG E Q S H S H V L S YVV L FM Q V K A V T K D S L Y N W D S R T W V HC E S M C SI V V V YS L S S P I S T I N S M Y W T F P I Y E L TN T S F P R Q T S M V V S M K S N A Q P S K V I F S S N T K D S PY I C L K L L S HI E L SN Y R Q S I YLW K TLP L S GKS P L M N S F S L R QI L L L L LY WSKVT LS V MG S PL K MT FY W K S L G P L L VF S LS M L L K L T L T 100 M L H P I P R A100 LM L L HW P I P P R L AA LG L L C WL E E100L HMP S R YLLH PS I P R AAC AG L L L LLCS YLW E E L PH SL R Y W S A L L G ALC LC L ELE L H LSLRSY Y S A C W L L S LY L L W L L L L L Consensus Match 50 Consensus Match 50 Consensus Match 50

0 0 0

Figure 2: Polymorphisms in MOLF STING affect evolutionary conserved residues and structure of the STING protein. (A) Comparison of motifs and structure of MOLF and B6 STING proteins; (B) Evolutionary tree of STING from different mouse subspecies; (C) Responses to DNA and DNA- pathogens in SPRETUS mice; (4) Alignment of STING from different species reveal conserved B residues affected by MOLF SNPs MOLF STING Predict Domain Muta.ons Aﬀected L47G, A48G LIG_CYCLIN1, LIR S53L 14-3-3 S103F LIG_BRCT:BRCA1 I114M, Y115C -N/A del 116-122 LIG_BRCT:BRCA1 Y126S -N/A N210D -N/A

Figure 14: MOLF amino acid changes disrupt evolutionary conserved sequences in STING N-terminus STING amino acid sequences of selected mammals downloaded from NCBI and input into DNASTAR software. A) DNASTAR alignment of STING N-terminal sequences from select mammals. MOLF STING and B6 STING amino acid sequences were loaded into Eukaryotic Linear Motif database (elm.eu.org) B) List of Eukaryotic Linear Motifs disrupted by MOLF amino acid changes compared to B6 STING.

CHAPTER 6 - MOLF STING is deficient in activating the IFNβ and NF-kB promoter elements in Luciferase assay.

Problem: We have found multiple polymorphisms of STING that confer amino acid changes. All of these amino acid changes are novel and have not yet been characterized in the literature. It is unknown if these amino acid substitutions confer a defect, or if the protein has any aberrant function when compared to the wild-type reference.

Approach: Overexpression of wild-type STING has been shown to induce elements of an IFNβ gene promoter. This indicates robust activation of the STING signaling up to transcription. We compare induction of IFNβ promoter elements in a

Luciferase reporter assay by the B6 STING, MOLF STING, and individual MOLF mutations placed into the wild-type B6 protein.

Chapter Summary: Luciferase Reporter Assay is a quantitative measure of the induction of a gene promoter. To determine the ability of MOLF STING to induce Interferon promoter elements, STING constructs with parental alleles, or single point mutants of STING on either background were co-transfected into 293T cells with Luciferase reporters linked to IFNβ promoter, NFkB promoter, or PRDII – IV. MOLF STING was severely attenuated in IFNβ induction, NFkB induction, and PRDII-IV induction.

Point mutagenesis of L47V-A48G and S53L mutations on the B6 background resulted in attenuated induction of the IFNβ promoter at levels similar to MOLF.

MOLF STING inheritance confers a defect, and has amino acid changes that disrupt residues that are conserved iin B6 mice and other mammals. Over- expression of STING in 293T induces its spontaneous dimerization and activation resulting IRF3 phosphorylation and induction of ISGs1. To determine MOLF STING ability to induce interferon, we cloned B6 and MOLF STING into the mammalian expression vector pEF-Bos, and co-transfected the vector along with a Luciferase vector inducible by the IFNβ promoter, then measured luminescence as a function of promoter induction. The luminescence measured is a function of promoter induction. The MOLF allele displayed a severe dysfunction in IFNβ induction compared to the B6 (WT) allele (Figure 15A).

We overexpressed MOLF STING at various doses. Regardless of the dose,

MOLF STING remained unable to competently induce the IFNβ promoter. To see if this phenotype extended to the ligation dependent induction of STING, we expressed STING at low amounts then induced activation with CDN treatment.

Activation of B6 STING with CDNs induced robust activation. However, MOLF

STING remained deficient in inducing the IFNβ promoter in response to all agonists.

2’3’cGAMP was the only ligand that produced a wild-type IFNβ response in

MOLF/Ei macrophages (Figure 3). However, in this assay 2’3’cGAMP was not more capable of inducing MOLF IFNβ responses compared to other CDNs. In this assay the background of the cell is common, while in the initial screen the macrophages of

MOLF and B6, confer unique cellular states. Thus, it maybe that other genetic elements on the MOLF background confer 2’3’cGAMP IFNβ responses. 94 Level STING expression rela3ve to induc3on of IFNβ promoter

IFNβ PROMOTER ACTIVITY OF STING MUTANTS

2.0

1.5

1.0 RLU RLU STING-HA STING-HA + 4ug cdA Firefly/Renilla

0.5

0.0 0 0 5 ug 5 ug 10 ug 2.5 ug 10 ug 2.5 ug 1.25 ug 1.25 ug

B6- STING HA MOLF-FIGURE STING HA 4

A A STING constructs B IFNβ promoter 0.8 IFNβ PROMOTER ACTIVITY OF STING MUTANTS STING 2.0 NF-kB elementB6 STING-HA of IFNβ 2 MOLF STING-HA 0.6 IRF3 elementB6 STING-HA of IFN + 4ugβ cdA STING + c-di-AMP MOLF STING-HA + 4ug cdA 1.5 0.4 1 + 4ug/mL c-diAMP

1.0 0.2 LUC/RLUC RU LUC+/RLUC RLU RLU RLU 0 Firefly/Renilla B6 B6 B6 S53L S53L S53L 0.0 0.5MOLF S103FY126SN210D MOLF S103FY126SN210D MOLF S103FY126SN210D

L47V A48G del116-122 L47V A48G del116-122 L47V A48G del116-122 S53LS53A S53LS53A S103FY126SN210D S103FY126SN210D 0.0 B6 STING No STINGB6 STING No STING IFNβ promoter0 NFkB0 element of0 IFNβ IRF30 element of IFNβ L47V A48G L47V A48G 5 ug 5 ug 5 ug 5 ug 10 ug 2.5 ug 10 ug 2.5 ug 10 ug 2.5 ug 10 ug 2.5 ug MOLF STING MOLF STING 1.25 ug 1.25 ug 1.25 ug 1.25 ug 2.0 STING B6- STING HA MOLF- STING HA B6- STING HAD MOLF- STING HA 1.5 BC E 1.0 0.6 B6 STING STING-HA LUC/RLUC RU 0.5 0.8 0.0 MOLF STINGSTING constructs

0.4 B6& B6 S53L S53L& * MOLF S103F Y126S N210D S53A& MOLF& I199N& S103F& Y126S& N210D& DMEM& Xgene9& 0.2 L47V A48G L47V&A48G& 0.4 * * * p-TBK1 LUC/RLUC RU 0.0 TBK1 I114M_Y115C del116-122 1448155&Δ1168122& * B6

LUC/RLUC RU p-IRF3 MOLF empty 0.0 B6 S53LB6 S53A IRF3 MOLF L53SMOLF L53A lipo B6 L47V A48G p-p38 No TX modified B6 cdiAMPcdiGMP DMXAA modified MOLF STING p38 2'3'cGAMP3'3'cGAMP MOLF V47L A48G L53S STING STING Figure 15: MOLF STING promotes reduced induction of the IFNβ promoter. GAPDH

A) Luciferase Assay in 293T cells of IFNβ promoter induction in response to B6 or MOLF STING transfection with or without c-di-AMP (cdA). B) Induction of the Interferon promoter by low level STING transfection (0.5ug) and CDN treatment. (Luciferase assay) A) representative of one as formatted. B) as 3+ experiments.

MOLF STING displays reduced induction of the IFNβ promoter, and thus a functional defect in signaling. To measure the contribution of the various SNPs to the defect, B6 STING clone was mutated to include the individual sets of SNPs found in the MOLF allele, and assayed the ability of the mutant STING vector to induce the

IFNβ promoter-Luciferase construct. B6 Tmem173 clones mutated to L47V+A48G or S53L show lack of induction of the full IFNβ promoter. (Figure 16A). These amino acid changes in the N-terminus are in the proposed transmembrane regions of STING. Expressing STING in low amount that require STING agonist to induce

IFN, did not reveal obvious further defects in the ligand inducible model of STING activation in 293T cells. (Figure 16B) Furthermore, inducible activation of parental

STING proteins showed that MOLF STING was defective to activation by all CDNs tested in 293Ts (Figure 15B).

To investigate the signaling pathway downstream of STING mutants in 293T cells, we overexpressed B6 STING, MOLF STING, and the single mutant constructs and probed by western blot for phosphorylation of IRF3, TBK1, and p65. The L47V-

A48G and S53L mutants, similar to MOLF STING, weakly induced IRF3 phosphorylation, and displayed weaker activation of TBK1 and p65 (Figure 16B).

The Vance lab has shown that mutation of some arbitrary N-terminal mutations in

STING abolished its ability to induce the IFNβ promoter without affecting c-di-GMP binding to STING. (Burdette et al. 2011) We show unique evolutionary determined residues, not found in the literature, that also affect STING-dependent induction of the IFNβ promoter. We have not done a binding assay to see if these mutations

affect CDN binding. However, MOLF N-terminal changes highlight the significance of the STING N-terminus in signaling.

IFNβ promoter NF-kB element of IFNβ ** ** ** IRF3 element of IFNβ 2

LUC+/RLUC RLU 0 B6 B6 B6 S53L S53L S53L MOLF S103FY126SN210D MOLF S103FY126SN210D MOLF S103FY126SN210D

L47V A48G del116-122 L47V A48G del116-122 L47V A48G del116-122

IFNβ promoter NFkB element of IFNβ IRF3 element of IFNβ

Figure 16: L47V-A48G, and S53L mutations confer the strongest individual effect on the defective regulation interferon induction downstream of the STING pathway.

A) Luciferase assay in 293 cells. Induction of IFNβ promoter after overexpression of single mutants of STING (indicated mutations are on the B6 background). B) WB of 293T lysate after overexpression of STING constructs. One-way ANOVA p-value ** < 0.001

Since L47V-A48G and S53L displayed the most severe defects in IFNβ promoter induction, we investigate whether reversion of these three residues on the

MOLF STING would restore ability to induce IFNβ promoter induction. MOLF L53S

STING mutation remained near as deficient as MOLF STING in IFNβ promoter induction (Figure 17). MOLF V47L-G48A-L53S STING activity was partially restored in this assay, still not as robust as B6 STING. B6 STING S53A mutation resulted in hyperactive IFNβ induction, while in contrast the S53A change in MOLF STING conferred no improvement in the ability of MOLF STING to induce the IFNβ promoter in 293T cells (Figure 17).

MOLF V47L-A48G-L53S STING was not sufficient to restore IFN-defect, yet the S103F, Y126S, and N210D mutants singly did not confer a defect. However, in the MOLF V47L-A48G-L53S STING mutant all the other MOLF mutation still confer a defect. It is possible that the mutations complement each other for to induce a global defect for which abrogates one or more nodes of signaling downstream of

MOLF STING.

Figure 17: MOLF N-terminal mutations to B6 partially restores function of STING. IFNβ promoter induction assay in 293T cells transiently expressing STING –HA (pEF-BOS vector) and IFNβ-Luc plasmid. B6, MOLF STING or mutants indicated on MOLF STING background (mutants S53A and S53L are on B6 background and included for comparison.) RLUs – IFNβ promoter induced Firefly luciferase signal normalized by co-transfected constitutively expressed Renilla luciferase signal. One-way ANOVA with Tukey test (p-value *p<0.05, **p<0.01, ***p<0.001) pool of N=2 experiments.

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Serine 53 of STING (Figure 14A) is highly conserved in mammalian species.

S53L mutation retards MOLF STING ability to induce IFNβ promoter induction in

293T. To assay if STING- Ser53 phosphorylation is necessary for signaling; we mutated Serine 53 to Alanine in B6 STING. Alanine substitution would not be phosphorylate-able but would preserve structure of B6 STING at this region. My expectation was that removal of this phosphorylation site would greatly reduce induction of the interferon promoter. However, the S53A mutation increased IFNβ promoter induction by 50%. (Figure 18). Thus, I hypothesized that the Ser53 phosphorylation may be a negative regulation of STING that may induce degradation of the protein. To determine if the phosphorylation causes degradation, we pulse-chased HEK 293 overexpressing B6 STING-HA or S53A with S35 methionine for 0, 1, or 2 hrs. STING-HA was immunoprecipitated and run on a western blot to quantify STING protein levels. No change was seen in B6-STING or

S53A mutant levels. (Figure 18)

An alternate mechanism by which the S53A mutation may increase signaling while S53L decreases, may be through aberrant association with 14-3-3 proteins.

The 14-3-3 motif may allow oligomerization of the STING molecules. S53A mutation may increase this interaction, while the S53L and L47V - A48G mutations disrupt it.

An alternative is to pull down B6 STING, S53L, S53A, and L47V - A48G and probe for interaction of 14-3-3 family proteins. There are seven proteins in the 14-

101

3-3 family, and they are all expressed in macrophages. 14-3-3 proteins serve as molecular switches that bind signaling proteins to switch localization or block interaction of potential binding partners while recruiting binding partners of an alternate pathway. In this way, 14-3-3 can negatively regulate a signal, induce a translocation and commence an alternate signaling pathway(Fu, Subramanian, and

Masters 2000; Van Der Hoeven et al. 2000; Shen et al. 2003). 14-3-3 proteins have been shown to differentially regulate MAPK pathways in similar ways. (Van Der

Hoeven et al. 2000) B6 STING upon activation translocates to a perinuclear space.

This translocation event is necessary for TBK1 activation and induction of the IFN response, as when STING translocation is blocked the interferon response is also abrogated(Ishikawa and Barber 2008) (Dobbs et al. 2015). I propose that MOLF

STING does not undergo this translocation due to changes on the N-terminus. Thus the molecular switch to a TBK1 signaling is blocked and then maybe the MAP Kinase signaling pathway is prolonged and intensified.

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Figure 18: S53A (structurally similar to Serine) mutation causes hyper- activation of the IFNβ promoter. (A) IFNβ promoter induction assay in 293T cells transiently expressing STING–HA (pEF-BOS vector) and IFNβ-Luc plasmid. B6, MOLF, S53L, and S53A STING compared in ability to induce the IFNβ promoter. (B) Pulse-Chase: 293 cells transiently overexpressing B6 STING-HA or S53A STING-HA were pulsed for 30 mins with 35S Methionine. Western Blot probes for HA levels 0hr, 2hr, and 4hr after pulse. One-way ANOVA Tukey test ***p<0.001. Data representative of A) 3+ and B) two experiments.

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MOLF N-terminal mutations: S53L, L47V, and A48G, have been shown to have the most detrimental affect on MOLF STING signaling. To see the relevance of

MOLF mutations on the human STING protein, we created S53L, L47V-A48G, and

S102F mutations on human wild-type STING. In our assay we found that S53L abolished human STING ability to induce the interferon promoter. Surprisingly, the

L47V-A48G mutation increased human STING ability to induce interferon (Figure

19). Accidentally, we created the S102F mutant of STING instead of the S103F found in MOLF. Interestingly, we found that the S102F human mutant also conferred a signaling defect. The S102F mutation disrupts a BRCA1:BRCT S-X-X-F motif to F-X-

X-F. Altogether we found that the S102F and S53L mutations have functional importance in human STING signaling. (Mohammad and Yaffe 2009; Yu et al. 2003)

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2.0 *** *** *** 1.5

1.0 RLU

0.5

0.0

S53L S102F no STING L47V-A48G Human STING

Figure 19: Recapitulation of MOLF mutations on human STING provides diverse effects on human STING induction of interferon promoter. IFNβ promoter induction assay in 293T cells transiently expressing STING–HA (pEF-BOS vector) and IFNβ-Luc plasmid. After 16-18hr of expression of constructs, Firefly Luciferase expression measured as luminence relative to tranfected Renilla Luciferase control. Human STING (ref), human STING (hS53L), and human STING (hS102F) were compared in ability to induce the IFNβ promoter. One way ANOVA with Tukey post test. Representative of N=3 experiments.

105

CHAPTER 7 - Characterization of Interferon responses in Congenic mice reveals further defect of MOLF STING.

Problem: The MOLF/Ei background is phenotypically divergent to the commonly used lab strains in literature. Any signaling network studied may have multiple genomic components leading to MOLF phenotypic outputs. Therefore, it would be extremely difficult to mechanistically investigate MOLF STING in the MOLF background, while unknown non-STING dependent variables may shift responses compared to B6.

Approach: We backcrossed MOLF onto the B6 background for up to 10 generations, while retaining the MOLF Tmem173 allele to create B6 mice congenic for MOLF STING – referred to as B6 StingMOLF.

Chapter Summary: Because MOLF mice diverged from commonly used lab strains around million years ago, their genome contains many polymorphisms that confer abundant variable phenotypes when compared to commonly used lab strains. To relate differences conferred by B6 or MOLF STING it was necessary to confine STING to a similar background. Therefore, we backcrossed MOLF/Ei to C57BL6 for multiple generations to create mice Congenic for MOLF STING on the B6 background

(B6.MOLF-Tmem173). Macrophages from B6 STINGMOLF mice were severely deficient in both IFNβ and IL-6 production to all STING dependent agonists.

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MOLF mice are genetically distinct from classically used lab strains. To study

MOLF STING on a common background we backcrossed MOLF STING onto B6, for at least seven generations (N7), to create B6 mice congenic for MOLF STING, before we began phenotyping. These mice serve to separate MOLF STING dependent interactions from many unknown possible interactions on the MOLF genomic background.

Peritoneal cells from a panel of N7 congenic mice was stimulated with STING activators: dAdT, c-di-AMP, c-di-GMP, 2’3’cGAMP, 3’3’cGAMP, and DMXAA; and RIG-I activator poly I:C. IFNβ cytokine levels were measured (Figure 20A). Overall, PECs of N7 mice with B6 STING responded with robust interferon responses to STING agonists, while peritoneal macrophages of N7 mice with MOLF STING gave low cytokine levels with all agonists except poly I:C control. Surprisingly, N7 peritoneal macrophages expressing MOLF STING did not respond to 2’3’cGAMP as did WT

MOLF peritoneal macrophages. Additionally, N7 STINGMOLF peritoneal macrophages were not hyper-responsive in IL-6 production to STING agonists (Figure 20B).

Therefore, some components of the MOLF response to CDNs are dependent on the

MOLF background, including the IL-6 hyper-responsiveness.

At the N7 stage, the congenic mice are on average 99.2% B6 in genetic composition. However, in this panel of mice with 6 mice per group we see complete penetrance of the phenotype. The responses in the mice segregated by Tmem173 inheritance, where mice inheriting the MOLF STING allele produced weak responses to CDNs.

107

108

400

300

B6/B6 200 STING *** STINGMOLF/MOLF

100 *** (IU/ml)

β *** *** IFN 50

lipo pI:C DMXAA c-di-AMP c-di-GMP 2'3' cGAMP3'3'cGAMP 16 hr stimulations

1.4 *** 1.2 1.0 B6/B6 0.8 STING 0.6 STINGMOLF/MOLF 0.6

0.4 IL-6 ng/ml)

0.2

0.0

lipo pI:C DMXAA c-di-AMP c-di-GMP 2'3' cGAMP3'3'cGAMP 16 hr stimulations

Figure 20: B6-StingMOLF IP Macs lack IFNβ to all STING dependent agonists. Thioglycollate elicited peritoneal macrophages from N7 StingB6/B6 and N7 StingMOLF/MOLF were treated with STING agonists c-di-AMP(4ug/mL), c-di-GMP (4ug/mL), 2’3’cGAMP (4ug/mL), 3’3’cGAMP (4ug/mL), dAdT (2ug/mL), and DMXAA(10ug/mL) for 16 hrs. Panel representative of one experiment in N7. Numerous biological replicates in N9, and N10 mice. Two-way ANOVA with Bonferroni post test.

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To further investigate MOLF STING responses to all agonists. B6 StingB6 and

B6 StingMOLF peritoneal macrophages were stimulated with STING agonists for 2hrs and activation of IRF3 and NFkB pathways were evaluated by Western Blot.

Activation of IRF3-TBK1 axis, p65 and p38 were variable in MOLF STING peritoneal macrophages, but overall much lower than the responses of their B6 StingB6 counterparts. (Figure 21 A, B) By indication of IRF3 activation MOLF STING seems to respond modestly to 2’3’cGAMP (Figure 21 B). However, IFNβ cytokine levels remain low (Figure 21 A).

On the B6 background MOLF STING retains selectivity in responses to CDNs.

Though weaker than B6 responses, we observe significant MOLF STING mediated

IRF3 phosphorylation downstream of 2’3’cGAMP, but not DMXAA. However, in both cases we observe poor induction of IFNβ mRNA and cytokine protein. This highlights a gap in knowledge downstream of the STING signaling pathway. It reveals that there is an unappreciated signaling component downstream of STING, which is defective in MOLF STING. This may be due to a signaling defect in MOLF

STING conferred by the N-terminal mutations, because at the STING C-terminus, where TBK1 and IRF3 are activated, B6 and MOLF sequences are identical.

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Figure 21: B6 StingMOLF congenics exhibit differential responses to STING agonists. Thioglycollate elicited peritoneal macrophages from StingB6/B6 and StingMOLF/MOLF were treated with STING agonists c-di-AMP(4ug/mL), c-di-GMP (4ug/mL), 2’3’cGAMP (4ug/mL), 3’3’cGAMP (4ug/mL), dAdT (2ug/mL), and DMXAA(10ug/mL) for 16hrs. A)IFNβ ELISA; B) Western Blot of NFkB, MAPK, and IRF3 activation after 2hr activation with agonists. A) two-tail t-test. (p-value *p<0.05, **p<0.01) Representative of N=3+ experiments. B) Representative of N=2 experiments

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A time course of peritoneal macrophages activated with 2’3’ cGAMP and

DMXAA was done to blot for phosphorylation of NF-kB, MAPKs, and IRF3 downstream of activation. MOLF STING shows weaker activation in response to either agonist, never peaking higher than B6 STING activation. MOLF STING activation to DMXAA is even weaker when compared to differences in 2’3’cGAMP activation. With 2’3’cGAMP, MOLF STING allows detectable phosphorylation of IRF3.

However, in all cases IFNβ cytokine production is barely detectable after stimulation of B6 StingMOLF macrophages. It may be that unknown functions of STING that lead to IFNβ production are also defective in MOLF STING. (Figure 22)

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Figure 22: p65 and IRF3 phosphorylation is reduced in B6 Sting MOLF IP macrophages (PECS). Western Blot time course of B6 StingB6/B6 and StingMOLF/MOLF peritoneal macrophages activated with A) 2’3’cGAMP (4ug/mL) or B) DMXAA (10ug/mL) for time course shown. Representative of A) N=3+ and B) N=3 experiments.

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In our forward genetic screen, the aim was to find genes in MOLF that conferred reduced IFNβ production to cytosolic DNA species. Inheritance of the

MOLF STING allele in a homozygous or heterozygous fashion correlates with a defect in IFNβ production in peritoneal macrophages to c-di-AMP or c-di-GMP

(Figure 9C,D). To further investigate the mechanism of defective activation of MOLF allele of Sting, we looked at the mode of inheritance of the trait by comparing levels of IFNβ in B6-StingMOLF/MOLF, B6-StingB6/B6, and B6-StingB6/MOLF hybrids.

Interestingly, in response to various STING-agonists, F1s exhibited low to intermediate levels of IFN (Figure 23A), which was indicative of a dominant negative mode of inheritance of the trait and similar to the mode of inheritance of parental MOLF Sting in F1s (Figure 1). Such inheritance is typical for interference between wild type and mutant allelic products such as might be formed by STING dimers (or higher order oligomers) in heterozygous mice.

Congenic mice that are heterozygous for STING, thus inheriting one allele of

MOLF STING are deficient in interferon production. Reduced IFNβ production in

B6-StingB6/MOLF macrophages in response to CDNs, is analogous to F1s responses

(Figure 1), and responses of F2s heterozygous for STING (Figure 9 C,D). However, haplo-insufficiency also affects STING dependent signaling (Figure 23B). Thus,

STING levels also affect signaling responses. Therefore, levels of B6 STING in the heterozygous condition may not be sufficient to initiate signaling when MOLF STING is deficient. On the other hand, MOLF STING may confer a dominant negative effect in the heterozygous condition if it can complex with B6 without inducing a full

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signal. Altogether, inheritance of one loss-of-function allele of STING is sufficient to affect STING-dependent IFNβ production. The human population has a 40% allelic frequency of alternate STING alleles. Many of these variants are loss-of function mutants, and thus can potentially affect STING-dependent immunity with the inheritance of one allele.

115 FIGURE 5

A ns B StingB/B StingM/M StingB/M M/M 200 300 Sting StingB/B

200 100 100 (IU/ml)

* β (IU/ml) ** β ** ** IFN 50 * 0 IFN

lipo pI:C 0 dAdT DMXAA lipo c-di-AMPc-di-GMP pI:C 2'3' cGAMP3'3'cGAMP DMXAA c-di-AMPc-di-GMP C57BL/6 B B6/B6 MOLF/MOLF -/-+/- 2'3' cGAMP3'3'cGAMP D Sting Sting B6-StingSting 100 B6-StingB6/MOLF B6-StingMOLF/MOLF C 80 STING -/- 60 Unst cGAMP40 dAdT DMXAA Unst cGAMPdAdT DMXAA Unst cGAMPdAdT DMXAA Rsad2 ! ! ! ! ! ! ! ! ! ! ! ! (IU/ml) 25 1.5 StingB6/B6 StingM/M Isg15 ! β ! ! ! ! ! ! ! "! ! ! ! Ifit1 ! 20! ! ! ! ! ! ! ! "! ! "! IFN Ccl4 ! 15! ! ! ! ! ! ! ! ! ! !

1.0 Cxcl10 ! 10! ! ! ! ! ! ! "! ! ! ! Ifit3 ! 5! ! ! ! ! ! ! "! "! ! "! Irf1 ! ! ! ! ! ! ! ! ! ! ! ! 0.5 0 Cmpk2 ! ! ! ! ! ! ! ! ! ! ! ! lipo Ifit2 ! ! ! ! ! ! ! ! "! "! ! "! IFNB mRNA RU cdAMP 0.0 Ifi204 ! ! ! ! ! ! cdGMP! ! ! ! DMXAA! ! Ifi47 ! ! ! ! ! ! ! 2'3'cGAMP! ! ! ! ! MOLF%STING%dependent%signaling%is%reduced%in% lipo Usp18 ! ! ! ! ! ! ! ! ! ! ! ! dAdT Gbp2 ! ! ! ! ! ! ! ! ! ! ! !

Intraperitoneal%MacropagescdiAMPcdiGMP %s9mulated%with%DMXAA Ccl3 ! ! ! ! ! ! ! ! ! ! ! ! 2'3'cGAMP3'3'cGAMP Oasl1Figure 23: Dominant negative inheritance of STING in B6 Sting! ! ! ! ! ! ! ! "! "! ! ! B6/MOLF can be E ! ! ! ! ! ! ! ! "! ! ! ! DMXAA% Irf7 explained by haploinsufficiency. Mx1 ! ! ! ! ! ! ! ! "! "! ! "!

Irg1 The effect seems to be haploinsufficiency rather than a dominant negative effect.! ! ! ! ! ! ! ! ! ! ! ! B6 MOLF B6/MOLF Tnf (A) IFN! β ! ELISA of N10 B6 Sting! ! ! ! ! , N10 B6 ! ! Sting! ! , and N10 B6 Sting! treated Pyhin1with STING agonists c! ! ! ! -di! -AMP(4ug/mL), c! ! ! ! -di-! GMP (4ug/mL), 2’3’cGAMP (4ug/mL), ! ! Ifi203 3’3’cGAMP (4ug/mL), dAdT (2ug/mL), and DMXAA(10ug/mL) for 16hrs. ! ! ! ! ! ! ! ! ! ! ! ! (B) IFNβ Gbp3 ! ! ! ! ! ! ! ! ! ! ! ! B6/- -/- Ifih1 ELISA as before, including Sting! ! ! ! ! ! ! , and Sting! ! ! . A) and B) representative of 3+ ! !

Oas1abiological replicates. ! ! ! ! ! ! ! ! ! ! ! ! Gbp5 ! ! ! ! ! ! ! ! "! "! ! "! Oasl2 ! ! ! ! ! ! ! ! ! ! ! ! HeterozygousIfi205 ! ! ! ! inheritance! ! ! ! ! "! !of"! Ccl5 ! ! ! ! ! ! ! ! ! "! ! !

Ddx58 ! ! ! ! ! ! ! ! ! ! ! ! 116 MOLF%STING%discriminates%between%MOLFTrim21 STING! ! ! ! confers! ! ! ! defect! ! ! ! Gbp7 ! ! ! ! ! ! ! ! ! ! ! "! via Ccl12Haploinsuﬃciency! ! ! ! ! ! ! ! "! "! "! "! Oas3 ! ! ! ! ! ! ! ! "! "! ! !

Oas1g ! ! ! ! ! ! ! ! ! ! ! "! CDN%agonists%Marcksl1 ! ! ! ! ! ! ! ! ! ! ! !

NFkB%and%IRF3%ac-va-on%is%reduced%in% Log2(FPKM+0.1) F MOLF%STING%Congenic%mice.% G 0 2 4 6 8 10 B6/B6 A STING STING MOLF/MOLF B6%STING%CONGENIC%PECs% MOLF%STING%CONGENIC%PECs% 2hrB6/B6%%%%%%% 2hr MOLF/MOLF%% add P- 2’3’$cGAMP$ Hrs%s-m% 0%–%1%–%2%–%3%–%4%–%6%–%8%% 0%–%1%–%2%–%3%–%4%–%6%–%8%% STING %%%.%STING% ERK

P%–%p65%

dAdT dAdT

media media

2'3' cGAMP 2'3' 2'3' cGAMP 2'3'

DMXAA DMXAA

3'3' cGAMP 3'3' 3'3' cGAMP 3'3'

c-di-AMP c-di-AMP

c-di-GMP c-di-GMP

P-p38 PDIRF3% P-p65

PDIRF3% P-IRF3

P-TBK1 GAPDH% GAPDH

CHAPTER 8 - Mapping genes that interact with MOLF STING on the MOLF background to confer responsiveness.

Problem: In our initial genetic screen 2’3’cGAMP was not yet discovered. Since, its discovery we have noted that MOLF/Ei macrophages are selectively responsive to

2’3’cGAMP. However, when MOLF STING is placed on the B6 background the IFNβ cytokine production component of this responsiveness is abolished.

Approach: Identify inheritance of other genetic components that modify MOLF

STING responsiveness by investigating MOLF STING responsiveness at the F1 and

F2 intercross level while keeping the MOLF allele fixed.

Chapter Summary: MOLF/Ei peritoneal macrophages selectively produce sufficient IFNβ to

2’3’cGAMP, while B6-Sting MOLF peritoneal macrophages remain extremely deficient in IFNβ production to all CDNs. Furthermore, treatment of BMDMs with concentrated L929 –conditioned media synergizes with and intensifies B6 STING

IFNβ production and restores MOLF STING responses. Altogether, this suggests that a complementary pathway exists that makes up for the MOLF STING defect. This pathway is likely naturally activated downstream of B6 STING, but not MOLF STING, ligation by CDNs. Also, this pathway may be activated in MOLF genetic background by another factor to allow some responsiveness in MOLF/Ei cells.

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In previous chapters, we show that MOLF STING is defective in induction of the IFN promoter when overexpressed, and confers a lack of

IFN cytokine production to CDNs when inherited in B6 mice. We discovered that MOLF STING responses differ on MOLF background compared to B6. MOLF/Ei and B6-Sting MOLF macrophage responses diverge. MOLF/Ei macrophages produces

IL-6 cytokines to most CDNs, and IFNβ to 2’3’cGAMP, while B6-Sting MOLF macrophages are hypo-responsive to all CDNs. We decided to begin another screen to investigate whether other genes on the MOLF background contributes to the defect.

To see the allelic affect of MOLF STING, we compared responses of MOLF

STING congenic mice to the parental C57BL/6 and MOLF/Ei strains. MOLF/Ei macrophages have attenuated interferon responses compared to B6. However,

MOLF macrophages somehow retain the ability to respond to 2’3’cGAMP with a relatively robust IFN production (Figure 24). MOLF/Ei macrophages are not completely deficient in responses to STING agonists; they produce ample Il-6 to all agonists but DMXAA. Somewhat in contrast to MOLF macrophages, StingMOLF macrophages are completely lacking in IFNβ and IL-6 production to all agonists.

Overall it seems that: 1) MOLF STING directly confers a complete lack of responsiveness to DMXAA, as neither MOLF/Ei or StingMOLF macrophages produced

IFNβ nor Il6 in response to DMXAA; 2) The ability of MOLF/Ei to distinguish between STING agonists and; 3) produce Il-6 responses to STING agonist is

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dependent on another interacting gene, as these properties disappear when MOLF

STING is isolated on the C57BL/6 background in the congenics.

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600 C57BL/6 STINGMOLF MOLF 400

200 *** ***

IFNß, IU/ml *** *** 100 ***

lipo DMXAA c-di-AMP 2'3' cGAMP pI:C transfected

*** 3

2 *** IL6, ug/ml, 1

0 lipo DMXAA c-di-AMP 2'3' cGAMP pI:C transfected

Figure 24: Analysis of DNA responses in C57BL/6, B6-StingMOLF, and MOLF/Ei macrophages suggests the existence of a 2nd genetic component on the MOLF background which permits 2’3’cGAMP IFNβ responses in MOLF macrophages. Peritoneal Macrophages from C57BL/6, B6-StingMOLF, and MOLF/Ei were stimulated for 16 hrs with 2’3’cGAMP (4ug/mL), c-di-AMP (4ug/mL), DMXAA (10ug/mL), or poly I:C (4ug/mL). 2’3’cGAMP, c-di-AMP, poly I:C were transfected lipofectamine 2000. DMXAA is overlaid. A) IFNβ ELISA and B) IL6 ELISA from supernatant of stimulated cells. Two-way ANOVA Bonferroni post test. Representative of N=3.

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As shown before, MOLF macrophage IFN and IL6 responses to CDNs and especially 2’3’cGAMP differ from StingMOLF macrophages. MOLF STING may have a crucial interacting partner in the MOLF background that confer these differences.

To test if a gene on the MOLF/Ei background interacts with MOLF STING to re- establish 2’3’cGAMP specific IFNβ responses, I crossed B6 StingB6/MOLF mice with

MOLF/Ei to create F1 (C57BL/6 x MOLF/Ei) mice that are heterozygous or homozygous for MOLF STING. The 2’3’cGAMP responses were higher in F1 macrophages homozygous for MOLF STING (F1 StingMOLF) than B6 StingMOLF macrophages. (Figure 25) F1 Sting B6/MOLF macrophages were intermediate for

2’3’cGAMP responses. Both F1 STINGB6/MOLF and F1 StingMOLF macrophages lacked c- di-AMP-dependent IFN responses as in the B6 StingMOLF macrophages. This data shows the selectivity for 2’3’cGAMP IFNβ responses conferred by the MOLF genomic background on to MOLF STING. This gene that confers responsiveness to

2’3’cGAMP, works in a trans and dominant fashion, which can make it easier to map by QTL analysis. Identifying this gene may also lead to a better mechanistic understanding of STING signaling.

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IFNβ production in IP macrophages from N9-B6 and F1 mice congenic for B6, MOLF STING or both.

N9 STING B6 #1 F1 STING MOLF #1 F1 STING Het #1 N9 STING B6 #2 F1 STING MOLF #2 F1 STING Het #2 N9 STING MOLF #1

400

200 (IU/ml)

β 60 IFN 40

No tx pI:C c-di-AMP 2'3' cGAMP

Figure 25: Comparison of IFNβ production in MOLF, B6-StingMOLF, and F1- StingMOLF reveals importance of genetic background for MOLF STING responsiveness. Responses of STING mice to CDNs. Peritoneal macrophages from B6 or MOLF STING congenic mice (N9 STINGB6, N9 STINGMOLF), and F1 mice (MOLFxB6-STINGB6/MOLF) homozygous for MOLF STING (F1 STINGMOLF) or heterozygous (F1 STINGHET). were stimulated for 16 hrs with 2’3’cGAMP (4ug/mL), c-di-AMP (4ug/mL), or poly I:C (4ug/mL) by transfection with lipofectamine 2000. IFNβ ELISA. Panel of mice used to generate mice in panel (Fig 29). Panel responses representative of two experiments.

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QTL analysis has been a useful resource in uncovering the MOLF STING allele. However, at the time of our initial mapping efforts, 2’3’cGAMP was not yet discovered. Therefore, MOLF macrophage 2’3’cGAMP responsiveness was not yet appreciated. Due to lack of MOLF STING dependent IFN or IL-6 production to

2’3’cGAMP in StingMOLF, we propose that either secondary CDN sensors or protein pathways enhance STING-dependent signaling to 2’3’cGAMP in the MOLF background. To investigate and map genes candidates on the MOLF background that interact with STING to confer IL6 and IFNβ production F1 were crossed to produce a panel of mice - F1 crosses (F1 StingMOLF x F1 StingMOLF) and (F1

StingB6/MOLF x F1 StingB6/MOLF). In this F2 panel, frequency of MOLF STING homozygousity is increased to produce more opportunities to find special gene-gene interactions that confer responsiveness to MOLF STING. The F2 panel of peritoneal macrophages from 40 mice was stimulated with 2’3’cGAMP, c-di-AMP, and DMXAA.

Il6 and IFNβ ELISAs were done with supernatants from activation. The responses gave a good quantitative spread that is needed for QTL analysis. (Figure 26) I noted a few F2 responders generated high IFN levels to 2’3’cGAMP but not c-di-AMP treatment. I also noted many F2 individuals that responded with both a strong IFNβ and Il-6 response to 2’3’cGAMP. These mice may be key to resolving the interacting gene. To genotype the mice, while also providing additional phenotypic data, RNA was isolated from the panel’s peritoneal macrophages treated 4h with 2’3’cGAMP.

This RNA was submitted for RNA-seq. MOLF or B6 specific markers will be extracted from the RNA-seq dataset to genotype the mice by fine mapping. 123

Phenotypes of IFNβ and Il6 production, and select ISGs from RNA-seq will be used as quantitative phenotypes to map loci that confer MOLF STING responsiveness. It will be an ongoing effort in the lab to determine these interacting partners.

124

Figure 26: Responses of F2-STINGMOLF/MOLF mice to CDNs reveals interaction of genes to confer 2’3’cGAMP dependent IFNβ production by MOLF STING. Spread of responses of F2 panel of mice. Peritoneal macrophages were isolated from 40 offspring of F1 crosses (F1 STINGMOLF x F1 STINGMOLF) and (F1 STINGHET x F1 STINGHET). Peritoneal macrophages were stimulated with 2’3’cGAMP (4ug/mL), c-di-AMP (4ug/mL), or DMXAA (10ug/mL). After 16 hrs Cytokine responses in supernatant measured by A) IFNβ ELISA, and B) Il6 ELISA. First panel shown of two F2 panels with identical conditions. 125

CHAPTER 9 - Localization of STING during signaling.

Problem: MOLF STING contains many amino acid changes in the N-terminal transmembrane domains that function to activate STING by an unknown mechanism after ligand binding. STING is retained at membrane proximal regions and is actively trafficked after activation. Whether MOLF STING polymorphisms perturb membrane compartment localization before or after signaling is unknown.

Approach: STING constructs were tagged with HA, GFP, and mCherry at the N- terminus or C-terminus. These constructs were transduced into STING knockout

MEFs and localization was determined relative to costains with organelle markers.

Chapter Summary: MOLF STING amino acid changes are in the N-terminal proximal, transmembrane regions of STING. Additionally, highly conserved regions of STING are disrupted in MOLF STING. STING activity is dependent on allosteric changes and on its subcellular location after binding CDNs. Expression of B6 or MOLF STING in

STING KO MEFs reveals ER localization of both proteins. Upon stimulation of MEFs with CDNs, MOLF STING is retained in the ER, while B6 STING translocates to uncharacterized punctate perinuclear structures.

126

127

The structure of the C-terminal, soluble fraction of STING (153-378) is clearly defined by crystallography submitted by multiple groups.18 Many of these studies depict STING C-terminal conformational changes that occur when STING binds a

CDN. STING binds CDNs in a binding pocket between its dimers. The dimer is stabilized and the C-terminal tail is exposed to where TBK1 can bind, autophosphorylate and activate IRF3. Our data, and others, elude that the C-terminus alone is not sufficient to perpetuate this response. The N-terminus, whose structure and function is unknown, is necessary for the activation of STING in vitro. The N- terminus in this regard may specify proper location of STING, recruitment of co- signaling molecules, and/or proper orientation of STING, in vitro, to undergo conformational changes to bind and respond to agonists. There are multiple models of STING N-terminal transmembrane topology. Models disagree on the number and location of the transmembrane passes (Figure 27). Models suggest that there are 2-

4 tm passes in any of 5 possible locations(Ouyang et al. 2012). All MOLF mutations except for N210D are in “proposed” transmembrane passes. When MOLF STING amino acid changes are input into the HMMTOP16,19 online modeling site these topological membrane passes shift compared to B6 STING (Figure 27). Specifically,

MOLF mutations strengthen the probability that the 46-67 amino acid stretch is a trans-membrane domain. This region was shown to be a low complexity region in

SMART domain modeling. It requires further inquiry to determine which MOLF amino acid mutations are cytosolic, trans-membrane, or luminal residues. These changes in MOLF STING residues at or near the membrane interface may impart

128

reduced interferon production through alternate migration of MOLF STING after activation.

129 MOLF N-terminal amino acid changes disrupt membrane topology A model of STING.

HMMTOP membrane topology maps of B6 STING and MOLF STING.

Most of the MOLF amino acid changes are in the 2nd and 3rd B transmembrane of STING and they drasGcally disrupt these TM and lumen sequences. iii (green) = cytosolic amino acid; MMM (red) = transmembrane amino acid; ooo (blue) = ER luminal amino acid

Figure 27: MOLF N-terminal mutations in STING disrupt membrane topology model of B6 STING.

HMMTOP membrane topology maps of A) B6 STING and B) MOLF STING.

iii (green) = cytosolic amino acid; MMM (red) = transmembrane amino acid; ooo (blue) = ER luminal amino acid

130

MOLF mice are a divergent wild derived mouse strain with many polymorphisms that influence many signaling pathways. STING add-back experiments in MEFs or BMDMs are important to confirm and elucidate the influences of MOLF STING mutations on signaling pathways in a common background. We have obtained Tmem173 -/- mice and isolated MEFs from these mice for the add-back studies.

STING translocates to an undefined perinuclear space upon activation. To visualize B6 versus MOLF expression in MEFs we cloned B6 and MOLF STING into lentiviral vectors. N-terminally tagged mCherry-STING constructs were generated.

Veit Hornung’s lab has produced mCherry tagged STING and has verified that the N- terminal tag does not affect signaling (Ablasser et al. 2013). Also, the Hornung lab has done a protease-digestion assay to show that the N-terminus of STING is cytosolic. (Veit - unpublished) B6 or MOLF mCherry STING was stably expressed in

MEFs with reduced expression vectors to avoid spontaneous activation. mCherry-

STING expressing MEFs were stimulated on coverslips with 2’3’cGAMP, dAdT, or

DMXAA for 2h, and imaged by confocal microscope.(Figure 28) B6 and MOLF mCherry STING both show ER localization in unstimulated cells. (Figure 28) After activation B6 STING shows more defined punctate patterning. (Figure 28). With optimal activation with 2’3’cGAMP B6 mCherry-STING moves to double membrane like vesicles, while MOLF mCherry STING is still quite diffuse. (Figure 28)

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Figure 28: MOLF STING show aberrant localization compared to B6 STING during activation. Localization of B6 or MOLF mCherry-STING stably transduced into MEFs after stimulation with dAdT, c-di-AMP, 2’3’cGAMP, or DMXAA. STING KO MEFs stably reconstituted with B6 or MOLF mCherry-STING; Stimulated for 2h with lipofectamine and: media, dAdT (2ug/mL), and 2’3’cGAMP (4ug/mL), or DMXAA (2ug/mL); Cells Fixed and Permeablized with 4% PFA and MeOH; Then co-stained for ER with Grp94 ab and Alexa 488 (Green). Images taken on confocal microscope. Images representative of the field, at least five images images taken per field, and A) 3+ experiments.

132

Since S53L and L47V, A48G amino acid changes imparted the greatest defect to IFN promoter induction in a 293T Luciferase reporter assay (Figure 16A), and are predicted to result in disruption of purported 14-3-3 and LC3 binding domains, respectively, we hypothesized that either of these amino acid changes would disrupt optimal translocation. Stimulation of STING knockout MEFs expressing S53L or

L47V-A48G mCherry STING revealed that L47V-A48G mutations severely reduce formation of STING puncta and displacement from the ER. S53L STING, however, often formed strange puncta even in unstimulated conditions, perhaps alluding to another mechanism of S53L action that is probably dependent on the context of other mutations. (Figure 29)

133 A B C F media 2’3’cGAMP dAdT DMXAA A B C No Tx F No Tx e 2h c-di-AMP

u 2h c-di-AMP media dAdT A l 1.0 1.0 2’3’cGAMP DMXAA a No Tx No Tx / v e e 0.82h c-di-AMP u u

R 2h c-di-AMP l 1.0 l 1.00.8

a 0.6 s a ' / B6 v e v n 0.80.4

u o R l R s 0.6 0.80.6 s a

r 0.2 ' B6 s a v n 0.4 ' e 0.0 o n mCherry STING P R s B6 0.6

o 0.4

r 0.2 c-di-AMP media s a s

' r e 0.0 n mCherry STING a P B6 Figure 29: Localization of L47Vo 0.40.2 - e c-di-AMP media s P A48G, and S53L mCherryNo Tx r -STING in Grp94 (ER) e 2h c-di-AMP a

u 0.2 e mCherry STING 0.0 stably transduced MEFs l 1.0 stimulated a No Tx P 6 F L v

Grp94 (ER) G

with CDNs e L 3 0.82h c-di-AMP B 8 5

u 4

B6 O mCherry STING R 0.0 l 1.0 M A S STING KO MEFs stably reconstituted a 0.6 s

' 6 F V L v L 7 G 3 n B 8 with 0.80.4A) B6, MOLF, B) L47-A48G or S53L 4 4 5

B6 O R MOLF o L M A S s / 0.6 s

r 0.2

' V mCherry-STING; Stimulated for 2h with mCherry7 STING a n 0.4 4 e 0.0 MOLF o L mCherry STING lipofectamine and: media, or c-di-AMP P s / MOLF r 0.2 mCherry STING c-di-AMP media (4ug/mL) Cells Fixed and Permeablized a

e 0.0 mCherry STING with 4% PFA and MeOH; Then coP MOLF - c-di-AMP media stained for ER with Grp94 ab and Alexa D 488 (Green). Images taken on confocal E G microscope. Images representative of B a D the field, at least five images images E No Tx G 100 e 2h c-di-AMP No Tx u mCherry STING a l Grp94 (ER) taken per field, and 1.0 A) 3+ or B) 2 a No Tx 100 c-di-AMP punct v experiments.e 0.82h c-di-AMP No Tx

u 80 mCherry STING R l Grp94 (ER) 1.0 a 0.6 c-di-AMP s punct ' v

n 0.80.4 80 R o MOLF Cherry s 0.6 s r ' 0.2 60 a L47V-A48G L47V-A48G n 0.4 e

o 0.0 MOLF hm mCherry STING Cherry s P

r 0.2L47V A48G 60 c-di-AMP media a L47V-A48G L47V-A48G wit e 0.0 40 hm mCherry STING P L47V A48G c-di-AMP media No Tx wit 40 e 2h c-di-AMP MEFs 20 u l 1.0 a No Tx v

e 0.82h c-di-AMP MEFs 20 u R l 1.0 a 0.6 s

' 0 v Percent S53L n 0.80.4 R o F L

s 0.6 s B6 8G

' 0 r 0.2 53 Percent

S53L OL a n 0.4 M A4 S e

o -

mCherry STING 0.0 F V L s P B6 8G r 0.2 S53L 7 53

c-di-AMP media OL a M L4 A4 S e -

mCherry STING 0.0 V P S53L 7 c-di-AMP media L4

134

CHAPTER 10 - RNA-seq and Pathway analysis of STING dependent responses in macrophages

Problem: MOLF STING confers lack of IFNβ production on the B6 background.

Investigation of responses downstream of STING activation reveals differential activation of signaling proteins and transcription factors. Furthermore, MOLF STING signaling is bimodal in the B6 and MOLF background. In the MOLF background there are STING dependent IL-6 responses, yet in B6 practically all responses are attenuated. This implies that MOLF STING can actively signal. However, the signaling downstream of MOLF STING is probably perturbed by the lack of activation of one or more signaling nodes.

Approach: Pathway analysis of the B6, MOLF, STING KO, and B6-StingMOLF transcriptome can reveal groups of genes downstream of a signaling pathway or transcription factor that are differentially upregulated. This can lead to insight into differential pathway activation by the alleles of STING activation.

Chapter Summary: Analysis of B6, MOLF, STING KO, and B6-StingMOLF macrophage reveals that the gene signatures of macrophages that express MOLF STING are shifted. MOLF

STING appears to be the major component determining pathways activated downstream of CDNs. However, there are some differences in pathways activated by MOLF STING activation on the B6 background versus the MOLF background.

Pathways that remain deficiently activated in the MOLF STING background may lead to the mechanism of the MOLF STING defect.

135

To investigate more extensively the functional consequences of MOLF STING effect between different agonists we performed genome-wide RNA-seq analysis of

RNA responses in macrophages from MOLF-congenic mice, littermate controls and parental lines activated with cGAMP, dAdT and DMXAA. (Figure 30) We constructed expression heat maps and initially focused our attention on annotated ISGs.

Although, cGAMP elicits slightly weaker production of IFN by MOLF macrophages

(compared to B6) (Figure 3A), we found it induced more robust up-regulation of

ISGs than DMXAA, which has a much weaker effect on ISG induction in MOLF macrophages than in B6 (Figure 30). Similar to DMXAA, stimulation with dAdT also resulted in weaker ISG expression than cGAMP. More broadly, the pattern of gene upregulation in MOLF and STINGMOLF macrophages was similar for all agonists thus further demonstrating that STING is the major principle component regulating DNA- responses in MOLF mice.

136

Figure 30: MOLF STING inheritance confers distinct ISG signature. Transcriptome analysis of peritoneal macrophages for B6, MOLF, STINGB6, or STINGMOLF stimulated for 4hrs with 2’3’cGAMP (4ug/mL), dAdT (4ug/mL), or DMXAA (10ug/mL). mRNA isolated converted to cDNA library using TRUseq kit, and sequence on MiSeq. Data analyzed and converted to RPKMs using tuxedo tools software suite. Heatmap representative select ISGs from RNA-seq RPKM data of B6, MOLF, STINGB6, and STINGMOLF, stimulated with 2’3’cGAMP, dAdT, or DMXAA. A) Heatmap levels of top 37 ISGs mRNAs from RNAseq FPKMs.

137

In order to quantify the differences we observed in our heat maps, we selected the 1000 most highly upregulated genes—including many non-ISGS—and plotted gene-specific responses, expressed as RPKM (Reads Per Kilobase sequenced per Million base of aligned reads) for each of the tested agonists. For macrophages stimulated with cGAMP we observed a close correlation (R2=0.676) in B6 and MOLF mice, with many of the same genes up-regulated to a similar degree. While a slightly lower correlation was still evident for stimulation with dAdT (R2=0.64), DMXAA clearly elicited very different responses (R2=0.12) in the two strains, supporting the conclusions drawn from our heat maps. Linear regression analysis of the most highly upregulated genes confirmed that in response to either mammalian 2’3’ cGAMP, or the synthetic B-DNA analog dAdT that induces 2’3’cGAMP synthesis by cGAS, expression of most ISGs (red dots) are slightly higher in B6. However, certain

ISGs are many fold higher in MOLF and StingMOLF macrophages (Figure 31).

To quantitatively confirm our heat map-based observation that B6 STING responds similarly to all agonists while MOLF STING appears to discriminate between them, we examined the correlation between cGAMP and DMXAA responses in B6, MOLF and MOLF-congenic mice. While the responses to DMXAA and cGAMP correlated very well (R2=0.729) in B6 macrophages, similar plots for MOLF and

MOLF congenic macrophages demonstrated much weaker correlations (R2=0.194,

0.416), with the curves skewing towards cGAMP-responses (Figure 31 B-D). Thus, our RNA-sequencing data clearly show that the MOLF STING allele generally confers reduced IFN responses, with MOLF STING congenic macrophages displaying

138

stronger phenotypic similarity to MOLF than to B6 macrophages. They also reveal that MOLF STING has a broader range of sensitivity towards STING ligands enabling it to discriminate between them and induce different responses, as shown by differential up-regulation of some genes by MOLF STING containing macrophages.

To investigate more extensively the functional consequences of MOLF STING effect between different agonists we performed genome-wide RNA-seq analysis of

RNA responses in macrophages from MOLF-congenic mice, littermate controls and parental lines activated with cGAMP, dAdT and DMXAA. (Figure 31) We constructed expression heat maps and initially focused our attention on annotated ISGs.

Although cGAMP elicits only weak production of IFN by MOLF macrophages (Figure

31A), we found it induced more robust up-regulation of ISGs than DMXAA, which has a much weaker effect on ISG induction in MOLF macrophages than in B6 (Figure

31B). Similar to DMXAA, stimulation with dAdT also resulted in weaker ISG expression than cGAMP. More broadly, the pattern of gene upregulation in MOLF and MOLF congenic macrophages was similar for all agonists thus further proving that STING is the principle component regulating DNA-responses in MOLF mice.

B6 and MOLF mice, with many of the same genes up-regulated to a similar degree.

139

While a slightly lower correlation was still evident for stimulation with dAdT

(R2=0.64), DMXAA clearly elicited very different responses (R2=0.12) in the two strains, supporting the conclusions drawn from our heat maps. Linear regression analysis of the most highly upregulated genes confirmed that in response to either mammalian 2’3’ cGAMP, or the synthetic B-DNA analog dAdT that induces 2’3’ cGAMP synthesis by cGAS, expression of most ISGs (red dots) are slightly higher in

B6. However, certain ISGs are many fold higher in MOLF and StingMOLF macrophages

(Figure 31). Altogether, MOLF STING potentiates an alternate signaling program in macrophages compared to B6 STING.

140 MOLF and B6 STING vary in upregula9on of ISGs in a ligand speciﬁc manner

cGAMP DMXAA cGAMP DMXAAcGAMP DMXAA 215 215 15 2 p<0.001 p<0.001 p<0.0001 cGAMP dAdT p<0.0001p<0.001 cGAMP dAdT 10 p<0.0001 A DMXAAcGAMP 210 10 10 DMXAAdAdT 2 10 B 10 10 DMXAA 2 C 2 40010 210 2 10 2 2 400 210 10 2 cGAMP DMXAA2 2 2 400 2 2 2 2 RSDA2 2 15 2 RSDA2 R =0.122 R =0.64 2 R =0.122 R =0.64R =0.676 RSDA2 R =0.64 RSDA2 cGAMP DMXAA cGAMP DMXAA R =0.676 RSDA2 R =0.12 IFIT1 p<0.001 R =0.676 RSDA2 IFIT1 IFIT1 215 15 25 p<0.0001 cGAMP IFIT1 5 dAdTIFIT3 25 2 5 IFIT1 5 IFIT3 IFIT3 2 10 p<0.001 2 RPKM IFIT1 2 IFIT3 5 10 5 DMXAA 2 RPKM 5 5 IFIT3 p<0.001 RPKM 200 IFIT3 2 2 p<0.0001 RPKM cGAMP5 IFIT3 IFIT3 102 5 dAdT 2 RPKM 200 p<0.0001 10 2 5 cGAMP 2 10 IFIT3 10 dAdTRSAD2 RPKM 200 2 400 2 2 10 2 DMXAA RSAD2 2 2 10 10 DMXAA RSAD2 2 10 0 2 10 2 2 400 2 0 10 2 2 2 2 R =0.64 RSDA2 400 2 2 0 2 R =0.12 IFIT1 2 2 2 IFIT1 R =0.6762 RSDA2 2 R =0.122 R =0.64 IFIT1RSDA2 R =0.64 RSDA2 IFIT1 R =0.676 5 R =0.12 0 0 RSDA2 2 R =0.676 RSDA2 0 IFIT1IFIT1 2 5 IFIT3 0 0 2 0 0 0 IFIT1 2 5 0 RPKM -5 IFIT3IFIT3 2 5 2 -5 5 0 IFIT1 2 2 25 50 2 IFIT3 IFIT3 RPKM 200 2 0 IFIT1 2 2 2 -5 22 5 MOLF (RPKM) RPKM 2 2 IFIT3 5 2 MOLF (RPKM) 2 RSAD2 RPKM

B6 IFIT3 MOLF (RPKM) 5 B6 IFIT3 5 MOLF (RPKM) RPKM 200 2 MOLF (RPKM)

B6 B6 MOLF (RPKM) 5 IFIT3

RPKM 2 200 MOLF (RPKM) 2

B6 B6 MOLF 2 MOLF RSAD2 MOLF (RPKM) MOLF MOLF 0 MOLF (RPKM) RSAD2 MOLF MOLF 2 0 IFIT1 2 0 2 IFIT1 -5 -5 -5 2 -5 IFIT1 -5 -5 2 0 0 -5 2 -5 2 -5 0 5 10 2 2 -5 2 0 5 10 0 2 -5 -5 0 5 10 15 2 0 20 2 2 2 -50 -5 0 0 5 5 10 10 15 2 2 2 2 -5 0 5 -510 0 2 2-5 -5 0 0 5 5 10 10 1522 2 0 2 2 2 -5 0 0 2 2 2 2 2 2 2 -5 2 02 25 2 10 2 2 0 2 2 2 2 2 2 2 2

2 2 2 2 2 MOLF (RPKM) 2 -5 2 0 2 2 2 2 2 2 MOLF (RPKM) 2 B6 UnstimcGAMPdAdTDMXAAUnstcGAMPdAdTDMXAAunstimcGAMPdAdTB6DMXAAunstcGAMPdAdTDMXAA MOLF (RPKM) B6(cGAMP)UN) MOLF(cGAMP)UN)diffin4act4cGAMP4(B6)MOLF)cGAMP DMXAA dAdT diff4DMXAA diff4dAdT dif)difDMXAA)cGAMP MOLF (RPKM) MOLF (RPKM)

UnstimcGAMPdAdTB6 DMXAAUnstcGAMPdAdTDMXAAunstimcGAMPdAdTDMXAAunstcGAMPdAdTDMXAA MOLF (RPKM) B6 B6(cGAMP)UN) MOLF(cGAMP)UN)diffin4act4cGAMP4(B6)MOLF)cGAMP DMXAA dAdT diff4DMXAA diff4dAdT dif)difDMXAA)cGAMP UnstimcGAMPdAdTDMXAAUnstcGAMPdAdTDMXAAMOLF (RPKM) unstimcGAMPdAdTDMXAAunstcGAMPdAdTDMXAA MOLF MOLF B6(cGAMP)UN) MOLF(cGAMP)UN)diffin4act4cGAMP4(B6)MOLF)cGAMP DMXAA dAdT diff4DMXAA diff4dAdT dif)difDMXAA)cGAMP B6 (RPKM) B6 B6 MOLF (RPKM) MOLF MOLF Ccl12 4 4 4 4 4 4 4 4 4 4 4 4 4 MOLF (RPKM) 4 4 4 B6 (RPKM) B6 (RPKM) B6 (RPKM) Ccl12 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 B6 (RPKM) B6 (RPKM)B6 (RPKM) MOLF MOLF Ccl12 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 B6 (RPKM) B6 (RPKM) Irf7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Irf7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -5 Irf7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -5 -5 2 Oas1g 4 4 4 4 4 4 4 4 4 4 4 4 -54 4 4 4 10242 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -5 Oas1g -5 1024 2 -5 0B6 5 10 2 -5 1024 -5 0 5 10 Oas1g 4 4 4 4 4 4 4 4 4 42 4 4 4 4 4 4 B6 -5 1024-5 0 5 10 1024 15 2 2 -5 2 2 2 -5 2MOLF0 5 10 15 2 2 2 2 Ddx58 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 MOLF B6 -5 2 0 5 10 1024 -5 0 5 10 Congenic Ddx58 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2-5 20 25 210 2 2 2 2 2 2 Congenic2 -5 2 MOLF0 2 5 2 10 2 15 2 22 22 22 Ddx58 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -5 0 5 10 2 2 2 2 2 Congenic Tnf 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 4 2 2 2 Tnf 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 UnstimcGAMPdAdTDMXAAUnstcGAMPdAdTDMXAAunstimcGAMPdAdTDMXAAunstcGAMPdAdTDMXAA 4 4 4 4 4 4 4 4 4 4 4 4 4 4Unstim4 cGAMP4 dAdTDMXAAUnstcGAMPdAdTDMXAAunstimcGAMPdAdTDMXAAunstcGAMPdAdTDMXAA 2 B6(cGAMP)UN) MOLF(cGAMP)UN)diffin4act4cGAMP4(B6)MOLF)cGAMP DMXAA dAdT diff4DMXAA diff4dAdT dif)difDMXAA)cGAMP 2 Tnf B6(cGAMP)UN) MOLF(cGAMP)UN)diffin4act4cGAMP4(B6)MOLF)cGAMP DMXAA dAdT diff4DMXAA diff4dAdT dif)difDMXAA)cGAMP 2 Trim21 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 R =0.729 2 R =0.416 4 4 4 4 4 4 4 4 4 4 Unstim4 cGAMP4 dAdT4 4 DMXAA4 Unst4 cGAMPdAdTDMXAAunstimcGAMPdAdTDMXAAunstcGAMPdAdTDMXAA R =0.729 Trim21 B6(cGAMP)UN) MOLF(cGAMP)UN)diffin4act4cGAMP4(B6)MOLF)cGAMP DMXAA dAdT diff4DMXAA diff4dAdT dif)difDMXAA)cGAMP B6 (RPKM) R =0.416 B6 (RPKM) 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 R =0.194 Ccl12 Trim21 4 4 4 4 4 4 4 4 4 4 Ccl124 4 4 B64 4 4 4 (RPKM)4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 R =0.194R =0.729 B6B6 (RPKM) (RPKM)B6 (RPKM) R =0.416 B6 (RPKM) 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oas1a4 Oas1a 4 4 4 4 4 4 Ccl124 4 4 4 4 4 4 4 4 4 B6 (RPKM) R =0.194 B6 (RPKM) 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Irf7 Oas1a 4 4 4 4 4 4 4 4 4 4 Irf74 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oas3 4 4 4 4 4 4 Irf74 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oas34 D E F 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1024 32 Oas1g 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 32 MOLF Oas3 Oas1g 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 10241024 4 B64 4 4 4 4 4 4 4 4 4 4 4 4 4 4 32 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Mx14 321024 DMXAA 32 32 1024 DMXAA Mx1 Oas1g DMXAA 32 MOLF B6 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DMXAA Congenic 1024 Ddx58 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 B6 32 MOLF

Mx1 DMXAA 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 32 Congenic Ddx58 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 MOLF DMXAA 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ccl4 Ccl4 Ddx58 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Congenic 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Tnf 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DMXAA Ccl4 DMXAA 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 Tnf 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Marcksl1 Marcksl1 Tnf 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 DMXAA 2 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 R =0.729 RPKM R =0.416 Trim21 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 Marcksl1 RPKM 2 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 R =0.729 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 R4 =0.194 R =0.416 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Trim21 2

Ccl3 RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ccl3 R =0.729 R =0.416 RPKM

Trim21 RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 R =0.194 Oas1a Ccl3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 R =0.194 4 4 4 4 4 4 4 4 4 4 4 4 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oas1a Ifitm3 Ifitm3 Oas1a 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Oas3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM 1 Ifitm3 RPKM 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 32 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Usp184 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Usp18 Oas3 4 4 4 4 4 4 4 4 4 4 4 4 Oas34 4 4 4 RPKM 11 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 32 1 Mx1 4 4 4 4 4 4 4 4 4 DMXAA 4 324 4 4 4 4 4 32 Usp18 DMXAA 1 32 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi2034 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 32 Ifi203 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Mx1 DMXAA 321 32

Mx1 DMXAA 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 DMXAA Ccl4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DMXAA Ifi203 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Irf1 DMXAA Irf1 4 4 4 4 4 4 4 4 4 4 4 4 Ccl44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Marcksl1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ccl4 Irf1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DMXAA DMXAA

RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit3 1 32 1024 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 4 4 4 4 32 4 4 4 4 10244 4 4 4 4 Ccl3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Marcksl1 Marcksl1 1 32 1024 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 1024 Ifit3 RPKM

RPKM 1 32 1024 RPKM Gbp2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 1024 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ccl3 Ccl3 RPKM RPKM Ifitm3 RPKM Gbp2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 cGAMP 1 cGAMP 32 RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

RPKM RPKM Oasl1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl1 cGAMP 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Usp18 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifitm3 Ifitm3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 RPKM RPKM RPKM RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl2 Oasl2 cGAMP cGAMP RPKM cGAMP 1 cGAMP 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM RPKM Ifi203 Usp18 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl2 4 4 4 4 4 4 4 4 4 4 Usp1814 4 4 4 4 4 1 cGAMP 1 cGAMP Gbp5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi203 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Irf1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp5 4 4 4 4 4 4 4 4 4 4 Ifi2034 4 4 4 4 4 1 1 Gbp7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Irf1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 4 4 32 1024 Gbp7 4 4 4 4 4 4 4 4 4 4 Irf14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 1024 Gbp3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 1024 Gbp3 4 4 4 4 4 4 4 4 4 4 Ifit34 4 4 4 4 4 4 RPKM4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 32 The1 logic might32 1024 1 32 1024in MOLF: The logic might 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 in MOLF: Ifi205 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Data forIfi205 hostcGAMP cGAMP versus pathogen cGAMPData for host cGAMP versus pathogen cGAMP 1 32 1024 Oasl1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 in MOLF: The logic might 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Data for host cGAMP versus pathogen1 cGAMP32 Ifi205 Gbp2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM Ifih1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifih1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM cGAMP RPKMRPKM 1 32 Oasl2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl1 4 4 4 4 4 4 4 4 4 4 be4 4 that4 4 we4 4 saw difference in heat maps and decided tocGAMP see howbe that quantitative we saw it differenceis. That iscGAMP cGAMP in heat maps and decided to see how quantitative it is. That is Ifih1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi47 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi47 4 4 4 4 4 4 4 4be4 that4 4 4 we4 4 saw4 4 difference in heat maps and decidedRPKM to see how quantitative it is.RPKM That is Gbp5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Oasl2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 cGAMP cGAMP Ifi47 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RPKM RPKM 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 cGAMP Isg15 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 whyOasl2 we didIsg15 correlation4 4 4 4 between4 4 4 4 4 parental4 4 4 4 strains4 4 4 for individualwhy agonists we did and correlation we observed between that parental strains for individualcGAMP agonists and we observed that 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp7 Gbp5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Isg15 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 why we did correlation between parental strains for individual agonists and we observed that Ifit2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp5 Ifit24 4 4 4 4 4 4 4 4 4 4 4 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp3 4 4 4 4 4 4 4 4 4 4 DMXAA4 4 4 4 is4 falling4 out. Then we moved on and compared parentsDMXAA AND iscongenic falling line out. for Then we moved on and compared parents AND congenic line for Gbp7 Ifit2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Cmpk2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp7 Cmpk24 4 4 4 4 4 4 4 4 4 4 4 44 4 4 4DMXAA4 4 4 4 4 4 4 4 4is4 4 falling4 4 4 4 out. Then we movedin onMOLF: and compared The logic parentsmight AND congenic line for 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Data for host cGAMP versus pathogen cGAMP Ifi205 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Gbp3 Cmpk2 4 4 4 4 4 4 4 4 correlation4 4 4 4 4 4 between4 4 DMXAA and cGAMP. And it looks like MOLFcorrelation and congenic between MOLF areDMXAA more and cGAMP. And it looks like MOLF and congenic MOLF are more 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 MOLF Pyhin1 Gbp3 Pyhin14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 in MOLF: The logic might Ifih1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DatacorrelationFigure 31: MOLF and B6 Sting for hostbetween cGAMP DMXAA and versus cGAMP. And congenic vary in upregulation of ISGs in pathogen it looks like MOLF cGAMP and congenic MOLF are morea Ifi205 Pyhin1 4 4 be4 4 that4 4 4 we4 4 saw4 4 4 difference4 4 4 4 in heat maps and decided to see how quantitative it is. That is Irg1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 in MOLF: The logic might Irg14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Data for host cGAMP versus pathogen cGAMP 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi205 MOLF diﬀeren9ally expresses ISGs to DMXAA Ifi47 sensitive to cGAMP than DMXAA. Then we reasoned that MOLFsensitive developed to cGAMPdifferential than DMXAA. Then we reasoned that MOLF developed differential 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifih1 Irg1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit1 4 4 4 4 4 4 be4 4sensitive that4 4 4 we4 4 to saw4 cGAMP4 4 difference than DMXAA. in heat Then maps we and reasoned decided that to MOLF see how developed quantitative differential it is. That is 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Isg15 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 why we did correlationIfih1 between parental strains for individual agonists and we observed that 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ligand specific manner. Ifi47 Ifit1 4 4 4 4 4 4 4 4 sensitivity4 4 4 4 4 towards4 4 4 host and pathogen cGAMPs and we did beyoursensitivity that experiment we towardssaw with difference different host and in pathogen heat maps cGAMPs and decided and we todid see your how experiment quantitative with itdifferent is. That is Rsad2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Rsad2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi47 4 4 4 4 4 4 4 4 4 sensitivity4 4 4 4 4 4 towards4 host and pathogen cGAMPs and we did your experiment with different 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 why we did correlation between parental strains for individual agonists and we observed that Isg15 Rsad2 4 4 DMXAA4 4 4 4 4 is4 falling4 4 4 4 out.4 4 4 Then4 we moved on and compared parents AND congenic line for B6, Ifi204 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi204 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 GAMPs. Cmpk2 Isg15 4 4 4 4 4 4 4 4 4 4 4 Transcriptome analysis of peritoneal macrophages for B6, MOLF, STING4 4 4 4 4 whyGAMPs. we did correlation between parental strains for individualor agonists and we observed that 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit2 Ifi204 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 GAMPs. Cxcl10 correlation betweenCxcl10 DMXAA4 4 4 4 and4 4 DMXAA4 cGAMP.4 4 4 4 is4 And 4 falling4 4 it4 looks out. likeThen MOLF we moved and congenic on and MOLFcompared are more parents AND congenic line for 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Pyhin1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit24 4 4 4 Cmpk2 Cxcl10 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 MOLF 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DMXAA is falling out. Then we moved on and compared parents AND congenic line for Ccl5 Ccl5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Irg1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 STING stimulated for 4hrs with 2’3’cGAMP (4ug/mL), dAdT (4ug/mL), or 4 4 4 4 4 4 4 4 correlation4 4 4 4 4 4 4 between4 DMXAA and cGAMP. And it looks like MOLF and congenic MOLF are more Pyhin1 Ccl54 4 4 44 4 sensitive4 4 4 4 4 4 4 4 4 4 4 4 to4 4 cGAMP4 4 Cmpk24 4 4 4 4 4 4 than4 4 DMXAA. Then we reasoned that MOLF developed differential Ifit1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 correlation between DMXAA and cGAMP. And it looks like MOLF and congenic MOLF are more Irg1 4 4 4 4 sensitivity4 4 4 4 4 4 4towards4 Pyhin14 4 4 4 4host4 4 4 and4 4 pathogen4 4 sensitive4 4 4 DMXAA (10ug/mL). mRNA isolated converted to cDNA library using TRUseq kit, 4 cGAMPs4 4to4 cGAMP4 and thanwe did DMXAA. your experiment Then we reasoned with different that MOLF developed differential Rsad2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifit1 4 4 4 4 4 4 4 4 4 4 4 4 Irg14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 sensitive to cGAMP than DMXAA. Then we reasoned that MOLF developed differential Ifi204 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 GAMPs. sensitivity towards host and pathogen cGAMPs and we did your experiment with different Rsad2 4 4 4 4 4 4 4 4 4 4 4 4 Ifit14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 and sequence on MiSeq. Data analyzed and converted to RPKMs using tuxedo tools 4 4 4 4 4 Cxcl10 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 sensitivity towards host and pathogen cGAMPs and we did your experiment with different 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Ifi204 Rsad2 4 4 4 4 4 4 4 4 GAMPs.4 4 4 4 4 4 4 4 Ccl5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Cxcl10 Ifi204 4 4 4 4 4 4 4 4 4 4 4 software suite.4 4 4 4 4 GAMPs. Ccl5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Cxcl10 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Ccl5 4 4 4 4 4 4 4 4 4 4 4 Pearson coefficents 4 4 4 4 4 to show linearity of B6 versus MOLF ISG responses to A) DMXAA, B) cGAMP, C) dAdT. Pearson coefficents to show linearity of MOLF ISG responses to D) DMXAA vs cGAMP, B6 ISG responses to E) DMXAA vs cGAMP and, StingMOLF ISG responses to F) DMXAA vs cGAMP.

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RNAseq analysis of transcripts upregulated in StingB6 and StingMOLF peritoneal macrophages after 2’3’cGAMP stimulation reveals significant differential regulation of genes. To determine the transcription factors most affected by the

MOLF STING signaling defect, genes significantly upregulated in StingB6 over

StingMOLF (Appendix I) were input into the gprofiler database. In Figure 32, the gprofiler output shows transcription factor family motifs most present in the geneset. It is apparent that IRF, AP-1, p65 (NFkB), and c-Rel (NFkB) transcription factor motifs are significantly represented in the promoters of the geneset. This shows that in the genes most upregulated in StingB6 and StingMOLF, differences in

IRF, AP-1, p65, and c-Rel family member activation may be responsible for differential activation of top genes in congenic macrophages. However, this is looking at a subset of highly activated genes. A holistic view of subtle gene network shifts may highlight difference in other signaling pathways. Overall, StingB6 and

StingMOLF show significant differences in activation downstream of 2’3’cGAMP stimulation.

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Comparison of B6 and MOLF STING dependent signaling in response to p-value Transcrip/on Factor Family 2’3’cGAMP reveals signiﬁcant decrease in IRF, NF-kB, and AP-1 1.92E-10 Factor: IRF; mo/f: BNCRSTTTCANTTYY; match class: 4 family transcripJon factor 3E-12 Factor: IRF; mo/f: BNCRSTTTCANTTYY; match class: 3 acJvaJon. 0.000000039 Factor: IRF; mo/f: BNCRSTTTCANTTYY; match class: 2 0.000000487 Factor: IRF; mo/f: BNCRSTTTCANTTYY; match class: 1

0.0222 Factor: AP-1; mo/f: NNTGACTCANN; match class: 1

0.0327 Factor: AP-1; mo/f: NNTGACTCANN; match class: 0

0.00385 Factor: NF-kappaB (p65); mo/f: GGGRATTTCC; match class: 4 Comparison of transcriptome of 2’3’cGAMP s/mulated C57BL/ 6B6 STING and C57BL/6MOLF STING PECs. 0.00174 Factor: NF-kappaB; mo/f: NNNNKGGRAANTCCCN; match class: 4

0.00146 Factor: NF-kappaB; mo/f: NNNNKGGRAANTCCCN; match class: 3 Signiﬁcantly upregulated genes in C57BL/6B6 STING over C57BL/ 6MOLF STING were input into gproﬁler database to determine 0.00000149 Factor: NF-kappaB; mo/f: NNNNKGGRAANTCCCN; match class: 2 transcrip/on factor enrichment in the dataset. 0.0000487 Factor: NF-kappaB; mo/f: NNNNKGGRAANTCCCN; match class: 1

0.00568 Factor: NF-kappaB; mo/f: NNNNKGGRAANTCCCN; match class: 0 Differences in ac/va/on of IRF and NF-kB family members appear to be the first principle differences conferred by the 0.0000855 Factor: c-Rel; mo/f: SGGRNTTTCC; match class: 4 alleles. 0.000506 Factor: c-Rel; mo/f: SGGRNTTTCC; match class: 3

0.000693 Factor: NF-kappaB; mo/f: GGGAMTTYCC; match class: 3

0.000000161 Factor: IRF; mo/f: RAAANTGAAAN; match class: 4

0.000000146 Factor: IRF; mo/f: RAAANTGAAAN; match class: 3

0.00734 Factor: IRF; mo/f: RAAANTGAAAN; match class: 1

0.000394 Factor: IRF; mo/f: RAAANTGAAAN; match class: 0

0.0000788 Factor: NF-kappaB; mo/f: NGGGGAMTTTCCNN; match class: 4

0.000209 Factor: NF-kappaB; mo/f: NGGGGAMTTTCCNN; match class: 3

0.00366 Factor: NF-kappaB; mo/f: NGGGGAMTTTCCNN; match class: 2

Figure 32: Comparison of B6 and MOLF STING dependent signaling in response to 2’3’cGAMP reveals significant decrease in IRF, NF-kB, and AP-1 family transcription factor activation. Peritoneal macrophages from B6-StingB6, and B6-StingMOLF mice were stimulated with or without 2’3’cGAMP for 4hrs. mRNA was submitted for RNA-seq in triplicate. Significantly upregulated genes in B6-StingB6 over B6-StingMOLF were input into gprofiler database to determine transcription factor enrichment in the dataset. List of significantly upregulated genes used to run analysis are contained in Appendix I. Source: biit.cs.ut.ee/gprofiler/

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A major ongoing hypothesis is that there is a major uncharacterized signaling node directly downstream of STING that is nullified in MOLF STING by disruption of a specific unknown protein-protein interaction. To investigate this, we stimulated peritoneal macrophages from C57BL/6B6 STING and C57BL/6MOLF STING littermates from 3 individual mice per group with 2’3’ cGAMP and then performed RNAseq on the samples. We then submitted our gene count data to our collaborators who specialize in “Genomic Analysis of Cell Signaling Systems”. A subset of this data is represented in (Figure 33). With some pathways MOLF STING confers near equally intense activation of the pathway (ie ERK signaling, Akt signaling). However, in some pathways analyzed MOLF STING confers negative regulation of the pathway compared to positive activation of the pathway with B6 STING (i.e. cAMP Main

Pathway, CREB Main pathway, and IP3 Main Pathway). This data set gives us clues on what signaling nodes may be different downstream of MOLF STING. A second signal from another signaling pathway may complement MOLF STING signaling to restore responsiveness to 2’3’cGAMP in MOLF/Ei macrophages, and StingMOLF

BMDMs stimulated with growth factor. For instance, ligation of a cellular growth receptor during 2’3’cGAMP stimulation may activate CREB, IP3, GSK3, and/or cAMP pathways to restore MOLF STING responsiveness. MOLF STING will be useful for determining a novel signaling node downstream of STING mediated by the N- terminus. This is evident in the 3-way comparison of StingB6, StingMOLF, and

MOLF/Ei peritoneal macrophages in Figure 33. Compared to StingB6 macrophages,

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some pathways that lack activation in StingMOLF, are activated in MOLF/Ei. These pathways include; GSK3, ILK, and IP3. An alternate gene product or signaling pathway may confer synergistic activation of these pathways to allow 2’3’cGAMP mediated IFNβ production on the MOLF/Ei background. That MOLF/Ei genetic element is the candidate that the next forward genetic screen will attempt to elucidate.

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Cellular Apoptosis Main Pathway B6 GSK3 Signaling Main Pathway STING STINGMOLF PTEN Main Pathway MOLF/Ei EGF Main Pathway

Ras Pathway (Cell-Cell Junctions)

PAK Pathway (Contractility Stress Fibres and Focal Adhesion)

TNF Signaling Pathway (Gene Expression and Cell Survival)

JAK-STAT Pathway (Akt Pathway)

Estrogen Main Pathway

Mitochondrial Apoptosis Main Pathway

MAPK Signaling Main Pathway

ILK Signaling Pathway (Migration Vasculogenesis)

ILK Signaling Pathway (EMT)

PAK Main Pathway

JAK-STAT Pathway (Gene Expression via STAT2)

ILK Signaling Main Pathway

cAMP Pathway (Myocardial Contraction)

Chemokine Main Pathway

PAK Pathway (Cell Survival)

PAK Pathway (Actin Organization)

Caspase Cascade Pathway (Activated Tissue Trans-glutaminase)

PAK Pathway (Lamelliopodia and Filopodia Outgrowth)

JAK-STAT Main Pathway

IP3 Main Pathway

TGF-Beta Main Pathway

CREB Main Pathway

Akt Signaling Main Pathway

ERK Signaling Main Pathway

cAMP Main Pathway

0 -50 50 Relative Pathway Activation100 150

Figure 33: Cell signaling pathway analysis of StingB6, StingMOLF, and MOLF/Ei macrophages stimulated 4hrs with 2’3’cGAMP. Peritoneal macrophages from StingB6, StingMOLF, and MOLF/Ei mice were stimulated with or without 2’3’cGAMP for 4hrs. mRNA was submitted for RNA-seq in triplicate. Transcripts submitted for pathway analysis by the “Group for Genomic Analysis of Cell Signaling Systems” Shemyakin-Ovchinnikov Institute. Data shown are select pathways differentially regulated between mouse strains. StingB6 and StingMOLF data is representative of N=3+ and MOLF data representative of N=2. Full dataset in appendix II 146

DISCUSSION

Previous work has shown the importance of STING as a signaling molecule that is central to the detection of many cytosolic nucleotide species. (Cai, Chiu, and

Chen 2014) STING signaling has been shown to be important in impeding viral and bacterial proliferation and dissemination. Because of STING’s importance in viral and bacterial immunity, many microbes have evolved mechanisms to subvert or modify STING signaling, thereby increasing host susceptibility to infection. (Konno and Barber 2014) There are many variant alleles of human STING that confer differential responses to DNA species derived from pathogens and the host. (Yi et al.

2013) Much has still to be learned regarding the effect of many of human STING variants on immunity, but it is probable that human STING variants evolved to overcome pathogen manipulation of host responses to permit infection.

Additionally, STING signaling has been found to be crucial downstream of the

DNA damage pathway, as overwhelming DNA damage can drive STING-dependent interferon from the presence of genomic DNA in the cytoplasm (Yu et al. 2015). This

STING-dependent DNA damage mediated interferon response has been found to be crucial for clearing irradiated tumors, (Deng et al. 2014) and for priming the immune response against tumor antigens. (Fu et al. 2015; Corrales et al. 2015; Woo et al. 2014) How STING allelic variations determine outcomes of DNA damage response and tumor clearance is unknown. This gives more importance to the

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investigation of how STING variations may have evolved, the mechanism of altered signaling responses, and the impact of these signaling responses on immunity.

Summary Findings

In our work, we describe a novel and unique murine allele of STING in the wild-derived mouse strain MOLF/Ei. MOLF STING was found to contain multiple

SNPs that conferred amino acid changes and an internal deletion of 6 amino acids.

Compared to C57Bl/6, MOLF/Ei macrophages are hyper-productive in IL-6 responses to cytosolic deoxynucleotides, yet defective in IFNβ production to pathogenic derived CDNs. Therefore, in MOLF/Ei macrophages, MOLF STING, much like the human H232 STING (Diner et al. 2013), distinguishes between endogenous and pathogenic CDNs to produce near wild-type IFNβ production to 2’3’cGAMP, but not c-di-AMP, c-di-GMP, 3’3’cGAMP, nor DMXAA (Conlon et al. 2013). However, when we isolate MOLF STING to the B6 background, MOLF STING dependent cytokine production becomes attenuated in response to all CDNs. MOLF mutations disrupt evolutionarily conserved sequences in mammalian STING. S53L and L47V-

A48G mutations confer the greatest defect in the ability of wild-type STING to induce the IFNβ promoter in a reporter assay, and L47V-A48G mutations appear to most affect the localization of STING during activation. Altogether, MOLF STING potentiates an alternate signaling response that confers an altered ISG profile in macrophages.

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In MOLF/Ei macrophages we observed a differential signaling response to infection with DNA viruses. Upon infection with viruses, MOLF macrophages produce greater pro-inflammatory IL-6 levels to infection, while they are severely attenuated in IFNβ production. mRNA profiling showed that transcripts of multiple other genes were alternatively expressed in MOLF macrophages. MOLF macrophages were hyper-producing IL-1 and IL-18 transcripts while displaying lower levels of ISGs such as viperin (rsad2) when compared to B6 macrophages infected with Listeria or HSV1. This showed that MOLF/Ei was responsive to cytosolic DNA species, yet was directed towards a more pro-inflammatory response rather than an IFN response to these pathogens.

Using an unbiased approach, we found STING as a strong candidate to be the causative agent of the lack of interferon responses to c-di-AMP and c-di-GMP. At the time of our primary screen, the endogenous CDN, 2’3’cGAMP had not yet been discovered. It was surprising to discover that MOLF/Ei produced IFNβ – the cGAS produced 2’3’cGAMP species. In addition to the IFNβ production to 2’3’cGAMP,

MOLF produced an even greater Il-6 production. Meanwhile, IRF3 phosphorylation in MOLF macrophages downstream of STING activation was relatively low.

Altogether, these data raise a few unanswered questions. First, a component of

HSV1 is DNA that should trigger cGAS dependent 2’3’cGAMP production. Why don’t we see appreciable HSV-induced IFNβ production in MOLF/Ei macrophages?

Second, MOLF STING amino acid changes are N-terminal and away from the C- terminal binding domain for STING dependent ligation of CDNs, so how does MOLF

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STING distinguish between CDNs? Last, MOLF/Ei produces IL-6 in response to all

CDNs except DMXAA, which infers that MOLF STING is responsive to these CDNs.

What is the molecular trigger to induce robust IFNβ production to 2’3’cGAMP exclusively?

STING dependent IL-6 production

We show that activation of MOLF macrophages with CDNs induces robust IL-

6 production. QTL mapping in the Poltorak lab, has revealed multiple loci conferring differential IL-6 production in macrophages of MOLF/Ei mice in response to TLR stimulation. The Why1 locus in MOLF/Ei, confers IL-6 hyper production to

TLR stimulation. This was found to be due to a lesion the gene Irak2c, which reduces its expression. Irak2c is a negative regulator of the TLR pro-inflammatory signaling pathway that reduces activation of MAPKs. Inheritance of the MOLF Why1 locus, results in: reduced levels of Irak2c transcripts, increased p38 activation, and increased levels of IL-6 cytokine. (Conner, Smirnova, and Poltorak 2009)

The significance of IRAK2 or p38 signaling downstream of STING has not been shown. It would be interesting to investigate if the Why1 locus plays a role.

However, preliminary mapping efforts of MOLF/Ei macrophages IL-6 hyper- responses downstream of STING show loci on chromosome 10 to confer the greatest positive effect on IL-6 production. The Why1 locus is on chromosome 6. Overall,

MOLF/Ei mice seem to have developed several mechanisms to promote IL-6 hyper- production. In the future, fine mapping of these loci can lead to better

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understanding of pro-inflammatory signaling downstream of STING. (Conner,

Smirnova, and Poltorak 2009, 2008)

Complementation

These questions are very interesting, yet can be complicated by the highly diversified MOLF genomic background. As MOLF Sting diverged from the common murine reference allele, it is likely that other genes co-evolved to complement changes in signaling networks. For example, when we isolated MOLF STING on the

B6 background, we lost 2’3’cGAMP IFNβ and Il-6 production, yet retained MOLF

STING ability to induce some IRF3 phosphorylation to 2’3’cGAMP activation. In contrast to StingMOLF mice, F1 (B6xMOLF) mice are responsive to 2’3’cGAMP.

Therefore, a gene or gene network on the MOLF background complements or synergizes with MOLF STING to produce IFNβ cytokine. Again, since there are so many divergences in the MOLF/Ei background, the best next course of action is an unbiased forward genetic screen to determine MOLF factors that confer 2’3’cGAMP induced IFNβ to MOLF STING. In our latest F2 panel, we generated intercrossed F1

StingMOLF (MOLF/Ei x B6 StingMOLF) mice, so that B6 and MOLF genes can independently assort on a MOLF STING homozygous background. The IFNβ production readouts in this panel varied from near undetectable levels, as in B6

StingMOLF macrophages, to IFNβ protein levels higher than B6 StingB6. This is further

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proof that genes on the MOLF/Ei are interacting with MOLF STING to produce a response, and can lead to elucidation of another pathway downstream of STING.

MOLF STING provides novel mutations in conserved regions that may highlight some important motifs in STING signaling. These mutations are predominantly in the N-terminus, a region that is necessary for signal transduction in a manner that is not fully understood. Recently many functional interactions with the STING N-terminus have been uncovered. Host proteins such as BTK, a tyrosine kinase (Lee et al. 2015); DDX41, a DNA sensor (Parvatiyar et al. 2012; Zhang et al.

2011; Lee et al. 2015); SCAP, an ER resident adapter (Chen et al. 2016); and AMFR, an ER resident E3 ligase (Wang et al. 2014), all interact directly with the N-terminal transmembrane domain of STING to facilitate STING activation and signaling in varying contexts. Various viral proteins bind the N-terminal transmembrane domain of STING to inhibit signaling. (Ma et al. 2015) The conserved sequences disrupted in MOLF STING may abolish one or more of these interactions.

Furthermore, the proposed accessory protein or pathway in MOLF/Ei that rescues the IFN-defect can lead to therapies that overcome viral inhibition of the N- terminus.

MOLF STING has 8 amino acid changes and a 6 amino acid deletion. The great number of evolution-pressured mutations allow for much to study, but also make mechanistic studies difficult. The primary reason for this being hard to study mechanistically is that it is hard to determine which mutations are functional – to mediate or disrupt protein-protein interactions, and which mutations are

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complementary – to retain structural integrity. Herein lies a major caveat of isolating single mutations of MOLF STING onto the B6 background. In some cases, a defect in function may occur by a single amino acid change, because the protein becomes misshapen or unstable. Alternatively, a mutation may only have phenotypic consequence with a concurrent distant mutation that acts in a concerted motion in a fully folded protein. With abundant mutations in MOLF STING, the permutation of pairing or associating mutations can be enormous, especially without an available structural model of the N-terminus as a guide.

The most interesting mutation found in MOLF STING was the S53L amino acid change in a residue that is highly conserved among species. This amino acid change alone in B6 STING blocked induction of the IFNβ promoter in a reporter assay, and an S53A residue in B6 STING-induced hyper-activation of the IFNβ promoter. Eukaryotic linear motif (ELM) analysis of the conserved region proposed that these changes disrupted activation of a 14-3-3 binding domain. 14-3-3's are a family of adaptor proteins that facilitate the interaction of proteins for signaling and trafficking. Recently, RIG-I and TLR-3, two PRRs upstream of IRF3-dependent IFN response, have been shown to depend on 14-3-3ε (Liu, Loo, et al. 2012)and 14-3-3z

(Funami et al. 2016) respectively for specific steps of translocation and activation. It is possible the S53 is a functional 14-3-3 site in STING necessary for a specific stage of activation.

It is surprising that L47V-A48G mutations produced a large defect in B6

STING. ELM indicates that L47V-A48G mutations can disrupt a LC3 interacting

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domain or a cyclin binding site. However, according to most TM mapping programs,

L47V-A48G seems to be buried in the transmembrane. Therefore, it may be that

L47V-A48G are complementary mutations to restore structural integrity. However, in recent reports the TM regions of AMFR and SCAP were found to interact with the

TM regions STING in the ER to facilitate signaling. It could be that L47V-A48G perturb these specific interactions in the ER membrane space. Additionally, a calcium flux is required to precede STING translocation. The cause of this calcium flux is unknown. One or more of these intra-membrane interactions can facilitate calcium flux.

The function of the 116-121 deletion in MOLF remains a mystery. The deletion is in the area where DDX41, a DNA sensor, binds STING. DDX41 binds DNA, c-di-AMP, or c-di-GMP to induce cooperative activation of STING. MOLF STING deletion may abrogate interactions with this DNA sensor, and possibly others.

Going forward immunoprecipitation studies with MOLF STING can answer these questions.

STING signalosomes

Upon activation, STING undergoes a complex program of activation. It interacts with various factors in a concerted reaction that propels it to a perinuclear location, then targets STING for degradation. In our imaging studies we observed that MOLF STING was largely retained in the ER after activation with CDNs. This was repeated with N-terminally and C-terminally tagged STING protein. In follow- 154

up studies, we also found that L47V-A48G mutations affected STING trafficking after stimulation. The imaging studies were done in the common background of B6

STING-/- MEFs. In the B6 background we do not expect much signaling from MOLF

STING. A good experiment would be to knockdown endogenous STING in MOLF/Ei

MEFs and add-back B6 or MOLF STING. One possibility would be that B6 would lose ability to translocate, while MOLF STING gains ability. This would indicate that complementary mutations in the MOLF background genes allow adapter binding of

MOLF STING, but excludes B6 STING. More likely, MOLF STING does not translocate in MOLF MEFs, but sufficient TBK1 and IRF3 is sustainably recruited to the ER to facilitate signaling.

The perinuclear organelle or signalosome in STING signaling is not well characterized. A close likeness to the STING perinuclear signalosome is the TLR-

TRIF-IRF3 perinuclear singalosome. (CHIANG et al. 2011; Fitzgerald et al. 2003)

TLR4 is internalized in a CD14 dependent manner to switch from MyD88 dependent pro-inflammatory signaling to TRIF dependent IFN signaling (Kurt-Jones et al. 2000;

Jiang et al. 2005). This signaling is dependent on the PLCγ2-IP3-Ca2+ flux downstream of CD14. In PLCγ2 KO macrophages TLR4 internalization blocked, and

IFNβ is severely attenuated. (CHIANG et al. 2011) However, attenuated IRF3 phosphorylation is still present in PLCγ2 KO. (CHIANG et al. 2011) This indicates that the signalosome may be important for other signaling functions in addition to optimal IRF3 phosphorylation. Furthermore, this can explain what we observe in

155

B6 StingMOLF macrophages where MOLF STING shows partially reduced IRF3 phosphorylation, yet little induction of IFNβ mRNA and protein.

Other TLR4 studies describe the perinuclear localization compartment to be

Rab11a positive vesicles of the endocytic recycling compartment (ERC). (Kagan

2010; Husebye et al. 2010) Delivery of the TLR4 and cargo not only allows access of the TLR4 –TRIF to enriched signaling complexes, but also delivers cargo, such as pathogen derived PAMPs, access to the MHCII loading compartments contained within. (Kagan 2010; Husebye et al. 2010) It would be interesting to see if STING translocates to the very same perinuclear bodies. Then there could be TLR4-STING induced synergy to promote autophagy, IFN signaling, cross presentation of antigen, and induced antigen presentation. This would be very impactful to the study of

STING dependent immunity, and it would be quite remarkable if MOLF STING were defective in this perinuclear localization, except when a sufficient second signal is provided.

Pathway analysis.

MOLF STING confers differential activation of pathways on the MOLF and B6 background. MOLF/Ei macrophages produce IFNβ approaching B6 levels with

2’3’cGAMP activation, but B6 StingMOLF macrophages are defective in producing

IFNβ. To determine signaling nodes downstream of STING that can confer differential outcomes in these states, RNA-seq was done of StingB6, StingMOLF, and

MOLF macrophages stimulated with or without 2’3’cGAMP for 4hrs. The transcripts 156

were submitted for pathway analysis. Notable pathways, which were activated in

MOLF/Ei and B6 StingB6, but not B6 StingMOLF, were the GSK3 pathway, IP3 pathway,

CREB, and cAMP main pathway. Interestingly, IP3 pathway activation leads to the calcium flux that is necessary for STING activation and translocation. The lack of IP3 pathway activation in B6 StingMOLF could be a feature of MOLF STING in ability to induce a PLCγ2 like pathway in the B6 background. CREB and cAMP pathway activation is likely in parallel and necessary to synergize with CBP/p300 activation that is necessary for activation of the IFNβ enhanceosome. Lack of activation of these pathways in B6 StingMOLF is indicative of poor signaling downstream of MOLF

STING on the B6 background. Poor GSK3 pathway activation in B6 StingMOLF may be a major pathway that defines the IFNβ production defect. GSK3β regulates and mediates IRF3 phosphorylation. (Lei et al. 2010) GSK3β also phosphorylates CBP to activate the CBP/p300 dependent IFNβ enhanceosome. (Wang et al. 2010) Necessity for GSK3 pathway activation in STING has not been reported. This can be a fruitful avenue to study.

CDKs and GSK3 family members are closely related in the kinome(Rayasam et al. 2009). CDK and GSK3 member share up to 86% homology in their ATP- binding sites, where many inhibitors bind(Rayasam et al. 2009). Consequently, many CDK inhibitors inhibit GSK3 family members and vice-versa. In past reports, we have shown that some CDK inhibitors block STING dependent signaling, while

BMDM growth and differentiation media - L1 supernatant - synergized with IFNβ

157

responses. GSK3β could be a potential target of these signals or inhibitors. GSK3β could be an additional signal necessary for MOLF STING activation.

B6 StingMOLF in vivo studies

Components of the cytosolic DNA sensing pathway, including STING, vary in expression in a tissue and cell type specific manner. In this thesis, STING activity in the macrophage and fibroblast cell compartments is reported. STING signaling compartments and cofactors may vary in other cells. STING activates IFNβ response downstream of the DNA damage response. DNAse2-/- mice cause embryonic lethality due to constitutive IFN production as IFNAR or STING knockout rescue lethality. (Ahn et al. 2012) To determine if MOLF STING defects in signaling could rescue lethality of DNAse2-/-, we crossed B6 StingB6/MOLF mice with DNAse-/-

+IFNAR-/- (DKO) mice or STING-/- +DNAse-/- +IFNAR-/- (TKO) mice to generate hemizygous F1s. We then intercrossed the STINGB/M +DNAse+/- +IFNAR+/- to see if

STINGMOLF/MOLF +DNAse-/- +IFNAR+/+ are born, and thus MOLF STING rescues the phenotype. Thus, far after a few generations we have not seen viable

STINGMOLF/MOLF +DNAse-/- mice born. This may mean that in vivo MOLF STING may be sufficient to detect host DNA. The IFNβ production may be lower, but is still sufficient to produce autoimmunity in the DNAse2-/- model. Going forward, we will check embryos before birth to check for presence of STINGMOLF/MOLF +DNAse-/- embryonic lethality.

158

Conclusion

We report here positional cloning of a novel allele of Sting that confers low type I IFN production due to mutations at the N-terminus of STING. While extensive analysis of the C-terminal domain of STING has provided critical insights into our understanding of its role in DNA responses, the N-terminal domain remains largely uncharacterized and has not yet even been crystallized. This is the first natural

STING mutant identified in mice that displays functional polymorphisms near the N- terminus. Our studies reveal the importance of the N-terminus of STING in mediating STING-signaling and STING mediated type I interferon production.

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APPENDIX I – Genes significantly differentially regulated between STINGB6 and

STINGMOLF peritoneal macrophages activated with 2’3’cGAMP

Geneset entered into gprofiler (Figure 32). Output of significantly differentially expressed genes (p-value<0.01) after Cuffdiff analysis of 2’3’cGAMP stimulated STINGB6 and STINGMOLF macrophages from three biological replicates.

Gene name Gene name (Mouse) (HUMAN) Gene description Cxcl9 CXCL9 chemokine (C-X-C motif) ligand 9 [Source:MGI Symbol;Acc:MGI:1352449] Ifnb1 IFNB1 interferon beta 1, fibroblast [Source:MGI Symbol;Acc:MGI:107657]

Socs3 SOCS3 suppressor of cytokine signaling 3 [Source:MGI Symbol;Acc:MGI:1201791]

Serpina3f,Serpina3g N/A N/A Iigp1 IIGP1 interferon inducible GTPase 1 [Source:MGI Symbol;Acc:MGI:1926259]

Gnpda1 GNPDA1 glucosamine-6-phosphate deaminase 1 [Source:MGI Symbol;Acc:MGI:1347054]

Il27 IL27 interleukin 27 [Source:MGI Symbol;Acc:MGI:2384409] AW112010 AW112010 expressed sequence AW112010 [Source:MGI Symbol;Acc:MGI:2147706]

Vaultrc5 VAULTRC5 vault RNA component 5 [Source:MGI Symbol;Acc:MGI:2673990] family with sequence similarity 26, member F [Source:MGI Fam26f FAM26F Symbol;Acc:MGI:2443082] Tmem173 TMEM173 transmembrane protein 173 [Source:MGI Symbol;Acc:MGI:1919762]

Gypc GYPC glycophorin C [Source:MGI Symbol;Acc:MGI:1098566] Fas FAS Fas (TNF receptor superfamily member 6) [Source:MGI Symbol;Acc:MGI:95484] v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) Maff MAFF [Source:MGI Symbol;Acc:MGI:96910] Cd40 CD40 CD40 antigen [Source:MGI Symbol;Acc:MGI:88336]

Cpa3 CPA3 carboxypeptidase A3, mast cell [Source:MGI Symbol;Acc:MGI:88479] basic leucine zipper transcription factor, ATF-like 2 [Source:MGI Batf2 BATF2 Symbol;Acc:MGI:1921731] Vcan VCAN versican [Source:MGI Symbol;Acc:MGI:102889]

Rnd3 RND3 Rho family GTPase 3 [Source:MGI Symbol;Acc:MGI:1921444] Cd69 CD69 CD69 antigen [Source:MGI Symbol;Acc:MGI:88343]

Hcar2 HCAR2 hydroxycarboxylic acid receptor 2 [Source:MGI Symbol;Acc:MGI:1933383]

Tmem171 TMEM171 transmembrane protein 171 [Source:MGI Symbol;Acc:MGI:2685751] Ier3 IER3 immediate early response 3 [Source:MGI Symbol;Acc:MGI:104814]

Socs1 SOCS1 suppressor of cytokine signaling 1 [Source:MGI Symbol;Acc:MGI:1354910] UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 [Source:MGI B4galt6 B4GALT6 Symbol;Acc:MGI:1928380] PHD finger protein 11B [Source:MGI Symbol;Acc:MGI:3645789]/PHD finger Phf11b PHF11A/PHF11B protein 11A [Source:MGI Symbol;Acc:MGI:1918441] Irf8 IRF8 interferon regulatory factor 8 [Source:MGI Symbol;Acc:MGI:96395] F630111L10Rik,P2ry14 N/A N/A 160

Gbp5 GBP5 guanylate binding protein 5 [Source:MGI Symbol;Acc:MGI:2429943]

Zyx ZYX zyxin [Source:MGI Symbol;Acc:MGI:103072] endoplasmic reticulum chaperone SIL1 homolog (S. cerevisiae) [Source:MGI Sil1 SIL1 Symbol;Acc:MGI:1932040] poly (ADP-ribose) polymerase family, member 8 [Source:MGI Parp8 PARP8 Symbol;Acc:MGI:1098713] ral guanine nucleotide dissociation stimulator [Source:MGI Ralgds RALGDS Symbol;Acc:MGI:107485] Isg20 ISG20 interferon-stimulated protein [Source:MGI Symbol;Acc:MGI:1928895]

Rgcc RGCC regulator of cell cycle [Source:MGI Symbol;Acc:MGI:1913464] guanine nucleotide binding protein (G protein), gamma 11 [Source:MGI Gng11 GNG11 Symbol;Acc:MGI:1913316] Mmp13 MMP13 matrix metallopeptidase 13 [Source:MGI Symbol;Acc:MGI:1340026]

Slamf8 SLAMF8 SLAM family member 8 [Source:MGI Symbol;Acc:MGI:1921998]

Gbp9 GBP9 guanylate-binding protein 9 [Source:MGI Symbol;Acc:MGI:3605620] Il18bp IL18BP interleukin 18 binding protein [Source:MGI Symbol;Acc:MGI:1333800]

Csrnp1 CSRNP1 cysteine-serine-rich nuclear protein 1 [Source:MGI Symbol;Acc:MGI:2387989]

Cxcl10 CXCL10 chemokine (C-X-C motif) ligand 10 [Source:MGI Symbol;Acc:MGI:1352450] Sdc4 SDC4 syndecan 4 [Source:MGI Symbol;Acc:MGI:1349164]

Stx11 STX11 syntaxin 11 [Source:MGI Symbol;Acc:MGI:1921982]

Icam1 ICAM1 intercellular adhesion molecule 1 [Source:MGI Symbol;Acc:MGI:96392] Nlrc5 NLRC5 NLR family, CARD domain containing 5 [Source:MGI Symbol;Acc:MGI:3612191] apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 [Source:MGI Apobec3 APOBEC3 Symbol;Acc:MGI:1933111] Tnf TNF tumor necrosis factor [Source:MGI Symbol;Acc:MGI:104798]

Ifi205 IFI205 interferon activated gene 205 [Source:MGI Symbol;Acc:MGI:101847]

Nfil3 NFIL3 nuclear factor, interleukin 3, regulated [Source:MGI Symbol;Acc:MGI:109495] Oasl1 OASL1 2'-5' oligoadenylate synthetase-like 1 [Source:MGI Symbol;Acc:MGI:2180849] interferon induced with helicase C domain 1 [Source:MGI Ifih1 IFIH1 Symbol;Acc:MGI:1918836] Otud1 OTUD1 OTU domain containing 1 [Source:MGI Symbol;Acc:MGI:1918448] T cell activation Rho GTPase activating protein [Source:MGI Tagap TAGAP Symbol;Acc:MGI:3615484] solute carrier family 28 (sodium-coupled nucleoside transporter), member 2 Slc28a2 SLC28A2 [Source:MGI Symbol;Acc:MGI:1913105] Mefv MEFV Mediterranean fever [Source:MGI Symbol;Acc:MGI:1859396]

Il1a IL1A interleukin 1 alpha [Source:MGI Symbol;Acc:MGI:96542] discoidin, CUB and LCCL domain containing 2 [Source:MGI Dcbld2 DCBLD2 Symbol;Acc:MGI:1920629] Ccl4 CCL4 chemokine (C-C motif) ligand 4 [Source:MGI Symbol;Acc:MGI:98261] N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 2 [Source:MGI Ndst2 NDST2 Symbol;Acc:MGI:97040] Gbp2 GBP2 guanylate binding protein 2 [Source:MGI Symbol;Acc:MGI:102772]

G530011O06Rik G530011O06RIK RIKEN cDNA G530011O06 gene [Source:MGI Symbol;Acc:MGI:3603513] membrane-spanning 4-domains, subfamily A, member 4C [Source:MGI Ms4a4c MS4A4C Symbol;Acc:MGI:1927656]

161

Spryd7 SPRYD7 SPRY domain containing 7 [Source:MGI Symbol;Acc:MGI:1913924]

Ccl12 CCL12 chemokine (C-C motif) ligand 12 [Source:MGI Symbol;Acc:MGI:108224] Irg1 IRG1 immunoresponsive gene 1 [Source:MGI Symbol;Acc:MGI:103206]

Cd274 CD274 CD274 antigen [Source:MGI Symbol;Acc:MGI:1926446] protein phosphatase 1, regulatory (inhibitor) subunit 15A [Source:MGI Ppp1r15a PPP1R15A Symbol;Acc:MGI:1927072] Gpr141 GPR141 G protein-coupled receptor 141 [Source:MGI Symbol;Acc:MGI:2672983]

AA467197,Mir147 N/A N/A

Il15 IL15 interleukin 15 [Source:MGI Symbol;Acc:MGI:103014]

Gm6377 GM6377 predicted gene 6377 [Source:MGI Symbol;Acc:MGI:3647255]

Ccl5 CCL5 chemokine (C-C motif) ligand 5 [Source:MGI Symbol;Acc:MGI:98262] Ifi47,Olfr56 N/A N/A

Irf1 IRF1 interferon regulatory factor 1 [Source:MGI Symbol;Acc:MGI:96590] furin (paired basic amino acid cleaving enzyme) [Source:MGI Furin FURIN Symbol;Acc:MGI:97513] B cell leukemia/lymphoma 2 related protein A1d [Source:MGI Bcl2a1d BCL2A1D Symbol;Acc:MGI:1278325] Il1rn IL1RN interleukin 1 receptor antagonist [Source:MGI Symbol;Acc:MGI:96547]

Oas1g OAS1G 2'-5' oligoadenylate synthetase 1G [Source:MGI Symbol;Acc:MGI:97429]

5730508B09Rik 5730508B09RIK RIKEN cDNA 5730508B09 gene [Source:MGI Symbol;Acc:MGI:1917867] Pcgf5 PCGF5 polycomb group ring finger 5 [Source:MGI Symbol;Acc:MGI:1923505]

Ccl3 CCL3 chemokine (C-C motif) ligand 3 [Source:MGI Symbol;Acc:MGI:98260] SLP adaptor and CSK interacting membrane protein [Source:MGI Scimp SCIMP Symbol;Acc:MGI:3610314] CASP8 and FADD-like apoptosis regulator [Source:MGI Cflar GM9845/CFLAR Symbol;Acc:MGI:1336166]/predicted pseudogene 9845 [Source:MGI Symbol;Acc:MGI:3704215] V-set and immunoglobulin domain containing 4 [Source:MGI Vsig4 VSIG4 Symbol;Acc:MGI:2679720] Gbp3 GBP3 guanylate binding protein 3 [Source:MGI Symbol;Acc:MGI:1926263] Il15ra IL15RA interleukin 15 receptor, alpha chain [Source:MGI Symbol;Acc:MGI:104644] tumor necrosis factor, alpha-induced protein 3 [Source:MGI Tnfaip3 TNFAIP3 Symbol;Acc:MGI:1196377] solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 Slc7a2 SLC7A2 [Source:MGI Symbol;Acc:MGI:99828] Adck1 ADCK1 aarF domain containing kinase 1 [Source:MGI Symbol;Acc:MGI:1919363]

Fgl2 FGL2 fibrinogen-like protein 2 [Source:MGI Symbol;Acc:MGI:103266] transmembrane and coiled-coil domains 3 [Source:MGI Tmco3 TMCO3 Symbol;Acc:MGI:2444946] Zfp36 ZFP36 zinc finger protein 36 [Source:MGI Symbol;Acc:MGI:99180] katanin p60 (ATPase-containing) subunit A1 [Source:MGI Katna1 KATNA1 Symbol;Acc:MGI:1344353] Pim1 PIM1 proviral integration site 1 [Source:MGI Symbol;Acc:MGI:97584] caspase 4, apoptosis-related cysteine peptidase [Source:MGI Casp4 CASP4 Symbol;Acc:MGI:107700] mitochondrial translational initiation factor 3 [Source:MGI mtif3 MTIF3 Symbol;Acc:MGI:1923616]

162

Il3ra IL3RA interleukin 3 receptor, alpha chain [Source:MGI Symbol;Acc:MGI:96553]

Plau PLAU plasminogen activator, urokinase [Source:MGI Symbol;Acc:MGI:97611] cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial [Source:MGI Cmpk2 CMPK2 Symbol;Acc:MGI:99830] Gca GCA grancalcin [Source:MGI Symbol;Acc:MGI:1918521]

Pianp PIANP PILR alpha associated neural protein [Source:MGI Symbol;Acc:MGI:2441908]

Oasl2 OASL2 2'-5' oligoadenylate synthetase-like 2 [Source:MGI Symbol;Acc:MGI:1344390]

Ccl7 CCL7 chemokine (C-C motif) ligand 7 [Source:MGI Symbol;Acc:MGI:99512]

Reep5 REEP5 receptor accessory protein 5 [Source:MGI Symbol;Acc:MGI:1270152]

Nt5c3 NT5C3 5'-nucleotidase, cytosolic III [Source:MGI Symbol;Acc:MGI:1927186]

Brd2 BRD2 bromodomain containing 2 [Source:MGI Symbol;Acc:MGI:99495] dehydrogenase/reductase (SDR family) member 9 [Source:MGI Dhrs9 DHRS9 Symbol;Acc:MGI:2442798] Gbp6 GBP6 guanylate binding protein 6 [Source:MGI Symbol;Acc:MGI:2140937] Ccrl2 CCRL2 chemokine (C-C motif) receptor-like 2 [Source:MGI Symbol;Acc:MGI:1920904]

Cd86 CD86 CD86 antigen [Source:MGI Symbol;Acc:MGI:101773]

Nrp2 NRP2 neuropilin 2 [Source:MGI Symbol;Acc:MGI:1100492] transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) [Source:MGI Tap1 TAP1 Symbol;Acc:MGI:98483] interferon-induced protein with tetratricopeptide repeats 2 [Source:MGI Ifit2 IFIT2 Symbol;Acc:MGI:99449] Tlr6 TLR6 toll-like receptor 6 [Source:MGI Symbol;Acc:MGI:1341296]

Rbm22 RBM22 RNA binding motif protein 22 [Source:MGI Symbol;Acc:MGI:1914060] nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, Nfkbie NFKBIE epsilon [Source:MGI Symbol;Acc:MGI:1194908] H2-K2 H2-K2 histocompatibility 2, K region locus 2 [Source:MGI Symbol;Acc:MGI:95906]

Lcp2 LCP2 lymphocyte cytosolic protein 2 [Source:MGI Symbol;Acc:MGI:1321402] Mov10 MOV10 Moloney leukemia virus 10 [Source:MGI Symbol;Acc:MGI:97054]

Ifi203 IFI203 interferon activated gene 203 [Source:MGI Symbol;Acc:MGI:96428] myeloid nuclear differentiation antigen like [Source:MGI Mndal MNDAL Symbol;Acc:MGI:3780953] Gyk GYK glycerol kinase [Source:MGI Symbol;Acc:MGI:106594]

Fbxw17 FBXW17 F-box and WD-40 domain protein 17 [Source:MGI Symbol;Acc:MGI:1923584]

Prkx PRKX protein kinase, X-linked [Source:MGI Symbol;Acc:MGI:1309999]

Cers6 CERS6 ceramide synthase 6 [Source:MGI Symbol;Acc:MGI:2442564]

Aff1 AFF1 AF4/FMR2 family, member 1 [Source:MGI Symbol;Acc:MGI:1100819] v-abl Abelson murine leukemia viral oncogene 2 (arg, Abelson-related gene) Abl2 ABL2 [Source:MGI Symbol;Acc:MGI:87860] SLIT-ROBO Rho GTPase activating protein 2 [Source:MGI Srgap2 SRGAP2 Symbol;Acc:MGI:109605] tumor necrosis factor, alpha-induced protein 2 [Source:MGI Tnfaip2 TNFAIP2 Symbol;Acc:MGI:104960] Snx10 SNX10 sorting nexin 10 [Source:MGI Symbol;Acc:MGI:1919232]

Etv6 ETV6 ets variant 6 [Source:MGI Symbol;Acc:MGI:109336]

Crlf3 CRLF3 cytokine receptor-like factor 3 [Source:MGI Symbol;Acc:MGI:1860086]

163

Anxa7 ANXA7 annexin A7 [Source:MGI Symbol;Acc:MGI:88031]

Cxcl2 CXCL2 chemokine (C-X-C motif) ligand 2 [Source:MGI Symbol;Acc:MGI:1340094] Pydc4 PYDC4 pyrin domain containing 4 [Source:MGI Symbol;Acc:MGI:3695276]

Ddhd1,Mir5131 N/A N/A dishevelled associated activator of morphogenesis 1 [Source:MGI Daam1 DAAM1 Symbol;Acc:MGI:1914596] microtubule-associated protein, RP/EB family, member 2 [Source:MGI Mapre2 MAPRE2 Symbol;Acc:MGI:106271] Dennd1b DENND1B DENN/MADD domain containing 1B [Source:MGI Symbol;Acc:MGI:2447812]

Mlkl MLKL mixed lineage kinase domain-like [Source:MGI Symbol;Acc:MGI:1921818]

Herc6 HERC6 hect domain and RLD 6 [Source:MGI Symbol;Acc:MGI:1914388]

Cd5l CD5L CD5 antigen-like [Source:MGI Symbol;Acc:MGI:1334419] AT rich interactive domain 5A (MRF1-like) [Source:MGI Arid5a ARID5A Symbol;Acc:MGI:2443039] G-protein signalling modulator 2 (AGS3-like, C. elegans) [Source:MGI Gpsm2 GPSM2 Symbol;Acc:MGI:1923373] Dr1 DR1 down-regulator of transcription 1 [Source:MGI Symbol;Acc:MGI:1100515]

Zbp1 ZBP1 Z-DNA binding protein 1 [Source:MGI Symbol;Acc:MGI:1927449] Gbp7 GBP7 guanylate binding protein 7 [Source:MGI Symbol;Acc:MGI:2444421]

Pyhin1 PYHIN1 pyrin and HIN domain family, member 1 [Source:MGI Symbol;Acc:MGI:2138243] leucine-rich repeats and calponin homology (CH) domain containing 1 Lrch1 LRCH1 [Source:MGI Symbol;Acc:MGI:2443390] chloride intracellular channel 4 (mitochondrial) [Source:MGI Clic4 CLIC4 Symbol;Acc:MGI:1352754] Samhd1 SAMHD1 SAM domain and HD domain, 1 [Source:MGI Symbol;Acc:MGI:1927468]

Rap2c RAP2C RAP2C, member of RAS oncogene family [Source:MGI Symbol;Acc:MGI:1919315]

Trim30d TRIM30D tripartite motif-containing 30D [Source:MGI Symbol;Acc:MGI:3035181] Rbl1 RBL1 retinoblastoma-like 1 (p107) [Source:MGI Symbol;Acc:MGI:103300] DNA-damage regulated autophagy modulator 1 [Source:MGI Dram1 DRAM1 Symbol;Acc:MGI:1918962] transformer 2 alpha homolog (Drosophila) [Source:MGI Tra2a TRA2A Symbol;Acc:MGI:1933972] Rhoh RHOH ras homolog gene family, member H [Source:MGI Symbol;Acc:MGI:1921984]

Gm14446 GM14446 predicted gene 14446 [Source:MGI Symbol;Acc:MGI:3650685] UDP-glucose ceramide glucosyltransferase [Source:MGI Ugcg UGCG Symbol;Acc:MGI:1332243] Kdr KDR kinase insert domain protein receptor [Source:MGI Symbol;Acc:MGI:96683]

Aim2 AIM2 absent in melanoma 2 [Source:MGI Symbol;Acc:MGI:2686159]

Mocos MOCOS molybdenum cofactor sulfurase [Source:MGI Symbol;Acc:MGI:1915841]

Phf6 PHF6 PHD finger protein 6 [Source:MGI Symbol;Acc:MGI:1918248]

Max MAX Max protein [Source:MGI Symbol;Acc:MGI:96921]

4933426M11Rik 4933426M11RIK RIKEN cDNA 4933426M11 gene [Source:MGI Symbol;Acc:MGI:2444661] Ccl9 CCL9 chemokine (C-C motif) ligand 9 [Source:MGI Symbol;Acc:MGI:104533] v-rel reticuloendotheliosis viral oncogene homolog A (avian) [Source:MGI Rela RELA Symbol;Acc:MGI:103290] Fndc3a FNDC3A fibronectin type III domain containing 3A [Source:MGI 164

Symbol;Acc:MGI:1196463]

Gch1 GCH1 GTP cyclohydrolase 1 [Source:MGI Symbol;Acc:MGI:95675] Setdb2 SETDB2 SET domain, bifurcated 2 [Source:MGI Symbol;Acc:MGI:2685139] tumor necrosis factor (ligand) superfamily, member 10 [Source:MGI Tnfsf10 TNFSF10 Symbol;Acc:MGI:107414] Aim1 AIM1 absent in melanoma 1 [Source:MGI Symbol;Acc:MGI:109544]

Tlr3 TLR3 toll-like receptor 3 [Source:MGI Symbol;Acc:MGI:2156367]

Slc35a5 SLC35A5 solute carrier family 35, member A5 [Source:MGI Symbol;Acc:MGI:1921352] laccase (multicopper oxidoreductase) domain containing 1 [Source:MGI Lacc1 LACC1 Symbol;Acc:MGI:2445077] C3 C3 complement component 3 [Source:MGI Symbol;Acc:MGI:88227]

Ccng2 CCNG2 cyclin G2 [Source:MGI Symbol;Acc:MGI:1095734]

Endod1 ENDOD1 endonuclease domain containing 1 [Source:MGI Symbol;Acc:MGI:1919196] ATP-binding cassette, sub-family B (MDR/TAP), member 1B [Source:MGI Abcb1b ABCB1B Symbol;Acc:MGI:97568] ubiquitin-like modifier activating enzyme 7 [Source:MGI Uba7 UBA7 Symbol;Acc:MGI:1349462] La ribonucleoprotein domain family, member 1 [Source:MGI Larp1 LARP1 Symbol;Acc:MGI:1890165] v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K (avian) Mafk MAFK [Source:MGI Symbol;Acc:MGI:99951] Il13ra1 IL13RA1 interleukin 13 receptor, alpha 1 [Source:MGI Symbol;Acc:MGI:105052]

Hk2 HK2 hexokinase 2 [Source:MGI Symbol;Acc:MGI:1315197]

Rnf14 RNF14 ring finger protein 14 [Source:MGI Symbol;Acc:MGI:1929668] Mtus1 MTUS1 mitochondrial tumor suppressor 1 [Source:MGI Symbol;Acc:MGI:2142572]

Plaur PLAUR plasminogen activator, urokinase receptor [Source:MGI Symbol;Acc:MGI:97612]

Ankrd17 ANKRD17 ankyrin repeat domain 17 [Source:MGI Symbol;Acc:MGI:1932101] nucleotide-binding oligomerization domain containing 1 [Source:MGI Nod1 NOD1 Symbol;Acc:MGI:1341839] AT rich interactive domain 5B (MRF1-like) [Source:MGI Arid5b ARID5B Symbol;Acc:MGI:2175912] protein phosphatase 1, regulatory (inhibitor) subunit 15b [Source:MGI Ppp1r15b PPP1R15B Symbol;Acc:MGI:2444211] Adap2 ADAP2 ArfGAP with dual PH domains 2 [Source:MGI Symbol;Acc:MGI:2663075]

Gm12250 GM12250 predicted gene 12250 [Source:MGI Symbol;Acc:MGI:3649299]

Slamf7 SLAMF7 SLAM family member 7 [Source:MGI Symbol;Acc:MGI:1922595]

Oas3 OAS3 2'-5' oligoadenylate synthetase 3 [Source:MGI Symbol;Acc:MGI:2180850]

Junb JUNB jun B proto-oncogene [Source:MGI Symbol;Acc:MGI:96647] immunity-related GTPase family M member 1 [Source:MGI Irgm1 IRGM1 Symbol;Acc:MGI:107567] Ttc39b TTC39B tetratricopeptide repeat domain 39B [Source:MGI Symbol;Acc:MGI:1917113]

Ccl2 CCL2 chemokine (C-C motif) ligand 2 [Source:MGI Symbol;Acc:MGI:98259] transmembrane 4 L six family member 19 [Source:MGI Tm4sf19 TM4SF19 Symbol;Acc:MGI:3645933] cyclin-dependent kinase inhibitor 1A (P21) [Source:MGI Cdkn1a CDKN1A Symbol;Acc:MGI:104556] predicted gene 6768 [Source:MGI Symbol;Acc:MGI:3648259]/nuclear receptor Ncoa4 GM6768/NCOA4 coactivator 4 [Source:MGI Symbol;Acc:MGI:1350932]

165

Sulf2 SULF2 sulfatase 2 [Source:MGI Symbol;Acc:MGI:1919293]

Mrpl39 MRPL39 mitochondrial ribosomal protein L39 [Source:MGI Symbol;Acc:MGI:1351620] interferon-induced protein with tetratricopeptide repeats 1 [Source:MGI Ifit1 IFIT1 Symbol;Acc:MGI:99450] Igsf8 IGSF8 immunoglobulin superfamily, member 8 [Source:MGI Symbol;Acc:MGI:2154090] oxidized low density lipoprotein (lectin-like) receptor 1 [Source:MGI Olr1 OLR1 Symbol;Acc:MGI:1261434] Nampt NAMPT nicotinamide phosphoribosyltransferase [Source:MGI Symbol;Acc:MGI:1929865]

Tpst1 TPST1 protein-tyrosine sulfotransferase 1 [Source:MGI Symbol;Acc:MGI:1298231] Rap guanine nucleotide exchange factor (GEF) 2 [Source:MGI Rapgef2 RAPGEF2 Symbol;Acc:MGI:2659071] Apaf1 APAF1 apoptotic peptidase activating factor 1 [Source:MGI Symbol;Acc:MGI:1306796] sprouty protein with EVH-1 domain 1, related sequence [Source:MGI Spred1 SPRED1 Symbol;Acc:MGI:2150016] Rhbdf2 RHBDF2 rhomboid 5 homolog 2 (Drosophila) [Source:MGI Symbol;Acc:MGI:2442473] Trim30a TRIM30A tripartite motif-containing 30A [Source:MGI Symbol;Acc:MGI:98178]

Crlf2 CRLF2 cytokine receptor-like factor 2 [Source:MGI Symbol;Acc:MGI:1889506] piezo-type mechanosensitive ion channel component 1 [Source:MGI Piezo1 PIEZO1 Symbol;Acc:MGI:3603204] MX dynamin-like GTPase 2 [Source:MGI Symbol;Acc:MGI:97244]/MX dynamin- Mx1 MX2/MX1 like GTPase 1 [Source:MGI Symbol;Acc:MGI:97243] transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) [Source:MGI Tap2 TAP2 Symbol;Acc:MGI:98484] choline/ethanolaminephosphotransferase 1 [Source:MGI Cept1 CEPT1 Symbol;Acc:MGI:2139793] Cd83 CD83 CD83 antigen [Source:MGI Symbol;Acc:MGI:1328316]

Stx3 STX3 syntaxin 3 [Source:MGI Symbol;Acc:MGI:103077] TCDD-inducible poly(ADP-ribose) polymerase [Source:MGI Tiparp TIPARP Symbol;Acc:MGI:2159210] interferon-induced protein with tetratricopeptide repeats 3 [Source:MGI Ifit3 IFIT3 Symbol;Acc:MGI:1101055] Epsti1 EPSTI1 epithelial stromal interaction 1 (breast) [Source:MGI Symbol;Acc:MGI:1915168]

Rab12 RAB12 RAB12, member RAS oncogene family [Source:MGI Symbol;Acc:MGI:894284] Tlk2 TLK2 tousled-like kinase 2 (Arabidopsis) [Source:MGI Symbol;Acc:MGI:1346023]

Zc3h7a ZC3H7A zinc finger CCCH type containing 7 A [Source:MGI Symbol;Acc:MGI:2445044]

Igtp IGTP interferon gamma induced GTPase [Source:MGI Symbol;Acc:MGI:107729] TRAF type zinc finger domain containing 1 [Source:MGI Trafd1 TRAFD1 Symbol;Acc:MGI:1923551] Kpna3 KPNA3 karyopherin (importin) alpha 3 [Source:MGI Symbol;Acc:MGI:1100863] DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Source:MGI Ddx60 DDX60 Symbol;Acc:MGI:2384570] Cd200 CD200 CD200 antigen [Source:MGI Symbol;Acc:MGI:1196990]

Pf4 PF4 platelet factor 4 [Source:MGI Symbol;Acc:MGI:1888711]

Lmo2 LMO2 LIM domain only 2 [Source:MGI Symbol;Acc:MGI:102811]

Irf5 IRF5 interferon regulatory factor 5 [Source:MGI Symbol;Acc:MGI:1350924] poly (ADP-ribose) polymerase family, member 14 [Source:MGI Parp14 PARP14 Symbol;Acc:MGI:1919489] DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 [Source:MGI Ddx58 DDX58 Symbol;Acc:MGI:2442858] 166

family with sequence similarity 111, member A [Source:MGI Fam111a FAM111A Symbol;Acc:MGI:1915508] Mx2 MX2 MX dynamin-like GTPase 2 [Source:MGI Symbol;Acc:MGI:97244]

Isg15 ISG15 ISG15 ubiquitin-like modifier [Source:MGI Symbol;Acc:MGI:1855694] Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal Cited2 CITED2 domain, 2 [Source:MGI Symbol;Acc:MGI:1306784] Tmod3 TMOD3 tropomodulin 3 [Source:MGI Symbol;Acc:MGI:1355315]

Dck DCK deoxycytidine kinase [Source:MGI Symbol;Acc:MGI:102726]

Plcl2 PLCL2 phospholipase C-like 2 [Source:MGI Symbol;Acc:MGI:1352756] DNA-damage regulated autophagy modulator 2 [Source:MGI Dram2 DRAM2 Symbol;Acc:MGI:1914421] Pnp PNP purine-nucleoside phosphorylase [Source:MGI Symbol;Acc:MGI:97365]

Jak2 JAK2 Janus kinase 2 [Source:MGI Symbol;Acc:MGI:96629] cytoplasmic polyadenylation element binding protein 4 [Source:MGI Cpeb4 CPEB4 Symbol;Acc:MGI:1914829] Cd180 CD180 CD180 antigen [Source:MGI Symbol;Acc:MGI:1194924]

Gm5431 GM5431 predicted gene 5431 [Source:MGI Symbol;Acc:MGI:3645205]

Irf7 IRF7 interferon regulatory factor 7 [Source:MGI Symbol;Acc:MGI:1859212] Ifi204 IFI204 interferon activated gene 204 [Source:MGI Symbol;Acc:MGI:96429] poly (ADP-ribose) polymerase family, member 12 [Source:MGI Parp12 PARP12 Symbol;Acc:MGI:2143990] Sp100 SP100 nuclear antigen Sp100 [Source:MGI Symbol;Acc:MGI:109561]

Cd164 CD164 CD164 antigen [Source:MGI Symbol;Acc:MGI:1859568]

Etnk1 ETNK1 ethanolamine kinase 1 [Source:MGI Symbol;Acc:MGI:1922570] Klf6 KLF6 Kruppel-like factor 6 [Source:MGI Symbol;Acc:MGI:1346318] immunity-related GTPase family M member 2 [Source:MGI Irgm2 IRGM2 Symbol;Acc:MGI:1926262] Rin2 RIN2 Ras and Rab interactor 2 [Source:MGI Symbol;Acc:MGI:1921280]

Bach1 BACH1 BTB and CNC homology 1 [Source:MGI Symbol;Acc:MGI:894680] nuclear receptor subfamily 3, group C, member 1 [Source:MGI Nr3c1 NR3C1 Symbol;Acc:MGI:95824] proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional Psmb8 PSMB8 peptidase 7) [Source:MGI Symbol;Acc:MGI:1346527]

167

168

APPENDIX II – Cell signaling pathway analysis of 2’3’cGAMP stimulated

macrophages.

Full list of Pathways from Figure 33.

169

STINGMOLF STINGB6 MOLF Unstim/ Unstim/ Unstim/ Pathway name 2’3cGAMP 2’3’cGAMP 2’3’cGAMP

AHR Main Pathway 4.53 4.62 2.70 AHR Pathway (C-myc expression via RELA) 4.53 4.62 2.19 AHR Pathway (Cath D expression via SP1) 4.06 3.80 2.70 AHR Pathway (CYP1A1 CYP1B1 CYP1A2 AHRR gene expression via POLR2B) 0.00 0.00 0.00 AHR Pathway (PS2 Gene expression via ESR1) 4.06 3.80 2.19 Akt Signaling Main Pathway 60.47 78.20 101.38 Akt Signaling Pathway (Acetylation of proteins) -0.81 -0.52 -0.20 Akt Signaling Pathway (Aggregation and Neurodegeneration) -2.18 0.62 0.64 Akt Signaling Pathway (Apoptosis) -0.81 0.05 0.46 Akt Signaling Pathway (AR mediated apoptosis) -0.81 -0.52 -0.20 Akt Signaling Pathway (Blocks Apoptosis) -0.81 -0.52 -0.20 Akt Signaling Pathway (Blood cell differentiation) -0.81 -0.52 -0.20 Akt Signaling Pathway (Cell Cycle Progression) -1.17 -0.79 -0.64 Akt Signaling Pathway (Cell Cycle) 0.46 1.45 -0.53 Akt Signaling Pathway (Cell Survival) 0.05 -0.24 0.73 Akt Signaling Pathway (Elevation of Glucose Import) -0.81 -0.52 0.84 Akt Signaling Pathway (Enhancement of Breast Epithelial) -0.81 -0.52 -0.20 Akt Signaling Pathway (ERK mediated apoptosis) -0.81 -0.52 -0.20 Akt Signaling Pathway (Genetic Stability) -0.81 -0.52 -0.20 Akt Signaling Pathway (Glucose Uptake) 1.58 0.00 -1.11 Akt Signaling Pathway (Glycogen Synthesis and Apoptosis) -0.35 -0.52 -0.20 Akt Signaling Pathway (Increased GLUT4 translocation) -0.81 -0.52 -0.94 Akt Signaling Pathway (Induction of Chromatin Condensation) -0.81 -0.52 -0.20 Akt Signaling Pathway (JNK mediated apoptosis) -0.81 -0.52 -0.20 Akt Signaling Pathway (Neuroprotection) -1.28 -0.52 -0.20 Akt Signaling Pathway (NF-kB dependent transciption) -0.05 0.43 1.03 Akt Signaling Pathway (NF-kB pathway) -0.81 -0.52 -0.20 Akt Signaling Pathway (NFAT degradation) -4.22 -0.26 -0.30 Akt Signaling Pathway (NO production) -0.81 -0.52 -0.74 Akt Signaling Pathway (p73 mediated apoptosis) -0.81 -0.52 -0.20 Akt Signaling Pathway (Promotes Adipogenesis) -0.81 -0.52 -0.20 Akt Signaling Pathway (Protein Synthesis) 0.94 1.15 1.80 Akt Signaling Pathway (Proto-Oncogenic and RTK-signaling) -0.81 -0.52 -0.20 Akt Signaling Pathway (Regulation of Cyclic Nucleotide) -0.81 -1.15 -0.20 Akt Signaling Pathway (Regulation of Na+ Transport) 0.00 0.00 -1.00 Akt Signaling Pathway (Splicing Regulation) -1.09 -1.02 -1.11 Akt Signaling Pathway (Survival Genes) -2.63 -1.46 -0.74 Akt Signaling Pathway (Synaptic Transmission) -0.81 -0.52 -0.20 Akt Signaling Pathway (Translation) 0.00 -0.36 0.07 Akt Signaling Pathway (Tumor Supression) 0.13 0.34 -1.57 ATM Main Pathway -5.18 -3.63 0.61 ATM Pathway (Apoptosis and Senescence) -0.62 -0.82 -1.08

170

ATM Pathway (Apoptosis) 0.00 -0.93 0.43 ATM Pathway (Cell Cycle Checkpoint Control) 1.05 0.57 0.42 ATM Pathway (Cell Survival) -1.82 -1.87 -1.50 ATM Pathway (Checkpoint Activation) 0.00 0.00 0.01 ATM Pathway (DNA repair) -2.11 -1.62 1.38 ATM Pathway (G2 M Checkpoint Arrest) -0.81 1.39 1.23 ATM Pathway (G2-Mitosis progression) -0.81 1.39 1.23 ATM Pathway (NF-kB Pathway) -1.49 -1.64 -2.22 ATM Pathway (Repair and Recombination) 0.00 -0.93 0.43 ATM Pathway (S-phase arrest) 0.00 0.00 1.34 ATM Pathway (S-phase progression) -0.95 -0.86 2.06 ATM Pathway (Synaptic Vesicle Transport) 0.00 0.00 0.01 BRCA1 Main Pathway -2.15 1.01 3.95 BRCA1 Pathway (Base Excission Repair) -0.95 0.42 1.38 BRCA1 Pathway (Cell Cycle Arrest DNA Repair Genes p21 WAF CIP1 14-3-3 GADD45) -1.57 -1.68 0.81 BRCA1 Pathway (Chromatin Remodeling) -0.04 -1.40 -1.70 BRCA1 Pathway (E2 Responsive Genes) 1.26 1.03 3.10 BRCA1 Pathway (G1 S arrest) -0.32 -0.04 2.05 BRCA1 Pathway (Growth Promoting Genes hTert S100A7) -1.57 -1.18 2.07 BRCA1 Pathway (Homologous Recombination Repair) -3.30 -1.26 0.88 BRCA1 Pathway (Mismatch Repair) -2.75 -2.05 -2.12 BRCA1 Pathway (NHEJ DSB Repair) -1.57 -2.14 1.65 BRCA1 Pathway (Nucleotide Excission Repair) -0.35 -0.86 0.76 BRCA1 Pathway (Transciption Coupled Repair) -1.57 -1.68 0.81 cAMP Main Pathway -7.54 16.86 -9.53 cAMP Pathway (Axonal Growth) 0.00 0.00 -1.53 cAMP Pathway (Cell Growth) -0.14 0.78 0.46 cAMP Pathway (Cell Proliferation) 0.46 -0.77 -3.73 cAMP Pathway (Cell Survival) -0.14 0.33 -1.07 cAMP Pathway (Chemotaxis) -0.14 0.78 0.46 cAMP Pathway (Cytokine Production) -0.14 0.78 -0.51 cAMP Pathway (Degradation of Cell Cycle Regulators) 0.98 -0.77 -2.83 cAMP Pathway (Endothelial Cell Regulation) -1.81 0.35 -1.19 cAMP Pathway (eNOS Signaling Cardiovascular Homeostasis) 0.46 -0.77 -4.27 cAMP Pathway (Fatty Acid Metabolism) -0.50 0.63 -2.13 cAMP Pathway (Gene Expression via NFKB2 CREBBP ELK1) 4.24 3.09 -4.30 cAMP Pathway (Glycogen Synthesis) 0.46 -0.77 -4.18 cAMP Pathway (Glycolisis) 1.73 0.41 -1.62 cAMP Pathway (Lipolysis) 0.46 0.09 -3.00 cAMP Pathway (Metabolic Energy) -3.41 -0.89 -1.25 171

cAMP Pathway (Myocardial Contraction) -4.89 3.08 -4.77 cAMP Pathway (Oncogenesis) 0.92 -0.77 -3.73 cAMP Pathway (Protein Retention) 0.04 -1.18 -3.73 cAMP Pathway (Regulation of Cytoskeleton) 0.46 -0.77 -3.29 Caspase Cascade Main Pathway -5.64 -7.34 -7.67 Caspase Cascade Pathway (Activated Tissue Trans-glutaminase) -9.17 -0.44 -5.58 Caspase Cascade Pathway (DNA Fragmentation) 0.84 -1.17 0.46 CD40 Main Pathway 5.85 6.76 1.12 CD40 Pathway (Cell Survival) 3.98 4.53 3.35 CD40 Pathway (Gene Expression Cell Adhesion Molecule via NFKB2) 1.31 0.06 -1.78 CD40 Pathway (Gene Expression Cell Survival via NFKB2) 1.31 0.06 -1.78 CD40 Pathway (Gene Expression Co-Stimulatory Molecules via NFKB2) 1.31 0.06 -1.78 CD40 Pathway (Gene Expression COX2 and Prostaglandins via NFKB2) 1.31 0.06 -1.78 CD40 Pathway (Gene Expression Immunoglobulin Class Switch via NFKB2) 1.31 0.06 -1.78 CD40 Pathway (Gene Expression Pro-Inflamatory Cytokines via NFKB2) 1.31 0.06 -1.78 CD40 Pathway (Gene Expression Procoagulant Activity via NFKB2) 1.31 0.06 -1.78 Cellular Apoptosis Main Pathway 1.84 -7.65 2.97 Cellular Apoptosis Pathway (Depolarization) -0.62 -0.82 -1.25 Cellular Apoptosis Pathway (DNA Fragmentation) 2.33 1.66 3.52 Cellular Apoptosis Pathway (Gene Expression BAX BID BAK Ras Noxa PUMA APAF1 Survivin BCL2 via TP53) -0.62 -0.82 -0.83 Cellular Apoptosis Pathway (Gene Expression FLIP CIAP2 BFL1 BCL2 via NFKB2) 1.31 0.06 -0.26 Chemokine Main Pathway 29.03 37.28 41.93 Chemokine Pathway (Cell Activation) -0.95 -0.36 -4.49 Chemokine Pathway (Gene Expression and Apoptosis via ELK1) -0.59 -1.03 -1.91 Chemokine Pathway (Internalization Degradation Recycling) -1.66 -0.98 -1.23 Chromatin Main Pathway 1.03 0.00 -0.49 Circadian Main Pathway 2.94 5.90 6.47 CREB Main Pathway -4.33 11.60 0.61 Cytokine Network Main Pathway 3.96 4.33 14.74 EGF Main Pathway 14.66 8.65 13.55 EGF Pathway (Actin Cytoskeletal Rearrangement) 4.98 1.50 3.49 EGF Pathway (Cell Motility) 0.11 -0.69 0.69 EGF Pathway (Cell Survival) -2.52 -1.85 -0.13 EGF Pathway (Cytoskeleton Regulation) 0.52 -2.58 -1.05 EGF Pathway (EGFR Endocytosis) -0.44 -1.38 -0.40 EGF Pathway (Gene Expression via FOS NFKB2 MYC STAT1 ELK1 STAT3 JUN) 10.51 8.48 12.38 EGF Pathway (IP3 Signaling) -0.44 -1.38 0.00 EGF Pathway (Rab5 Regulation Pathway) 0.00 0.00 0.00

172

ErbB Family Main Pathway -1.28 -4.05 -2.27 ErbB Family Pathway (Anti-Apoptosis) -0.35 -0.46 -0.52 ErbB Family Pathway (Gene Expression via JUN FOS ELK1) 3.49 0.89 -0.79 ErbB Family Pathway (Translation) 0.00 0.00 -0.53 ERK Signaling Main Pathway 21.42 43.79 58.50 ERK Signaling Pathway (Cell Survival) 0.00 -0.65 -1.82 ERK Signaling Pathway (EGFR signaling) -0.16 -0.34 -1.46 ERK Signaling Pathway (Gene Expression via CAPN6 TP53 FOS ATF1 MYC ELK3 MYLK ETS1 SRF HIST1H3B CREB3 STAT3 NFKB2 HMGN1 ESR2 ELK1 PAX6 JUN) 1.59 1.89 4.08 ERK Signaling Pathway (Translation) -1.52 -1.49 -3.74 Erythropoietin Main Pathway 2.23 0.75 0.71 Erythropoietin Pathway (Anti-Apoptosis) -1.00 -0.97 -0.39 Erythropoietin Pathway (BCLXL Gene Expression via STAT5B) 1.10 1.26 -1.66 Erythropoietin Pathway (Cell Cycle Progression) 1.02 2.75 -1.24 Erythropoietin Pathway (Cell Survival) -2.27 -1.38 1.36 Erythropoietin Pathway (Gene Expression Neuroprotection via NFKB2) 0.28 -0.91 -1.29 Erythropoietin Pathway (GPI Hidrolysis and Ca2+ influx) 0.00 0.00 -0.44 Estrogen Main Pathway -0.07 5.51 5.42 Estrogen Pathway (Anti-Apoptosis) -3.51 -2.26 -1.23 Estrogen Pathway (Gene Expression via FOS JUN ELK1 SP1 POLR2B CREB3 NFKB2) 1.52 2.60 0.11 Estrogen Pathway (Vasodilatation) 0.00 0.00 -0.55 FLT3 Signaling Main Pathway -7.26 -3.93 -1.84 FLT3 Signaling Pathway (Transcription via ELK3 MAPK12 CREB3 STAT2) -2.02 -1.55 0.95 FLT3 Signaling Pathway (Translation) 0.16 -0.02 -1.06 Glucocorticoid Receptor Signaling Main Pathway -20.88 -22.39 -37.68 Glucocorticoid Receptor Signaling Pathway (Cell Cycle Arrest) -0.52 -1.06 -1.08 Glucocorticoid Receptor Signaling Pathway (Cell Cycle Progression) 1.56 -0.02 1.15 Glucocorticoid Receptor Signaling Pathway (Gene Expression via CREB3 STAT5B SLC22A2 POU2F1) 0.27 -1.06 -0.55 Glucocorticoid Receptor Signaling Pathway (Histone Deacetylation) 0.49 0.42 0.46 Glucocorticoid Receptor Signaling Pathway (Inflammatory Cytokines) -2.62 -2.57 -4.78 GPCR Main Pathway 14.33 13.39 17.17 GPCR Pathway (Gene Expression via JUN NFKB2 ELK1 SRF FOS CREB3) 12.59 8.37 12.42 Growth Hormone Signaling Main Pathway -1.20 -1.98 -4.62 Growth Hormone Signaling Pathway (Cell Survival) -2.62 0.31 -0.90 Growth Hormone Signaling Pathway (Gene Expression via SRF ELK1 STAT5B CEBPD STAT1 STAT3) -0.89 -1.52 -4.94 Growth Hormone Signaling Pathway (Glucose Uptake) -2.31 0.78 0.84 Growth Hormone Signaling Pathway (Protein Synthesis) 0.44 0.00 0.00 GSK3 Signaling Main Pathway 8.83 0.62 9.76

173

GSK3 Signaling Pathway (b-CTNN Degradation) 2.96 2.92 4.86 GSK3 Signaling Pathway (Gene Expression via CTNNB1) 3.24 4.68 13.14 GSK3 Signaling Pathway (Glycogen Synthesis) 2.26 2.53 5.96 GSK3 Signaling Pathway (Protein Synthesis) 2.26 2.53 6.41 Hedgehog Signaling in Mammals Main Pathway 0.97 1.91 2.83 HGF Main Pathway 5.81 2.73 7.29 HGF Pathway (Anoikis) 0.49 1.67 2.74 HGF Pathway (Cell Adhesion and Cell Migration) 4.09 3.79 3.17 HGF Pathway (Cell Cycle Progression) 3.91 2.15 4.46 HGF Pathway (Cell Scattering) -2.51 0.77 0.18 HGF Pathway (Cell Survival) -2.51 0.77 0.18 HGF Pathway (Gene Expression via JUN STAT3 FOS ELK1 ETS2) 5.83 4.71 7.23 HGF Pathway (IP3 Pathway) 0.38 0.41 1.46 HGF Pathway (PKC Pathway) 1.84 0.84 2.52 HGF Pathway (Regulation of Cytoskeleton Cell Polarity and Cell Motility) -0.57 -0.85 0.42 HIF1Alpha Main Pathway -2.66 -4.08 -6.58 HIF1Alpha Pathway (Gene Expression via JUN CREB3) -2.66 -4.08 -2.58 HIF1Alpha Pathway (HIF1a Degradation) 0.00 0.00 0.00 HIF1Alpha Pathway (NOS Pathway) -0.84 -3.14 -2.04 HIF1Alpha Pathway (p53 Hypoxia Pathway) -0.84 -3.14 -6.04 HIF1Alpha Pathway (VEGF Pathway) -0.84 -3.14 -2.04 IGF1R Signaling Main Pathway 13.93 10.65 13.80 IGF1R Signaling Pathway (Apoptosis) 1.00 0.45 0.65 IGF1R Signaling Pathway (Cell Migration) 3.51 2.32 2.27 IGF1R Signaling Pathway (Cell Proliferation Diferentiation and Apoptosis) 0.45 0.05 -1.99 IGF1R Signaling Pathway (Cell Survival) -0.35 -0.46 -0.97 IGF1R Signaling Pathway (Gene Expression Proliferation Growth Survival via NFAT5 CREB3) -1.82 -3.79 -1.44 IGF1R Signaling Pathway (Glucose Uptake) 1.22 0.96 -0.46 IGF1R Signaling Pathway (Glycogen Synthesis) -0.29 -0.46 -1.24 IGF1R Signaling Pathway (Protein Synthesis) 1.63 0.65 3.08 IL-10 Main Pathway 3.15 6.39 0.92 IL-10 Pathway (IL-10 Responsive Genes Transcription of BCLXL Cyclin-D1 D2 D3 Pim1 c-Myc and P19(INK4D) via STAT3) -2.19 0.42 -7.18 IL-10 Pathway (Inflammatory Cytokine Genes Expression via STAT3) -2.19 0.42 -7.18 IL-10 Pathway (Stability Determination) 0.95 0.53 2.80 IL-10 Pathway (Translational Modulation) 2.62 2.59 -0.68 IL-2 Main Pathway -0.82 -0.07 6.92 IL-2 Pathway (Actin Reorganization) 3.68 3.23 1.83 IL-2 Pathway (Apoptosis Inhibition) -2.22 -0.49 -0.19 IL-2 Pathway (Apoptosis) -2.28 -2.86 -2.09

174

IL-2 Pathway (Gene Expression via FYN LYN RPS6KB1 PTK2B) 1.50 3.81 2.61 IL-2 Pathway (IL2 Gene Expression via POLR1E EGR1 HMGA1 ELF1 POU2F1 MYC) 0.25 1.09 8.39 IL-2 Pathway (Protein Synthesis) 1.58 3.21 2.88 IL-6 Main Pathway -3.20 -0.19 -9.74 ILK Signaling Main Pathway -0.82 7.09 9.23 ILK Signaling Pathway (Actin Polymerization Cytoskeletal Reorganization) 0.07 -0.47 -0.81 ILK Signaling Pathway (Apoptosis) -0.75 -0.35 -1.04 ILK Signaling Pathway (Cell Adhesion) 0.00 0.00 0.00 ILK Signaling Pathway (Cell Cycle Proliferation) -0.35 2.23 0.87 ILK Signaling Pathway (Cell Migration Retraction) -0.35 2.23 0.87 ILK Signaling Pathway (Cell Motility) 0.54 -0.47 -0.81 ILK Signaling Pathway (Cytoskeletal Adhesion Complexes) 4.06 6.80 2.97 ILK Signaling Pathway (Epithelial Mesenchymal Transition Tubulo- Interstitial Fibrosis) 0.29 6.67 3.56 ILK Signaling Pathway (Epithelial Mesenchymal Transition) 1.14 -0.64 3.16 ILK Signaling Pathway (G2 Phase Arrest) -0.35 2.23 0.87 ILK Signaling Pathway (Induced Cell Proliferation) -1.82 -0.83 -1.90 ILK Signaling Pathway (Loss of Occludin Barrier Dysfunction) 3.44 2.24 1.46 ILK Signaling Pathway (Migration Vasculogenesis) 0.29 6.67 3.56 ILK Signaling Pathway (MMP2 MMP9 Gene Expression Tissue Invasion via FOS) -1.94 -1.73 3.24 ILK Signaling Pathway (Opsonization) 0.47 0.00 0.00 ILK Signaling Pathway (Regulation of Intermediate Filaments) 0.00 0.00 0.00 ILK Signaling Pathway (Regulation of Junction Assembly at Desmosomes) 0.00 0.00 0.00 ILK Signaling Pathway (Tissue Morphogenesis) -1.94 -1.73 3.24 ILK Signaling Pathway (Tumor Angiogenesis) 0.00 0.00 1.44 ILK Signaling Pathway (Wound Healing) 0.00 0.00 0.00 Interferon Main Pathway -3.89 -7.69 -3.50 Interferon Pathway (Gene Expression via HIST1H3B CREB3) -2.10 -2.62 -3.67 Interferon Pathway (Transciption) -0.90 2.25 5.26 Interferon Pathway (Translation) -0.16 -0.63 0.48 IP3 Main Pathway -1.82 9.19 10.47 IP3 Pathway (Gene Expression via CREB3 NFATC2 MEF2D) -1.51 -5.34 -0.69 JAK-STAT Main Pathway 22.59 33.44 41.26 JAK-STAT Pathway (Akt Pathway) -2.77 2.24 -0.24 JAK-STAT Pathway (Gene Expression via MYC) 0.64 3.23 2.73 JAK-STAT Pathway (IFN-Inducible Gene Expression via STAT2 STAT1) 2.51 2.73 6.45 JAK-STAT Pathway (JAK Degradation) -2.96 -2.92 -4.86 JAK-STAT Pathway (Nml SOCS BCL-XL p21 Myc Nos2 Gene Expression via STAT2) 21.13 28.67 35.45 JNK Main Pathway 18.07 20.11 38.70 175

JNK Pathway (Gene Expression Apoptosis Inflammation Tumorigenesis Cell Migration via SMAD4 STAT4 HSF1 TP53 MAP2 DCX ATF2 NFATC3 SPIRE1 MAP1B TCF15 ELK1 BCL2 JUN PXN NFATC2) -1.05 -3.87 -0.19 JNK Pathway (Insulin Signaling) -0.40 0.88 -2.97 MAPK Family Main Pathway 22.70 21.87 39.55 MAPK Family Pathway (Chromatin Remodelling) -1.41 -3.65 -3.69 MAPK Family Pathway (Cytoskeleton) -0.06 0.10 -1.72 MAPK Family Pathway (Gene Expression via ATF2 JUN ELK1 NFKB2 CREB3) -1.13 -2.07 -2.79 MAPK Family Pathway (Translation) -2.04 -2.48 -2.95 MAPK Signaling Main Pathway 7.37 13.10 25.07 MAPK Signaling Pathway (Cell Motility Inflammation Apoptosis Osmoregulation) -1.82 -1.71 -4.98 MAPK Signaling Pathway (Cell Survival) 0.27 -1.53 -0.95 MAPK Signaling Pathway (Gene Expression Apoptosis Inflammation Tumorigenesis via MYC HSF1 STAT2) 1.65 3.00 6.57 MAPK Signaling Pathway (Gene Expression Cell Proliferation Cell Survival Tumorigenesis Differentiation Development via PXN CREB3 RPS6KA5 RPS6KA6) 1.24 -0.23 -4.19 Mismatch Repair in Eukaryotes Main Pathway -2.89 1.77 -1.99 Mitochondrial Apoptosis Main Pathway 0.13 5.75 5.05 Mitochondrial Apoptosis Pathway (Apoptosis) 3.61 4.98 4.28 Mitochondrial Apoptosis Pathway (Depolarization) -0.56 0.00 0.43 Mitochondrial Apoptosis Pathway (DNA Fragmentation) 2.33 3.64 3.06 Mitochondrial Apoptosis Pathway (Gene Expression via TP53) -0.56 0.00 0.43 mTOR Main Pathway -12.12 -8.13 -9.59 mTOR Pathway (Actin Organization) 1.01 3.56 5.94 mTOR Pathway (Autophagy) 0.55 0.69 1.83 mTOR Pathway (Cap-dependent Translation) -0.28 -0.27 0.54 mTOR Pathway (Inflammation Stress Resistance) 0.28 -0.91 -1.29 mTOR Pathway (Lipid Synthesis) -1.22 1.90 3.14 mTOR Pathway (Metabolism Stress Response and Apoptosis) -1.64 -1.22 -4.04 mTOR Pathway (Microtubule Organization) 1.01 4.15 5.94 mTOR Pathway (Mitochondria Proliferation and Function) -0.55 -3.14 -2.49 mTOR Pathway (mRNA Biogenesis) 0.00 0.00 0.00 mTOR Pathway (Ribosome Biogenesis) 0.00 0.00 0.00 mTOR Pathway (Translation Elongation) -2.17 -3.34 -2.71 mTOR Pathway (VEGF Pathway) 0.55 0.00 1.85 NGF Main Pathway -1.13 -0.73 -1.02 NGF Pathway (Actin Polymerization Neurite Outgrowth and Differentiation) 0.43 0.00 0.00 NGF Pathway (Apoptosis) 0.00 0.00 -0.01 NGF Pathway (Gene Expression via MYC ELK1 CREB3 NFKB2) 1.50 2.73 2.31 NGF Pathway (Neurite Outgrowth and Differentiation) -2.95 0.01 -4.35 176

NGF Pathway (Neuronal Survival) -4.17 -0.95 -2.86 Notch Signaling Main Pathway -2.83 1.59 0.65 Notch Signaling Pathway (gamma Secretase) 0.00 0.00 -0.59 Notch Signaling Pathway (Gene Expression Chromatin Remodeling via RBPJ) -2.83 1.59 1.23 Notch Signaling Pathway (Transcription of Target Genes Hairy E-SpI via RBPJ) -2.83 1.59 1.23 p38 Signaling Main Pathway 55.99 58.34 86.67 p38 Signaling Pathway (Actin Cytoskeleton Reorganiztion) -0.06 0.10 -0.75 p38 Signaling Pathway (Apoptosis) 0.00 0.00 0.00 p38 Signaling Pathway (Gene Expression Cell Motility Inflammation Apoptosis Osmoregulation via MEF2D TP53 CREB1 ATF2 JUND ETV1 NFKB2 AP2A1 MAX FOSL1 CEBPG ELK1 CDC25C JUNB STAT1 SP1 DDIT3 ELK4 CEBPA) -0.18 -2.19 -3.18 p38 Signaling Pathway (Translation) -1.35 -1.16 -1.80 p53 Signaling Main Pathway -1.99 -5.33 -3.62 p53 Signaling Pathway (Apoptosis) 0.60 -0.46 2.12 p53 Signaling Pathway (Breast Cancer) 0.09 -1.00 1.77 p53 Signaling Pathway (Cancer) 0.60 -0.46 2.12 p53 Signaling Pathway (Cell Cycle Arrest) 0.09 -1.00 1.77 p53 Signaling Pathway (Cell Growth Accumulation) 0.09 -1.00 1.77 p53 Signaling Pathway (DNA Repair) 0.09 -1.00 1.77 p53 Signaling Pathway (Exosome Mediated Secretion) 0.09 -1.00 1.77 p53 Signaling Pathway (Gene Expression Anti-Apoptosis via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Gene Expression Cell Cycle and Cell Growth via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Gene Expression Cell Fate and Development via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Gene Expression Cell Signaling via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Gene Expression DNA Replication and Repair via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Gene Expression ECM and Adhesion via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Gene Expression Infection and Immune Response via TP53) 0.60 -0.46 2.12 p53 Signaling Pathway (Inhibition of Angiogenesis and Metastasis) 0.09 -1.00 1.77 p53 Signaling Pathway (Inhibition of IGF1R mTOR Pathways) 0.00 0.00 0.00 p53 Signaling Pathway (Normal Cell Cycle Progression) 0.60 -0.46 2.12 PAK Main Pathway 24.14 31.39 58.17 PAK Pathway (Actin Organization) 17.83 26.40 56.49 PAK Pathway (Cell Survival) 17.83 26.40 56.49 PAK Pathway (Contractility Stress Fibres and Focal Adhesion) 1.53 -4.36 -0.78 PAK Pathway (Lamelliopodia and Filopodia Outgrowth) 18.89 29.21 55.12 PAK Pathway (Paxillin Disassembly) 0.00 -0.59 0.00

177

PAK Pathway (Transciptional Activation and Filopodia Formation) 0.51 -0.23 -0.17 PPAR Main Pathway -2.03 -2.51 -4.25 PPAR Pathway (Adipocyte Differentiation Glucose Homeostasis and Macrophage Function) 0.11 -2.13 -0.78 PPAR Pathway (Fatty Acid Metabolism Lipid Homeostasis and Skin Proliferation) 0.11 -2.13 -0.78 PPAR Pathway (Peroxisome Proliferation Hepatocarcinogenesis Fatty Acid Metbolism and Lipid Homeostasis) 0.11 -2.13 -0.78 PTEN Main Pathway -2.87 -10.49 -23.11 PTEN Pathway (Adhesion or Migration) -2.62 -7.22 -16.16 PTEN Pathway (Angiogenesis and Tumorigenesis) -3.67 -1.91 -13.68 PTEN Pathway (Apoptosis) -4.22 -5.29 -1.98 PTEN Pathway (Ca2+ Signaling) -3.67 -1.91 -13.68 PTEN Pathway (Cell Cycle) -2.06 -5.35 -14.45 PTEN Pathway (Cell Survival) 0.09 -0.48 -0.41 PTEN Pathway (Differentiation) -2.31 -5.73 -14.73 PTEN Pathway (DNA Repair) -5.13 -6.48 -16.25 PTEN Pathway (Genomic Stability) -5.13 -6.48 -14.59 PTEN Pathway (Growth) -2.06 -5.35 -14.45 PTEN Pathway (Migration) -6.45 -8.28 -17.20 PTEN Pathway (Neuronal Survival) 1.82 0.94 0.54 PTEN Pathway (Proliferation CyclinA2 Geminin AuroraA PLK1) -1.66 1.25 -1.82 PTEN Pathway (Proliferation) -4.46 -6.18 -14.72 PTEN Pathway (Protein Synthesis) 0.00 0.00 0.00 PTEN Pathway (Senescence Ets2 p16) -1.66 1.25 -1.82 PTEN Pathway (Synaptic Transmission) -5.13 -6.48 -14.59 RANK Signaling in Osteoclasts Main Pathway 2.11 0.25 -2.20 RANK Signaling in Osteoclasts Pathway (Expression of Osteoclastic Genes via JUN NFAT5 NFKB2 MITF FOS) 2.35 -0.81 0.57 RANK Signaling in Osteoclasts Pathway (Inhibition of Death) 2.26 7.08 3.15 RANK Signaling in Osteoclasts Pathway (Resorption) 0.38 2.35 -2.84 Ras Main Pathway 9.24 13.80 16.69 Ras Pathway (Apoptosis) 0.23 3.96 2.28 Ras Pathway (CDC42 Pathway) -0.54 -0.43 -0.56 Ras Pathway (Cell-Cell Junctions) 1.95 -4.01 -0.31 Ras Pathway (Gene Expression Cell Proliferation Cell Survival Differentiation Development Cell Cycle Control Cell Motility Tumorigenesis via ELK4 ATF2 MEF2D STAT2 CREB3 CCNA1 MYC ELK1 JUN CDK4) -0.25 -4.67 1.67 Ras Pathway (Golgi Trafficking and Vesicle Formation) 1.99 2.60 1.79 Ras Pathway (Increased T-cell Adhesion) 2.08 1.09 0.92 Ras Pathway (Receptor Endocytosis) -0.57 -0.85 0.42 Ras Pathway (RhoA Pathway) -0.54 -0.43 -0.52

178

SMAD Signaling Network Main Pathway 0.41 0.26 -4.53 STAT3 Main Pathway 52.81 52.67 80.35 STAT3 Pathway (Anti-Apoptosis) 0.00 -1.05 1.14 STAT3 Pathway (G1 to S Cell Cycle Progression) 0.00 0.50 -0.47 STAT3 Pathway (Growth Arrest and Differentiation) 33.02 29.90 26.92 TGF-Beta Main Pathway 17.64 28.95 40.69 TGF-Beta Pathway (Epithelial Mesenchymal Transdifferentiation) -3.41 -0.49 -2.96 TGF-Beta Pathway (Post Transciptional G1 Arrest) 0.51 1.27 -0.57 TGF-Beta Pathway (Transciption Arrested Growth Apoptosis) 1.55 1.28 0.95 TGF-Beta Pathway (Transciption Cell Growth and Mobility and Angiogenesis) 1.55 1.28 0.95 TNF Signaling Main Pathway 5.61 0.83 8.81 TNF Signaling Pathway (Apoptosis) 3.34 4.15 3.97 TNF Signaling Pathway (Gene Expression and Cell Survival via FOS NFKB2 JUN ELK1 ATF6) -0.82 -5.91 -2.78 TRAF Main Pathway 11.54 10.14 30.24 TRAF Pathway (Apoptosis) 2.71 4.15 7.45 TRAF Pathway (Cell Survival) -3.41 -0.49 -1.19 TRAF Pathway (Direct Antimicrobial Response and Cell-Mediated Immunity and Apoptosis of Host Cell) 0.28 -0.91 -1.29 TRAF Pathway (Gene Expression via FOS JUN) 1.55 1.28 0.48 Ubiquitin-Proteasome Dependent Proteolysis Main Pathway 3.68 2.89 5.96 VEGF Main Pathway 3.59 5.56 -1.87 VEGF Pathway (Actin Reorganization) 2.42 2.91 -1.24 VEGF Pathway (Cell Migration) 2.42 2.91 -1.24 VEGF Pathway (Cell Survival) -3.41 0.08 -1.06 VEGF Pathway (Focal Adhesion Turnover) 1.73 2.18 1.58 VEGF Pathway (Gene Expression and Cell Proliferation via MAPK7) -0.11 -0.03 -3.74 VEGF Pathway (Nitric Oxide Production) 0.00 0.00 -1.07 VEGF Pathway (Prostaglandin Production) 1.53 2.30 -0.99 WNT Main Pathway -2.32 -4.41 -7.36 WNT Pathway (Cell Fate Proliferation Differentiation Adhesion and Survival) -3.03 -3.85 -3.87 WNT Pathway (Cell Survival) -1.60 -3.16 -3.94 WNT Pathway (Cytoskeletal Rearrangement) 1.77 1.17 0.74 WNT Pathway (Gene Expression via CREB3) -1.82 1.74 3.16 WNT Pathway (NFAT Pathway) -0.56 -0.34 -1.33 WNT Pathway (PKC Pathway) 3.53 4.69 4.27

179

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