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. 2003 Jun;13(6b):1307–1317. doi: 10.1101/gr.1016503

Cytokine-Related Genes Identified From the RIKEN Full-Length Mouse cDNA Data Set

Vladimir Brusic 1,2,10, Rekha S Pillai 1, Diego G Silva 3,4, Nikolai Petrovsky 2,3; RIKEN GER Group5,8; GSL Members6,9, Christian Schönbach 7
PMCID: PMC403723  PMID: 12819128

Abstract

To identify novel cytokine-related genes, we searched the set of 60,770 annotated RIKEN mouse cDNA clones (FANTOM2 clones), using keywords such as cytokine itself or cytokine names (such as interferon, interleukin, epidermal growth factor, fibroblast growth factor, and transforming growth factor). This search produced 108 known cytokines and cytokine-related products such as cytokine receptors, cytokine-associated genes, or their products (enhancers, accessory proteins, cytokine-induced genes). We found 15 clusters of FANTOM2 clones that are candidates for novel cytokine-related genes. These encoded products with strong sequence similarity to guanylate-binding protein (GBP-5), interleukin-1 receptor-associated kinase 2 (IRAK-2), interleukin 20 receptor α isoform 3, a member of the interferon-inducible proteins of the Ifi 200 cluster, four members of the membrane-associated family 1-8 of interferon-inducible proteins, one p27-like protein, and a hypothetical protein containing a Toll/Interleukin receptor domain. All four clones representing novel candidates of gene products from the family contain a novel highly conserved cross-species domain. Clones similar to growth factor-related products included transforming growth factor β-inducible early growth response protein 2 (TIEG-2), TGFβ-induced factor 2, integrin β-like 1, latent TGF-binding protein 4S, and FGF receptor 4B. We performed a detailed sequence analysis of the candidate novel genes to elucidate their likely functional properties.


Cytokines are polypeptides secreted by immune cells that function as humoral regulators modulating functional activities of cells and tissues including cellular interactions and processes. Cytokine genes often encode functional variants through alternative splicing, resulting in proteins that may have slightly different biological activities (Ibelgaufts 1999). Cytokines are typically mutually unrelated, but some may be grouped in families.

Activated cytokine-producing cells often release many different cytokines, resulting in complex networks of interacting signals. Cytokines are crucial in immune cell activation, cell-to-cell communication, signal transduction, mitosis, cell survival, death, and transformation. In addition to their involvement in initiation and regulation of immune responses, cytokines are also important in embryogenesis, organ development, and neuronal processes (Ibelgaufts 1999). Clinical uses of cytokines include application in hematopoiesis (Smith 1990), tumor immunotherapies (Cao et al. 1998; Rosenberg 2001; Homey et al. 2002), bone marrow transplantation (Miyazaki and Kato 1999), gene therapy (Jeschke et al. 2001), and vaccine formulations (Knutson and Disis 2001).

The function of a particular cytokine can be assessed in vivo in transgenic or gene knock-out animals by studying the effects of an excess or lack of a particular cytokine gene product. It is of great importance for cytokine research, therefore, that the counterparts of human cytokine, cytokine receptors, and cytokine-related genes are identified in animals (e.g., the mouse) suitable for genetic manipulation. Because of the remarkable similarity between human and mouse genomes and their closely related biochemical, physiological, and pathological pathways, the mouse is an extremely useful animal model for immunological studies.

The FANTOM2 collaboration (Okazaki et al. 2002) involved functional annotation of a set of 60,770 RIKEN mouse cDNA clones (FANTOM2 clones). The FANTOM2 data set, the most complete picture of the mouse transcriptome to date, provides an ideal opportunity for identification of novel mouse genes, including cytokine-related genes. The annotation process included data from microarray expression, mapping and protein interactions, and sequence similarity search analyses. The annotation was performed by large-scale, computerized annotation combined with manual curation by human experts. We analyzed the FANTOM2 annotations, specifically looking for novel and existing mouse cytokines and cytokine-related genes. In this study, we focused on genes related to interferons (IFN), interleukins (IL), growth factors (epidermal growth factors [EGF], fibroblast growth factors [FGF], and transforming growth factors [TGF]), and small inducible cytokines (SIC). Some cytokines, such as tumor necrosis factors (TNF), belong to superfamilies that also include non-cytokines. Because this study was based on matching cytokine names from descriptions of database entries, cytokine families that belong to broader superfamilies were excluded from the analysis. Further study is required for a comprehensive analysis of all known cytokines and cytokine-related genes that have not been included in this work. The FANTOM2 clones contained more than 100 previously identified cytokine and cytokine-related genes. We also identified several candidates for novel mouse cytokine-related genes.

RESULTS

Of the 60,770 FANTOM2 clones, 256 had annotations that contained a cytokine name. A total of 222 clones had high-sequence identity (>96% over 50% length of the reference sequence) to 108 known mouse genes, as listed in the major databases. Of these, clones represented 27 known interferon-related genes (Table 1). The known mouse genes in the FANTOM2 set comprised 1 interferon (IFN-γ), 2 IFN receptors (IFNαβ receptor, and IFNαβ receptor 2), 20 inducible/activatable genes, and 4 other IFN-related genes. Search of the protein translation of the GenBank database (Benson et al. 2002, as of September 2002) revealed that there were at least 15 nonredundant entries of various mouse IFNs, 10 IFN receptors, 190 IFN inducible or activatable proteins, and 60 IFN regulatory proteins.

Table 1.

The List of 54 Clones Representing 27 Known Interferon-Related Genes in Mouse Found in the FANTOM2 Data Set

Clone ID Mouse strain cDNA library Gene name
1 5830458K16 C57BL/6 Thymus 28-kD interferon α responsive protein
1810013G24 C57BL/6 Pancreas (5830458K16RIK protein) homolog (Mus musculus)
2 0710005K22 C57BL/6 Brain Genes associated with retinoid-IFN-induced mortality 19
0710005K22 C57BL/6 Brain
2700054G14 C57BL/6 Embryo11
2410016H22 C57BL/6 ES
3 A430075E22 C57BL/6 N0 Thymus Interferon (α and β) receptor
4 1100001K09 C57BL/6 18-day embryo Interferon (α and β) receptor 2
2510009P07 C57BL/6 Embryo 13-Liver
5 E430025C02 NOD (NOD)N2 Thymic cells Interferon-activated gene 202A (noncoding RNA)
6 A730048F03 C57BL/6 N7 Cerebellum Interferon-activated gene 205
7 A430077M01 C57BL/6 N0 Thymus Interferon-inducible protein 203 (IFI-203) (interferon-inducible protein P203)
A530058P16 C57BL/6 Aorta and vein
A530093G11 C57BL/6 Aorta and vein
E430077M01 NOD (NOD)N2 Thymic
8 9030603A05 C57BL/6 Colon Interferon concensus sequence-binding protein
9830117K07 C57BL/6 Bone
9 A830021G01 C57BL/6 N10 Cortex Interferon-dependent positive-acting transcription factor 3 γ
10 F830002I10 NOD Activated spleen from NOD.Cz Interferon γ
F830032O20 NOD Activated spleen from NOD.Cz
11 0610039K12 C57BL/6 Kidney Interferon γ induced GTPase
E430012H16 NOD (NOD)N2 Thymic cells
12 5730557A06 C57BL/6 Embryo 8 Interferon γ inducible protein 30
13 2010205D02 C57BL/6 Small intestine Interferon γ inducing factor-binding protein
1110003K24 C57BL/6 18-day embryo
2310040F23 C57BL/6 Tongue
2310075I12 C57BL/6 Tongue
2310047H18 C57BL/6 Tongue
14 0610011H23 C57BL/6 Kidney Interferon inducible protein 1
15 9830146E22 C57BL/6 Bone Interferon regulatory factor 2
E430001A19 NOD (NOD)N2 Thymic
16 F730005C13 C57BL/6 B6 derived Interferon regulatory factor 4
F830028K14 NOD Activated spleen
17 A130064P06 C57BL/6 N16 Thymus Interferon regulatory factor 6
E230028I05 C57BL/6 P2 oviduct
18 0610039E12 C57BL/6 Kidney Interferon regulatory factor 7
1110062H15 C57BL/6 18-day embryo
A430014N21 C57BL/6 N0 Thymus
F830035B02 NOD Activated spleen
19 2010008K16 C57BL/6 Small intestine Interferon-induced 35-kD protein homolog (IFP 35) homolog (Mus musculus)
20 2010002M12 C57BL/6 Small intestine Interferon-induced protein with tetratricopeptide repeats 1
F830004H17 NOD Activated spleen
21 A130072L05 C57BL/6 N16 Thymus Interferon-induced protein with tetratricopeptide repeats 2
A630085F01 C57BL/6 N3 Thymus
22 5730412C13 C57BL/6 Embryo 8 Interferon-induced protein with tetratricopeptide repeats 3
5031412D17 C57BL/6 Ovary & Uterus (Preg. 11 day) (IFIT-3) glucocorticoid-attenuated response gene 49
23 F830009I11 NOD Activated spleen interferon-inducible GTPase
24 A430077M01 C57BL/6 N0 Thymus Interferon-inducible protein 203 (Mus musculus)
25 0610030F02 C57BL/6 Kidney Interferon-related developmental regulator 2
26 2900034J12 C57BL/6 Hippocampus Interferon-stimulated protein (15 kD)
27 1600023I01 C57BL/6 Placenta Interferon-stimulated protein (20 kD)
2010006M08 C57BL/6 Small intestine
2010009P14 C57BL/6 Small intestine
2010107M23 C57BL/6 Small intestine

A total of 77 clones represented 39 known interleukin-related genes (Table 2). The known mouse genes in the FANTOM2 set comprised 11 interleukins (IL) or chains thereof (IL-1δ, IL-1ε, IL-6, IL-7, IL-1F9, IL-16, IL-17B, IL-20, IL-23p19, and IL-25), 19 interleukin receptors (ILR-1I, ILR-1-1, ILR-2α, ILR-2β, ILR-2γ, ILR-4α, ILR-6α, ILR-7, ILR-9, ILR-10β, ILR-12β1, ILR-12β2, ILR-13α1, ILR-13α2, ILR-15α, ILR-17, ILR-18-1, ILR-21, and ILR-23αp19), and 9 other IL-related genes. Search of the protein translation of the GenBank database revealed that there are at least 40 nonredundant entries of mouse ILs (including variants and subunits), 50 IL receptors, and 80 other IL-related proteins. These results are in agreement with an earlier finding (Staudt and Brown 2000), which stated that only a half of human ILs, mainly those widely expressed, were found in the EST libraries. The ILs reliant on the activation of immune cells, namely IL-2, IL-3, IL-5, IL-9, IL-11, IL-12β, IL-14, and IL-17, were lacking in the EST libraries.

Table 2.

The List of 77 Clones Representing 39 Known Interleukin-Related Genes Found in the FANTOM2 Data Set

Clone ID Mouse strain cDNA library Gene name
1 2210418I04 C57BL/6 Stomach Interleukin 1 family, members 5 (δ)
4632413N13 C57BL/6 Skin (Neonate 0 day)
2310041K07 C57BL/6 Tongue
2310063B08 C57BL/6 Tongue
2 1110033G16 C57BL/6 18-day embryo Interleukin 1 family, member 6 (ε)
9330131B06 C57BL/6 Diencephalon
9330171A08 C57BL/6 Diencephalon
3 A330066C15 C57BL/6 Spinal cord Interleukin 1 receptor accessory protein
B230304B05 C57BL/6 C.quadrigemina
C630028F01 C57BL/6 Hippocampus
4 F630041P17 NOD NOD-derived Interleukin 1 receptor antagonist
4632427H17 C57BL/6 Skin
5 C130072K02 C57BL/6 E16 Head Interleukin 1 receptor, type I
E230023C01 C57BL/6 P2 oviduct
6 B230327C20 C57BL/6 C.quadrigemina Interleukin 1 receptor-associated kinase
D830047P08 C57BL/6 N16 heart
7 9030010H19 C57BL/6 Colon Interleukin 1 receptor-like 1
8 A530048E20 C57BL/6 Aorta and vein Interleukin 10 receptor, β
9 1500012D04 C57BL/6 Cerebellum Interleukin 10-related T cell-derived inducible factor
10 A430092F05 C57BL/6 N0 Thymus Interleukin 12 receptor, β 1
11 A430056E01 C57BL/6 N0 Thymus Interleukin 12 receptor, β 2
A530070O09 C57BL/6 Aorta and vein
12 C430004G12 C57BL/6 E7 whole body Interleukin 13 receptor, α 1
G430044I06 C57BL/6 Mixeda
13 F830008L10 NOD Activated spleen Interleukin 13 receptor, α 2
14 D630025K20 C57BL/6 N0 kidney Interleukin 15
A530045E03 C57BL/6 Aorta and vein
15 A530048H17 C57BL/6 Aorta and vein Interleukin 15 receptor, α chain
A630048I20 C57BL/6 N3 Thymus
16 A130095H14 C57BL/6 N16 Thymus Interleukin 16
17 9830138F10 C57BL/6 Bone Interleukin 17 receptor
A530085G20 C57BL/6 Aorta and vein
18 1110006O16 C57BL/6 18-day embryo Interleukin 17B
19 E030029C19 C57BL/6 N0 lung Interleukin 18 receptor 1
F630101C07 NOD NOD-derived
20 E030025K24 C57BL/6 N0 lung Interleukin 18 receptor accessory protein
21 E430021B09 NOD (NOD) N2 Thymic Interleukin 2 receptor, α chain
A130010M06 C57BL/6 N16 Thymus
22 5430409O05 C57BL/6 Head (Neonate 6 day) Interleukin 2 receptor, β chain
9530010F16 C57BL/6 U.bladder
23 A130027N13 C57BL/6 N16 Thymus Interleukin 2 receptor, γ chain
24 7530415H19 C57BL/6 Eyeball (adult) Interleukin 20
25 A430056E07 C57BL/6 N0 Thymus Interleukin 21 receptor
26 A830047G15 C57BL/6 N10 Cortex Interleukin 23, α subunit p19
27 1110033L24 C57BL/6 18-day embryo Interleukin 25
2210408F01 C57BL/6 Stomach
2010016N09 C57BL/6 Small intestine
5830455M19 C57BL/6 Thymus
28 9330197K12 C57BL/6 Diencephalon Interleukin 4 receptor, α
A630083M23 C57BL/6 N3 Thymus
E430003K23 NOD (NOD) N2 Thymic
29 F830019G17 NOD Activated spleen from NOD.Cz Idd3 Interleukin 6
30 9530096I01 C57BL/6 U.bladder Interleukin 6 receptor, α
B230372K20 C57BL/6 C.quadrigemina
C230031H18 C57BL/6 N0 Cerebellum
31 A430091K05 C57BL/6 N0 Thymus Interleukin 7
A530098I02 C57BL/6 Aorta and vein
A630007F02 C57BL/6 N3 Thymus
D430026C11 C57BL/6 E13 lung
32 A430089D02 C57BL/6 N0 Thymus Interleukin 7 receptor
A530022C19 C57BL/6 Aorta and vein
A630041A17 C57BL/6 N3 Thymus
A630076J23 C57BL/6 N3 Thymus
33 E430023N04 NOD (NOD) N2 Thymic Interleukin 9 receptor
34 2700043J07 C57BL/6 Embryo 11 Interleukin enhancer-binding factor 2
3000002D13 C57BL/6 Embryo 12-head
6230405A16 C57BL/6 Head (embryo 11)
6330441H14 C57BL/6 Medulla
B130010B06 C57BL/6 Partenogenetic
35 5730447J21 C57BL/6 Embryo 8 Interleukin enhancer-binding factor 3
C130034D23 C57BL/6 E16 Head
E430021F21 NOD (NOD) N2 Thymic
36 3200002B17 C57BL/6 Embryo (14+17)-Head Interleukin-four induced gene 1
E330015O10 C57BL/6 P2 ovary
37 E130317D06 C57BL/6 N0 eyeball Nuclear factor, interleukin 3, regulated
38 C130076J06 C57BL/6 E16 Head Mus musculus IL-1F9 (IL1F9)
39 1110054H05 C57BL/6 18-day embryo Mus musculus similar to Interleukin enhancer-binding factor 1
a

Mixed library (AL,AK,AD,AF,AG,AC,AE,AQ,AP,AH,AA,AO,AB,AI,AJ,AN,AM)

A total of 61 clones represented 23 known growth factor (EGF, FGF, and TGF)-related genes (Table 3). The known mouse genes in the FANTOM2 set comprised 5 growth factors (EGF, FGF 14, FGF 15, TGFβ, and TGFβ 68 kD) and 18 other growth factor-related genes. Search of the protein translation of the GenBank database revealed that there are at least 140 nonredundant entries of various mouse growth factor (EGF, FGF, and TGF)-related genes (including variants and sub-units).

Table 3.

The List of 61 Clones Representing 23 Known Growth Factor-Related Genes Found in the FANTOM2 Data Set

Clone ID Mouse strain cDNA library Gene name
1 D730020G24 C57BL/6 L2 mammary Epidermal growth factor
D430013M15 C57BL/6 E13 lung
E430016A08 NOD (NOD) N2 Thymic cells
2 1300005M11 C57BL/6 Liver Epidermal growth factor receptor (Epidermal growth factor receptor isoform 3) homolog (Mus musculus)
1300008I23 C57BL/6 Liver
1300003K07 C57BL/6 Liver
3 C630023M09 C57BL/6 Hippocampus Epidermal growth factor receptor pathway substrate 15
4 9430061P05 C57BL/6 Upper body Epidermal growth factor receptor pathway substrate 15, related sequence
5 9430070A01 C57BL/6 E12 Upper body Epidermal growth factor receptor pathway substrate 15, related sequence
6 9830147J04 C57BL/6 Bone Epidermal growth factor receptor pathway substrate 15, related sequence
9830168E22 C57BL/6 Bone
7 5230401N02 C57BL/6 Xiphoid Epidermal growth factor-containing fibulin-like extracellular matrix protein 1
8 0610011K11 C57BL/6 Kidney Epidermal growth factor-containing fibulin-like extracellular matrix protein 2
9 0610042A18 C57BL/6 Kidney Eukaryotic translation initiation factor 3 subunit 2 (EIF-3 β) (EIF P36) (TGF-β receptor interacting protein) (TRIP-1) homolog (Mus musculus)
2410126D05 C57BL/6 ES
2700045C12 C57BL/6 Embryo 11
4930503E24 C57BL/6 Testis
10 1190024F07 C57BL/6 18-day embryo Fibroblast growth factor (acidic) intracellular-binding protein
2010004G08 C57BL/6 Small intestine
D930040K23 C57BL/6 E15 head
2810001F12 C57BL/6 Embryo 10 + Embryo 11
2010005N21 C57BL/6 Small intestine
3010027N18 C57BL/6 Embryo 12-Head
G430069F17 C57BL/6 Mixeda
11 5730551C10 C57BL/6 Embryo 8 Fibroblast growth factor 15
D030003J18 C57BL/6 Embryo 9
D230045H09 C57BL/6 E12 eyeball
12 0610042J09 C57BL/6 Kidney Fibroblast growth factor inducible 14
13 2810484E09 C57BL/6 Embryo 10 + Embryo 11 Fibroblast growth factor-regulated protein 2
1110020J11 C57BL/6 18-day embryo
14 9630030D05 C57BL/6 N16 Latent transforming growth factor β-binding protein 1
E330034H12 C57BL/6 P2 ovary
C730041O04 C57BL/6 SV40T antigen
9830146M04 C57BL/6 Bone
E230007P04 C57BL/6 P2 oviduct
15 1810044G20 C57BL/6 Pancreas Latent transforming growth factor β-binding protein 3
A230048E14 C57BL/6 Hypothalamus
B430105M07 C57BL/6 Adipose
16 E130107B17 C57BL/6 N0 eyeball MEGF 12 homolog (Mus musculus)
17 E130317D06 C57BL/6 N0 eyeball Nuclear factor, interleukin 3, regulated
18 1300004O05 C57BL/6 Liver TGFβ-inducible early growth response
A730095A08 C57BL/6 N7 Cerebellum
A430010P14 C57BL/6 N0 Thymus
19 1100001I05 C57BL/6 18-day embryo Transforming growth factor β 1-induced transcript 1
A530093G23 C57BL/6 Aorta and vein
D430029G15 C57BL/6 E13 lung
E030027O16 C57BL/6 N0 lung
20 1110002A05 C57BL/6 E13 lung Transforming growth factor β-regulated gene 1
2610001N22 C57BL/6 Embryo 10
2610029J18 C57BL/6 Embryo 10
21 D230045E03 C57BL/6 E12 eyeball Transforming growth factor, β-induced, 68 kD
22 1700105F20 C57BL/6 Testis Transforming growth factor, β receptor I
4930438I19 C57BL/6 Testis
1700105F20 C57BL/6 Testis
23 5730530B02 C57BL/6 Embryo 8 Hypothetical 30.1-kD protein (TGF β inducible nuclear protein TINP1) (hairy cell leukemia protein) (Homo sapiens)
6720481N02 C57BL/6 Extra testis (embryo 12)
2310004E21 C57BL/6 Tongue
2810027P06 C57BL/6 Embryo 10 + Embryo 11
3200001B21 C57BL/6 Embryo (14 + 17)-Head
0610010G24 C57BL/6 Kidney
5730530B02 C57BL/6 Embryo 8
a

Mixed library (AL,AK,AD,AF,AG,AC,AE,AQ,AP,AH,AA,AO,AB,AI,AJ,AN,AM)

Finally, 30 clones represented 19 known small inducible cytokine-related genes in mouse (Table 4). Search of the protein translation of the GenBank database revealed that there are at least 38 nonredundant entries of various small inducible cytokines in mouse.

Table 4.

The List of 30 Clones Representing 19 Known Small Inducible Cytokine-Related Genes Found in the FANTOM2 Data Set

Clone ID Mouse strain cDNA library Gene name
1 1810065I01 C57BL/6 Pancreas Small inducible cytokine A1
2 2310074D07 C57BL/6 Tongue Small inducible cytokine A11
2310011L12 C57BL/6 Tongue
3 2700043D19 C57BL/6 Embryo 11 Small inducible cytokine A12
4 1600023B02 C57BL/6 Placenta Small inducible cytokine A27
1600002M16 C57BL/6 Placenta
5 4921506E08 C57BL/6 Testis Small inducible cytokine A28
6 1010001B04 C57BL/6 Heart Small inducible cytokine A5
7 0610030C20 C57BL/6 Kidney Small inducible cytokine A6
2010308M07 C57BL/6 Small intestine
8 8430433J09 C57BL/6 Lung (embryo 16) Small inducible cytokine A7
9 1810063B20 C57BL/6 Pancreas Small inducible cytokine A8, MCP-2
10 1810031C02 C57BL/6 Pancreas Small inducible cytokine A17
11 A430095C13 C57BL/6 N0 Thymus Small inducible cytokine A20
12 A930010L22 C57BL/6 Retina Small inducible cytokine B, member 5
13 A630052H15 C57BL/6 N3 Thymus Small inducible cytokine B (Cys-X-Cys), member 9
A630095J17 C57BL/6 Thymus
14 A430052M02 C57BL/6 N0 Thymus Small inducible cytokine B (Cys-X-Cys), member 11
A430101A17 C57BL/6 N0 Thymus
C730001K22 C57BL/6 SV40T antigen transgenic mouse
15 4631412M08 C57BL/6 (EST_a) Skin (Neonate 0 day) Small inducible cytokine B (Cys-X-Cys), member 13
16 1110012L01 C57BL/6 (EST_a) 18-day embryo Small inducible cytokine B (Cys-X-Cys), member 14
1110031L23 C57BL/6 18-day embryo
1200006I23 C57BL/6 Lung
2810410F08 C57BL/6 Embryo 10 + Embryo 11
3300001J19 C57BL/6 Embryo 17-Head
17 3110001P14 C57BL/6 Embryo 13-Head Small inducible cytokine B, member 15
18 A430076J21 C57BL/6 N0 Thymus C, member 1 (lymphotactin)
19 6430571D05 C57BL/6 Olfactory brain Small inducible cytokine D, 1
A430078J23 C57BL/6 N0 Thymus

Clones representing 17 known mouse cytokine-related genes were derived from cDNA libraries from the non-obese diabetic (NOD) mouse. All of the remaining cytokine-related clones were derived from the C57BL/6 mouse strain. Six known genes were only found from the NOD mouse libraries. Clones representing a total of 11 known mouse genes were found in both C57BL/6 and NOD mouse cDNA libraries. The clones representing known cytokine-related genes were derived from a broad variety of RIKEN cDNA libraries. These libraries were generated from various tissues and developmental stages. Because this analysis focused on name matching from both the FANTOM2 data set and GenBank database, we can estimate that FANTOM2 clones cover ∼20% of existing known cytokine-related genes. Cytokines induce cascades of cytokine networks as well as of regulatory molecules. Often it is unclear whether the induced molecule acts as a cytokine or has some other regulatory role. In this study, we consider all clones as cytokine related and did not discriminate between the two groups.

We have also identified 15 candidate novel cytokine or cytokine-related genes (Table 5), including 7 candidate novel mouse IFN-related genes (13 clones), 3 candidates for novel IL-related genes (5 clones), and 4 candidates for novel growth factor-related genes (3 TGF- and 1 EGF-related) (16 clones). All of the clones representing candidate novel genes were derived from the C57BL/6 mouse and showed ≤96% identity over >50% length of the reference sequence.

Table 5.

The List of Candidate Novel Cytokine Related Genes Represented in the FANTOM2 Data Set

Clone ID DDBJa cDNA library Similarityb Similar gene name
1 1110004C05c 18-day embryo 89.8, 100.0 (145 AA) Interferon-inducible protein (Rattus norvegicus). Identical to 1110004C05RIK protein (Q9CQW9)
1810060G19 AK003507 Pancreas 89.1, 100.0
1810061A10 Pancreas 89.8, 100.0
2 4930507H06c AK076846 Testis 73.1, 75.9 (145 AA) Interferon-inducible protein (Rattus norvegicus). Identical to 4933438K12RIK protein (Q9D3R8)
4933438K12 Testis 64.8, 89.0
3 A330075D07c AK039626 Spinal cord 52.8, 81.8 (145 AA) Interferon-induced transmembrane protein 2 (interferon-inducible protein 1-8D) (Homo sapiens)
4 1110036C17c AK004121 18-day embryo 72.9, 78.1 (145 AA) Homolog to interferon-inducible protein
5 2310061N23c AK010014 Tongue 78.0, 97.6 (84 AA) α-interferon inducible protein (fragment) (Mesocricetus auratus)
6 A430075K09c AK040191 N0 Thymus 84.9, 100.0 (425 AA) Interferon-activatable protein 205 (IFI-205), also known as D3 protein (Mus musculus). Contains PAAD/DAPIN/Pyrin domain (Pfam 02758) and the associated HIN-200/IF120x domain.
9830003L02 Bone 77.3, 77.3
7 5330409J06c Pituitary gland 69.0, 93.6 IF-induced GUANYLATE-BINDING PROTEIN 5 (Homo sapiens)
9530054H01 AK030414 U. bladder 65.1, 29.3
E030025M22 N0 lung 69.0, 93.6 (551 AA)
8 B430113A10c AK080873 Adipose 87.6, 100.0 (123 AA) Hypothetical Toll/Interleukin receptor TIR domain structure containing protein
9 4732448K15c Skin (Neonate 10 day) 68.8, 100.0 (598 AA) Interleukin-1 receptor-associated kinase 2 (Homo sapiens)
9130227N12 AK028733 Cecum 75.5, 24.2
6330415L08 Medulla oblongata 64.9, 100.0
10 E230031K19c AK054215 P2 oviduct 76.3, 93.3 (556 AA) Interleukin 20 receptor α isoform 3 (Homo sapiens)
11 9830142A17 Bone 75.2, 99.8 (512 AA) Transforming growth factor-β-inducible early growth response protein 2 (TGFβ-inducible early growth response protein 2) (TIEG-2) (Kreppel-like factor 11) (Homo sapiens)
C330038O18 ES cells 75.0, 99.8
C730023J20 SV40T antigen transgenic 75.4, 99.8
D430010O15c AK084913 E13 75.0, 99.8
A230003P18 Hypothalamus 75.2, 99.8
D630011E14 N0 kidney 75.0, 99.8
E230024J15 P2 oviduct 75.2, 99.8
E430037H02 (NOD)N2 Thymic cells 75.1, 99.8
12 4921501K224c AK076631 Testis 93.7, 100.0 (237 AA) DJ977B1.4.1 (isoform 1) (TGIF2) (TGFβ-induced factor 2) (TALE family homeobox)) homolog (Homo sapiens)
5730599O09 Embryo 8 89.9, 100.0
13 B930011D01c AK047014 N10 Cerebellum 88.2, 84.4 (153 AA) Integrin, β-like 1 (with EGF-like repeat domains) homolog (Mus musculus)
14 2310046A13c Tongue 88.3, 26.7 Latent transforming growth factor-β-binding protein 4S homolog (Homo sapiens)
E330012C08 AK054297 P2 ovary 88.8, 96.5 (1505 AA)
A730031D03 N7 Cerebellum 95.4, 32.5
15 9130025L13c AK033606
Cecum 77.1, 99.5 (423 AA) FGF receptor 4B (Homo sapiens)
9530005F04 U. bladder 62.8, 99.5

Thirty-one clones representing fourteen candidate new genes

a

DDBJ accession no. of the representative transcript

b

Similarity was defined by two parameters, first the percent identity and second the percent of the length of the reference sequence (similar-gene) having similarity to the FANTOM2 clone. The number in the parentheses in the SIMILARITY field shows the length of the alignment or the maximum length of multiple alignment. All clones were derived from the mouse strain C57BL/6, except for clone E430037H02, which was derived from NOD mouse

c

Representative transcripts

Interferon-related Novel Gene Candidates

The clones 1110004C05, 4930507H06, A330075D07, and 1110036C17 represent four putative novel members of the membrane-associated family 1-8 of interferon-inducible proteins (Fig. 1). Sequences of these clones showed similarity to a variety of interferon-induced genes and proteins in human (Lewin et al. 1991), rat (Hayzer et al. 1992), cattle (Pru et al. 2001), and trout (SPTR Q8QFL3). Members of the 1-8 family of interferon (IFN)-inducible genes encode proteins thought to modulate cellular growth and adhesion wound healing, and inflammatory responses. It was hypothesized that they may function as membrane transport proteins (Pru et al. 2001). The 1-8 proteins participate in multimeric complexes involved in the transduction of homotypic and antiproliferative adhesion signals. Sequences of all four clones of novel candidate members of the 1-8 family contain a novel (not found in motif databases), motif DxxxWSxxxxxxxNxxCLGxxAxxxSxKSRDRKMVGDxxGAxxxxxTA that is conserved across species (fish, rat, mouse, and human). Furthermore, sequence of the clone 1110004C05 (Fig. 1) contains the motif YxxxxCLxI that conserves critical residues found in the active sites of human E2 ubiquitin conjugating enzymes (Pru et al. 2001). Sequences of the clones 4930507H06 and 1110036C17 contain similar motif FxxxxCLxI, whereas the remaining clone A330075D07 does not contain the corresponding motif. Each of the four candidate new members of the 1-8 family showed highest similarity to a distinct database entry and therefore each represents a distinct transcript (data not shown). The amino acid differences between the members of the 1-8 family at carboxy- and amino-termini may contribute to subtle functional differences or cellular localization.

Figure 1.

Figure 1

The alignment of conceptual translations of the four candidate novel mouse genes of the family 1-8 of IFN-inducible proteins with representative sequences from rainbow trout (Q8QFL3) and rat (Q9R175). A conserved motif characteristic for the members of interferon-induced 1-8 family of proteins in mice, rats, humans, cattle, and trout is shown with the alignment. Capital letters in the consensus motif stand for identical residues, + for conserved residues (aliphatic—I,V,L, or M; positively charged—K or R; aromatic—F,Y, or W), and x for any residue. The ubiquitin conjugating enzyme E2 active site motif in 1810061A10 [25] and a similar motif in 4930507H06 are shaded. SPTR stands for SWISS-PROT/TrEMBL database [15], and RI stands for RIKEN clone.

Sequence of the clone 2310061N23 showed similarity to the gene for the IFN-α-inducible protein p27-h found in hamster cardiomyopathic tissue (Denovan-Wright et al. 2000) and human gene TLH29 (Q9H2X8) from liver tissue (Fig. 2). The p27-h gene showed increased expression in cardiac and skeletal muscle tissue undergoing active necrosis. These sequences are similar (data not shown) to the human IFN-α-inducible protein p27 (GenBank NM_005532) shown to have increased expression in breast carcinoma (Rasmussen et al. 1993). The p27 protein is relatively small (11 kD) and highly hydrophobic. The p27-like mRNA is present in multiple tissues treated with the TNF, and it was speculated that these genes may be linked to inflammation related to the necrotic processes. However, the specific function of p27 has yet to be determined.

Figure 2.

Figure 2

Candidate novel gene represented by clone 2310061N23 similar to the fragments of IFN-α inducible proteins in human (Q9H2X8) and hamster (Q9QXH3). (+) Conserved replacements.

Clone A430075K09 represents a potential new member of a group of structurally related interferon-inducible proteins, the Ifi 200 cluster, that regulate biological activities of IFNs. The IFNs are immune response modulating cytokines that exhibit antiviral effects, induce expression of class I or class II major histocompatibility complex proteins, and help activate macrophages, natural killer cells, and neutrophils. Sequence of the clone A430075K09 shows similarity (84.9%ID, 425 amino acids, 100% of the total length) to the Ifi 205, the new member of the Ifi 200 family (Table 5 and Fig. 4, supplemental research data; available online at www.genome.org). Little is known about the precise function of the Ifi 205. However, it contains the PAAD/DAPIN/Pyrin domain (Pfam 02758), which is present in other members of the Ifi 200 family, indicating a possible protein–protein interaction site. This domain was predicted to have the same six α-helices-containing fold as the well-described Death Domain (Pfam 00531). The death domain is present in the TNFR-1 and FAS/APO1 receptors involved in cell death (Hofmann and Tschopp 1995). The potential functions of our candidate novel Ifi 205 factor, therefore, is the inhibition of cell proliferation and the arrest of cell growth, possibly through modulation of the activity of several transcription factors (Gribaudo et al. 1999).

Sequence of the clone 5330409J06 showed a weak similarity (Table 5 and Fig. 5, supplemental research data) to the guanylate-binding protein 5—GBP5 (P26376), published only as a database entry (GenBank AF288815). To our knowledge, the function and structure of GBP5 has not been described or published. In humans, the members of the GBP family are the most abundant class of proteins induced by IFN-γ (Prakash et al. 2000). GBPs have the ability to hydrolyze guanosine triphosphate (GTP) to both mono- (GMP) or di-phosphate (GDP) products (Schwemmle and Staeheli 1994) involved in cellular signal transduction. However, little is known about either the specific biological function of GBPs or their cellular localization (Vestal et al. 2000). Members of the GBP family were reported to have antiviral properties (Anderson et al. 1999), play a role in the macrophage activation processes (Han et al. 1998), and inhibit proliferation of endothelial cells (Guenzi et al. 2001). The clone 5330409J06 contains GBP (Pfam 002263) and GBP-C (Pfam 02841) domains. It is therefore a strong candidate for a novel gene—the mouse guanylate-binding protein mGBP-5.

Interleukin-Related Novel Gene Candidates

Sequence of the clone B430113A10 contains Toll/Interleukin receptor TIR domain structure and shows similarity (88% identity in 123 AA overlap) to the hypothetical 14.3-kD protein in long-tailed macaque (Table 5 and Fig. 6, supplemental research data). The TIR domain (Pfam 01582) is an intracellular-signaling domain found in MyD88, interleukin 1 receptor, and the Toll receptor. The Toll/Interleukin receptor domain is associated with a family of proteins that are involved in early responses to injury and infection (e.g., interleukin-1 receptor) (Slack et al. 2000). The Toll-like receptors in insects and mammals mediate innate defensive responses against a variety of microbial products (Anderson 2000). The clone B430113A10 thus represents a hypothetical protein potentially involved in immune responses.

Sequence of the clone 4732448K15 is similar (69% identity in full-length sequence of 598 AA) to the human IL-1 receptor-associated kinase-2 (IRAK-2), a proximal mediator of IL-1 signaling (Table 5 and Fig. 7, supplemental research data). IRAK-2 binds to the IL-1 receptor and is involved in IL-1-dependent signaling (Muzio et al. 1997). The clone 4232448K15 contains a Death Domain (Pfam 00531) (Hofmann and Tschopp 1995) present in many apoptosis pathway proteins. It also contains a catalytic Protein Kinase domain (Hanks and Hunter 1995) that has a conserved catalytic core common in both serine/threonine and tyrosine protein kinases. It is therefore a strong candidate for defining a novel gene—the mouse interleukin-1 receptor-associated kinase 2—mIRAK-2.

Sequence of the clone E230031K19 is weakly similar (76.3% identity over 93.3% length) to the human interleukin 20 receptor α isoform 3 (Table 5 and Fig. 8, supplemental research data). The conceptual translation (amino acids 1–292, PFAM 01108) contains a tissue factor motif (Muller et al. 1996) that is involved in blood coagulation. IL-20 was reported to be overexpressed in transgenic mice that have skin abnormalities similar to psoriatic lesions in humans (Dumoutier and Renauld 2002).

Growth Factor-Related Novel Gene Candidates

The FANTOM2 clone D430010O15 is a representative of a cluster (TIEG2-like) that showed similarity to human TGFβ-inducible early growth response protein 2 (TIEG2) (Table 5 and Fig. 9, supplemental research data). The conceptual translation of 9830142A17 showed a slightly higher similarity (75% identity) to the human, than to mouse TIEG2 (O89091) (74% identity). TIEG2 is ubiquitously expressed in human tissues, enriched in pancreas and muscle (Cook et al. 1998). Similarly, the TIEG2-like FANTOM2 clones were derived from eight different libraries. The TIEG2-like clone has a conserved zinc finger domain. TIEG2 is involved in cell growth, and its overexpression in hamster ovary cells inhibits cell proliferation (Cook et al. 1998).

Sequence of the clone 4921501K24 showed similarity— 93.7% identity over the full length—(Table 5 and Fig. 10, supplemental research data) to TGF-β-induced factor 2 (TGIF2), a homeobox gene of the TALE superclass, thought to play a role in the development and progression of some ovarian tumors through a gene-amplification mechanism (Imoto et al. 2000). TGIF2 was shown to repress TGF-β-responsive transcription through interaction with a TGIF-binding site in DNA (Melhuish et al. 2001).

Little is known about the integrin β-like 1 homolog (with EGF-like repeats), that showed similarity to the clone B930011D01 (Table 5 and Fig. 11, supplemental research data). The EGF-like domain is characteristic for extracellular domain of membrane-bound proteins (InterPro entry IPR000561). This product may possibly function as a cell-adhesion receptor.

The conceptual translation of clone E330012C08 showed similarity (Table 5 and Fig. 12, supplemental research data) to the human latent TGF-β-binding protein 4S, LTBP-4 (O75412). The LTPB-4 is a high molecular weight calcium-binding protein with EGF-like repeats. The cDNA of the LTBP-4 was found in several different forms generated by alternative splicing (Saharinen et al. 1998), consistent with the suspected frame shift positions found in the clone E330012C08. TGF-β proteins are multifunctional growth factors excreted as inactive precursors forming large protein complexes. These complexes include, among others, LTBPs which are believed to regulate both local tissue deposition of TGF-β and the related signaling (Sterner-Kock et al. 2002). The disruption of LTBP-4 in mice leads to ailments such as severe pulmonary emphysema, cardiomyopathy, and colorectal cancer (Sterner-Kock et al. 2002).

Finally, the conceptual translation of clone 9130025L13 showed similarity (Table 5 and Fig. 13, supplemental research data) to the human FGF receptor 4B—FGFR4b (Q9BXY2). FGF receptors are involved in multiple hormonal and proliferative processes of hormonal cells (Yu et al. 2002). A variant of the FGF receptor 4, related to the FGFR4b was found to increase tumor cell motility (Bange et al. 2002).

DISCUSSION

The mouse is the most popular model organism for both basic and applied immunological studies, including the study of cytokines. Our analysis of the FANTOM2 data set focused on the subset of transcripts that showed similarity to the cytokine-related products, and which had cytokine names or abbreviations in the name or description fields of the corresponding database entries (the cytokine-name criterion). Our study is only partial because databases include cytokine-related entries of genes and proteins that do not have an explicit cytokine name in the name or description fields. However, this study is representative because it focused on a well-defined subset of cytokine-related genes from which we can make an estimate of the coverage of cytokine-related genes in the FANTOM2 data set.

In comparison with database entries that conform to the cytokine-name criterion, we estimate that the ratio of cytokine-related products represented in the FANTOM2 data set is of the order of 20% of the total known cytokine-related products. This estimate is not surprising, because cytokines are inducible and often tissue-specific products. In addition, even when induced, the expression level of cytokines is often very low. The RIKEN libraries, although diverse, may not represent the optimal conditions required for induction of cytokines and their related genes. The regulatory processes involving cytokines are complex processes involving activation and suppression of multiple genes (Doly et al. 1998; Munshi et al. 1999; Vanden Berghe et al. 2000). This illustrates the need for creating more specific cDNA libraries that dynamically capture gene expression profiles for the study of the cellular processes involving cytokines.

It is not always possible, particularly in large-scale studies, to derive clear conclusions and explanations of the data. Our selection of the threshold that distinguishes novel from the known genes or gene candidates is arbitrary. The clone representing integrin β-like homolog (Fig. 11, supplemental research data) has only 10 amino acid differences to its reference sequence. Such clones may, therefore, represent variants or merely the inter-strain differences of the same transcript. Three of the clones representing the 1-8 family of IFN-inducible proteins (Fig. 1) do not appear to be full length and may represent poor-quality sequence data. We should also be aware of the possible problems with the reference data. The Ifi 205 reference sequence (Fig. 4, supplemental research data) has a triple repeat of the sequence AGSTAQ, which is missing in the corresponding FANTOM2 clone. This may possibly represent a sequencing artifact in the reference sequence. Conversely, the FANTOM2 clone having sequence similarity to human latent TGF-β binding protein (Fig. 12, supplemental research data) contains P+R-rich regions that are not present in the reference sequence and may represent artifacts. Mapping FANTOM2 clones to the mouse genome sequence at Ensembl (Hubbard et al. 2002) indicated that some of the FANTOM2 clones represented clear hits, whereas others, (especially Ifi 200 clones) had multiple hits. The multiple hits results may be explained by possible artifacts or poor sequence data, but may also represent the complexity of transcriptional regulation of inducible genes warranting further investigation.

Computational analysis has been applied previously to identification of novel cytokines from EST libraries, such as cardiotrophin-like cytokine (Shi et al. 1999). We have shown that this process can be enhanced by using the elimination strategy that involves multiple consecutive steps as follows: generation of multiple cDNA libraries, large-scale computational annotation, human curation, identification of relevant subsets of sequences, and individual clone analysis. This process involves a number of elimination steps—it is therefore critical that the loss of information is kept at a reasonable level. Too stringent criteria for elimination of clones from further analysis would lead to a small number of clones having a high-quality and high-confidence annotation. On the other hand, this would lead to the elimination of clones that represent results of complex transcriptional events, modifications, or variants. Too lenient criteria for the inclusion of clones for further analysis may lead to the proliferation of artifacts and errors into sequence data. To minimize the proliferation of errors, we excluded a number of clones from the analysis, including those that contained stop codons (data not shown).

We identified 15 candidates for novel cytokine-related genes. Because many cytokine-related genes do not contain cytokine names in their description, this should represent only a fraction of the novel cytokine-related candidate genes captured by the FANTOM2 library. Further strategies will involve making comprehensive lists of cytokine-related genes followed by comparison of these genes with the existing cDNA libraries and future cDNA libraries designed for capturing specific gene expression during the immune processes.

METHODS

The FANTOM2 set of full-length mouse cDNA clones, referred to as clones in this text, contains 60,770 sequences with the total length of almost 120 million base pairs and more than 12 million conceptually translated amino acids. The FANTOM2 clones were functionally annotated using automatic computational annotation followed by expert human curation (Okazaki et al. 2002). The overall process of identification of novel candidate genes is shown in Fig. 3).

Figure 3.

Figure 3

The overall process of identification and functional annotation of cytokine-related FANTOM2 clones.

Sequence Analysis

The sequences of the cDNA clones were compared with the genetic, protein, motif and protein domain databases. The genetic databases used for the analysis of clones included GenBank (Benson et al. 2002), LocusLink (Wheeler et al. 2002), UniGene (Wheeler et al. 2002), Mouse Genome Database (MGD) (Blake et al. 2002), and TIGR databases (Quackenbush et al. 2001). The comparisons to the genetic databases were performed using the nucleotide to nucleotide sequence local alignment algorithm BLASTN (Altschul et al. 1990). The protein databases used for the analysis of FANTOM2 clones included SWISS-PROT and TrEMBL (Bairoch and Apweiler 2000), and PIR (Wu et al. 2002). The comparisons were performed using the program FASTY (Pearson et al. 1997) that compares a DNA sequence with protein sequences in a database. FASTY translates a DNA sequence in three frames and aligns the translations to each sequence in the protein database, permitting gaps and frame shifts. Finally, the FANTOM2 clones were compared with the SCOP (Lo Conte et al. 2002), Pfam (Bateman et al. 2002), and InterPro (Apweiler et al. 2001) databases for identification of potential protein domains and functional sites, and to the MDS (Maximum Density Subgraph) Motif Database (Kawaji et al. 2002) for the presence of potential protein motifs. Unless specified otherwise, the gene products references throughout the text correspond to SWISS-PROT and TrEMBL (SPTR) primary accession numbers.

Functional Annotation and Identification of FANTOM2 Clones

Functional annotation of clones was performed in three steps as follows: data mining, manual annotation, and sequence analysis of the selected clones followed by literature text searching. First, the data-mining step involved a large-scale sequence comparison using BLASTN or FASTY against all of the selected databases. The results of these searches were accessible to the annotators via the FANTOM2 graphical interface, providing a convenient tool for the second step, the manual annotation. The most likely coding sequences (CDS) were determined by comparison of the results of four prediction programs (ProCrest, Decoder, rsCDS, and NCBI cds). Additional tools, such as ClustalW (Thompson et al. 1994) were available for facilitation of the functional annotation. The annotation results were then summarized and searched by keywords cytokine, interferon, IF, IFN, interleukin, IL, transforming growth factor, TGF, epidermal growth factor, EGF, fibroblast growth factor, and FGF. The clones that showed 50%–95% identity to the known cytokine-related genes were considered as candidate novel genes and were further analyzed using the programs BLAST or FASTA. The clone A530093G11 was also included in analysis, because its conceptual translation showed 96% identity over the 50% of the reference sequence length. Finally, literature searches in the PubMed (Wheeler et al. 2002) were performed for identification of key publications. The possible functions of the candidate novel genes were elucidated from literature sources.

Acknowledgments

Diego G. Silva is the recipient of a scholarship from the Canberra Hospital Salaried Specialists Private Practice Fund.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1016503.

Footnotes

[Supplemental material is available online at www.genome.org]

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