Characterization of the type I interferon locus and identification of novel genes

Genomics 84 (2004) 331 – 345 www.elsevier.com/locate/ygeno

Characterization of the type I interferon locus and identification $ of novel genes Matthew P. Hardy, a,b Catherine M. Owczarek, a,* Lars S. Jermiin, c Mikael Ejdeba¨ck, b and Paul J. Hertzog a a

Center for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia b Department of Biochemistry and Biotechnology Institute, Trinity College, University of Dublin, Dublin 2, Ireland c School of Biological Sciences and Sydney University Biological Informatics and Technology Center, Heydon-Laurence Building A08, University of Sydney, Sydney, New South Wales 2006, Australia Received 25 April 2003; accepted 3 March 2004 Available online 6 May 2004

Abstract The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-q1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense. D 2004 Elsevier Inc. All rights reserved. Keywords: Interferon type I; Human chromosome 9; Mouse chromosome 4; Genomic locus; Embryonic development

The type I interferons (IFNs) are a family of cytokines with pleiotropic activities that include inhibition of viral replication and cell proliferation and activation of the immune system [1]. These properties give the IFNs important physiological and pathological roles in infections and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of multiple IFN-a subtypes, as well as single IFN-h and -N subtypes [2,3]. A further human type I IFN, designated IFN-n and expressed only in epidermal keratinocytes, has similar biological functions and binds to the same receptor as other type I IFNs [4]. In mice, the same IFN subtypes exist, except for IFN-N and IFN-n [5]. A type I IFN-like cytokine called Limitin/IFN-like 1 has recently been identified in mice that bind the type I IFN receptor [6,7]. The trophoblast $ Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AY190044 – AY190047. * Corresponding author. Fax: +61-3-9594-7211. E-mail address: [email protected] (C.M. Owczarek).

0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.03.003

(H ) and delta (y) IFNs are produced by the trophectoderm and are involved in the maternal recognition of pregnancy in ruminants and pigs, respectively. In addition to their antiluteolytic properties, they also have antiviral, antiproliferative, and immunomodulatory effects on the endometrial epithelium, giving them a key role in development and early gestation [8]. Similar proteins have not to date been identified in humans or mice [9,10]. The human type I IFN gene cluster is distributed over approximately 400 kb on the short arm of Homo sapiens (HSA) chromosome 9 (9p21) [5,11,12]. The type I IFN gene cluster consists to date of 26 genes: 13 IFNAs, a single IFNB and IFNW, and 11 IFN pseudogenes [5,12]. The locations of a number of breakpoints within the type I IFN locus have been deduced; these have been associated with gliomas and leukemia, thus implicating the IFNs as tumor-suppresser genes [12]. The mouse type I Ifn gene cluster has not been well characterized, although individual Ifns have been localized to a cluster of similar size on the centromereproximal region of Mus musculus (MMU) chromosome 4 in a region of conserved synteny with HSA chromosome 9p21

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[5,13 –16]. Eleven functional interferon-a (Ifna) genes and one Ifn pseudogene have been identified in the mouse genome [5,17,18]. Of these functional genes, Ifna1, Ifna4, Ifna5, Ifna6 (also known as Ifna6-P and Ifna8), Ifna11, and Ifna7 have been physically mapped as a single group of genes by several authors [15,18,19]. A detailed annotation of the genomic loci containing the type I IFN gene cluster in humans and mice is an important step toward elucidating the full spectrum of the type I IFNs and will give further insight into their biological functions and pathological significance. Mice are also the animal model system closest to humans that can be genetically modified for in vivo functional studies. We present here a detailed analysis and annotation of the entire mouse type I Ifn locus and part of the human IFN locus. We identified a homologue of the mouse Ifnl1 (IFNlike1, Limitin) gene, Ifnl2, and cloned two further novel mouse Ifna genes, Ifna12 and Ifna13. In addition, we identified a novel IFN in humans and mice, designated IFN-q1, whose structure and mRNA expression patterns suggest that it may have a function distinct from those of the other members of the human and mouse type I IFN family.

Results Annotation of the type I interferon genomic locus To obtain a more complete annotation of the genes and potential transcriptional units in the mouse type I Ifn gene cluster, f430 kb of mouse genomic DNA located on MMU chromosome 4 at 42.6 cM (Fig. 1A) was analyzed in detail using the NIX suite of programs. Mouse Ifn genes, including Ifna1, Ifna2, Ifna3 (IfnaA), Ifna4, Ifna5, Ifna6, Ifna7, Ifna8 (IfnaB), Ifna9, Ifna10 (Ifna6-T), Ifna11, and Ifnb (IFN-h), were identified; these genes were subsequently confirmed by BLASTN and BLASTX analysis and through comparison of the respective cDNA sequences with the genomic sequence. Ifnl1/Limitin [6] was also detected in the mouse type I Ifn locus f10 kb 5V of Ifna7 (Fig. 1A). Of all type I Ifn genes within the mouse cluster on MMU chromosome 4, Ifnb was the most 5V (Fig. 1A). This gene, and all genes/transcriptional units as far as Kiaa1354, are transcribed in the same orientation (Fig. 1A); all genes and transcriptional units downstream of this,

but not including Ifnp6 and Ifne1, are transcribed in the opposite direction. In addition to the known functional mouse type I Ifn genes, two novel Ifna genes (Ifna12, Ifna13), a second Ifnl/Limitin (Ifnl2), six Ifn pseudogenes (designated Ifnp1 to 6), and five genes of unknown function were also identified by NIX analysis (Figs. 1A and 1B). Of the six Ifnp genes, one, designated Ifnp2, has previously been described [17]; the rest are novel pseudogenes. The Ifnp4 pseudogene had 91% nucleotide identity with human IFNW1 but contained multiple stop codons, suggesting that this may once have been a mouse orthologue of human IFNW1. A previously undescribed predicted gene was located at the extreme 3V end of the mouse type I Ifn locus. This has been designated IFN-q1 (Ifne1) (Figs. 1A and 1C), is intronless and transcribed toward the 5V end of MMU chromosome 4, and encodes an open reading frame of 192 amino acids. RepeatMasker analysis (excluding ambiguous bases) of the repetitive elements within the entire mouse type I Ifn locus (Figs. 1B and 1C and data not shown) indicated that they comprised f42% of the genomic sequence. Putative non-Ifn genes within the mouse type I Ifn locus The most 3V of the three putative non-Ifn genes was the mouse orthologue of human KIAA1354 (a cDNA isolated from human brain; GenBank Accession No. XM_027604) designated Kiaa1354 (Fig. 1B). Both human KIAA1354 and mouse Kiaa1354 are intronless and 4.0 –4.5 kb in size; they have 91% nucleotide identity and encode predicted polypeptides of f616 amino acids. In addition, a CpG island of f800 bp and putative polyadenylation signal sequences (AATAAA) were present at the 5V and 3V ends, respectively, of mouse Kiaa1354 and human KIAA1354 (Fig. 1B). The fact that human and mouse KIAA1354 genes are highly conserved and are present in positions of conserved synteny on HSA chromosome 9 and MMU chromosome 4 (data not shown) suggests that these genes are bona fide transcriptional units and not simply processed pseudogenes, although further studies will be required to confirm this. BLAST/ SwissProt analysis of these putative polypeptides revealed six conserved h-sheet-forming Kelch motifs, which are known to associate to form a h-propeller domain involved in protein –protein interactions and certain enzymatic activities [20,21]. A multiexon gene of unknown function spanning f20 kb of genomic DNA between Ifna13 and

Fig. 1. (A) Schematic representation of MMU chromosome 4 and an expanded representation of the mouse type I Ifn locus, with distances shown in kilobases. The Ifn genes and pseudogenes are denoted by closed and open triangles, respectively. The apex of each triangle points toward the direction of transcription. Arrows denote non-Ifn genes. The vertical lines represent the genomic contigs used to perform the sequence analysis. (B) Results of the NIX analysis of the genes from Ifnp1 to Ifnp3 found on a genomic contig. Ifn genes, pseudogenes, and non-Ifn genes are denoted as above to the right of the NIX output. The following programs were used to identify transcriptional units by NIX: (i) GRAIL/cpg; (ii) Fex; (iii) Hexon; (iv) MZEF; (v) GeneMark; (vi) GRAIL/exons; (vii) Genefinder; (viii) GENSCAN; (ix) FGenes; (x) HMMGene; (xi) BLAST/Trembl; (xii) BLAST/SwissProt; (xiii) BLAST/Unigene; (xiv) BLAST/mrna; (xv) GENSCAN/polya; (xvi) RepeatMasker. Outputs to the right of the central vertical black bar are analyses on the sense strand; those to the left are those on the antisense strand. (C) Results of the NIX analysis of the genes from Ifna5 to Ifne1 found on the telomeric genomic contig. Gene annotations and NIX program usage are as above.

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Ifna12 (Fig. 1B) contained 38% similarity in its exons to a mouse cDNA encoding a protein with similarity to the hypothetical protein DJ1198H6.2 (GenBank Accession No. LOC195555). However, no orthologue was found in a similar region on the HSA type I IFN cluster and no

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recognizable motifs or domains were identified upon BLAST/SwissProt analysis of the putative polypeptide. A mitochondrial ribosomal protein L48 pseudogene (Mrpl48) (GenBank Accession No. LOC269553) was located between Ifna12 and Ifna9 (Fig. 1B).

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Cloning of mouse Ifna12 and Ifna13 and the identification of IFN-like 2 (Ifnl2) Two new members of the Ifna gene family, designated Ifna12 and Ifna13, were detected within the type I Ifn locus (Fig. 1B). Both predicted proteins had greater than 90% amino acid identity to a consensus IFN-a amino acid sequence [5]. To determine whether these putative genes could encode cDNAs, we performed RT-PCR using genespecific primers and a mouse embryonic fibroblast cDNA library. Ifna12 was amplified as a 797-bp PCR product; Ifna13 was amplified as an 819-bp PCR product (Figs. 2A and 2B). No PCR products were generated when minus reverse transcriptase controls were performed using the same gene-specific primers, confirming that the amplified PCR products were amplified cDNAs and not the result of genomic DNA contamination (Figs. 2A and 2B). Direct sequencing of the RT-PCR products also confirmed the authenticity of the amplified cDNAs (data not shown). The nucleotide sequences of the isolated mouse Ifna12 and Ifna13 cDNAs have been deposited with GenBank (Accession Nos. AY190046 and AY190047). Like all type I Ifn genes, Ifna12 and Ifna13 are intronless. The 5V

untranslated regions of other reported Ifna cDNAs are only 67– 68 nucleotides long [16,22]; it is therefore unlikely that any further 5V cDNA sequence exists for either Ifna12 or Ifna13. The predicted proteins encoded by Ifna12 and Ifna13 (designated IFN-a12 and IFN-a13, respectively) are 189 amino acids in length and comprise a 23-aminoacid leader peptide sequence and 166 amino acids of mature protein. IFN-a12 and IFN-a13 each differ from the consensus mouse IFN-a amino acid sequence by only a few amino acids (Fig. 2C); hence, they can be classified as novel IFN-a family members. An intronless transcriptional unit (GenBank Accession No. LOC230400) f3 kb 5V of mouse Limitin/Ifnl1 (Fig. 1A) was located near the center of the mouse type I Ifn gene cluster. This transcriptional unit contains a 549-bp open reading frame encoding a mature protein of 159 amino acids with a leader peptide of 23 amino acids and is identical to Limitin/interferon-like 1, except for seven amino acid substitutions. We have therefore designated this putative gene Ifnl2 (interferon-like 2). An alignment of the amino acids of Limitin/interferon-like 1 and interferon-like 2 is shown in Fig. 2D. We also identified a 700-bp region f3 kb 3V of Limitin/Ifnl1 with 96% nucleotide identity to Limitin/Ifnl1

Fig. 2. RT-PCR analysis of (A) Ifna12 and (B) Ifna13. Gene-specific primers were used to amplify these cDNAs from mouse embryonic fibroblast cDNA. Minus reverse transcriptase and Gapdh controls are indicated. (C) Alignment of the amino acid sequences of mouse IFN-a1 and the novel IFN-a12 and IFNa13. The consensus IFN-a sequence is shown above for reference. Hashes (#) indicate conserved amino acids and dots (.) indicate amino acid identity. (D) Alignment of the amino acid sequences of Limitin/IFNL1 and IFNL2. Leader sequences are italicized. Identical amino acids are shaded dark gray and similar amino acids are shaded light gray.

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(Fig 1A). A complete open reading frame could not be obtained, due to gaps in the sequence available, but the first 15 amino acids had 100% identity to those of Limitin/

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interferon-like 1 itself. It is possible that this region encodes a second Limitin/interferon-like 1 homologue (Ifnl3). Given the close proximity of these genes and their high identity, it

Fig. 3. Schematic representation of HSA chromosome 9, including an expanded representation of the centromeric end of the human type I IFN locus with the graphical output of the NIX analyses of the same genomic region. The programs used to identify transcriptional units by NIX and the names of the IFN genes and pseudogenes are given in the legend to Fig. 1. Outputs to the right of the central vertical black bar are analyses on the sense strand; those to the left are those on the antisense strand. The position of the IFNK gene outside the type I IFN locus is also shown.

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is likely that these genes arose by gene duplication from a common ancestor. In support of this we identified a 500-bp region proximal to the 5V end of Limitin/Ifnl1 that was also duplicated in the proximal 5V flanking regions of the other Limitin/Ifnl1 homologues. Cloning of human IFNE1 and mouse Ifne1 To determine whether a human orthologue of mouse Ifne1 existed, we analyzed f150 kb of genomic DNA in the region of conserved synteny on HSA chromosome 9p21. Most or all gene prediction programs detected the type I IFN genes previously localized [12] on HSA 9p21, including IFNA1, IFNA2, IFNA6, IFNA8, and IFNA13 (Fig. 3); these were confirmed by BLASTN and BLASTX analysis and comparison of the respective cDNA sequences against the genomic sequence. We also detected five a and N IFN pseudogenes (IFNAP11, IFNAP23, IFNWP2, IFNWP12, and IFNWP19) (Fig. 3). A small unit designated MXI1, which was repeated four times throughout the telomeric end of the type I IFN gene cluster, had weak (f40%) amino acid similarity to MAX-interacting protein-1 (GenBank Accession No. XM_129299) but is probably a pseudogene

since it had no open reading frames greater than 20 amino acids. All genes from IFNWP19 to IFNWP12 are transcribed toward the centromere of HSA chromosome 9; all genes telomeric of IFNAP11 are transcribed toward the telomere. A predicted transcriptional unit with identity to mouse Ifne1 was also identified at the extreme centromeric end of the HSA type I IFN locus. This predicted human gene, also designated IFN-q1 (IFNE1) (Fig. 3), is intronless and is transcribed toward the telomere of HSA chromosome 9; it encodes a putative open reading frame of 208 amino acids (IFN-q1). The relative position of the IFNE1 gene within the type I IFN locus are also conserved between humans and mice (Figs. 1A and 3). To determine whether human IFNE1 and mouse Ifne1 encoded cDNAs, we amplified fragments encoding human IFNE1 (642 bp) and mouse Ifne1 (664 bp) from human WISH cell cDNA and mouse embryonic fibroblast (MEF) cDNA, respectively (Figs. 4A and 4B). No PCR products were amplified in minus reverse transcriptase reactions, indicating that the PCR had amplified cDNA and not genomic DNA. The nucleotide sequences of the cloned human IFNE1 and mouse Ifne1 cDNAs were identical to their respective genomic DNA sequences and have been

Fig. 4. Expression analysis of human and mouse IFNE1. RT-PCR analysis of (A) human IFNE1 and (B) mouse Ifne1 expression in a variety of primary cells, cell lines, and tissues. Gene-specific primers were used to amplify these cDNAs and minus reverse transcriptase controls were also performed. A Gapdh control is indicated. Specific internal oligonucleotides were used to increase the level of detection by hybridization to the RT-PCR-generated cDNAs. (C) Northern blot analysis of poly(A)+ mRNA from a number of mouse cell lines showing the distribution of the 1.5-kb mouse Ifne1 mRNA transcript. Blots were sequentially hybridized with a 664-bp mouse Ifne1 and a Gapdh cDNA probe. (D) Northern blot analysis of poly(A)+ mRNA from whole-mouse embryos between E8 and E12 of development and from placentas of pregnant female mice at E10, E14, and E18 days gestation. The same cDNA probes were used as above. (E) Northern blot analysis of poly(A)+ mRNA from L929 cells stimulated with SFV for 0 and 12 h. Blots were sequentially hybridized with a 664-bp mouse Ifne1 and a Gapdh cDNA probe. The Northern blots were then exposed to a Phosphor screen and the radioactivity in each hybridizing band was quantified using a PhosphorImage analyzer with MacBas v2.5 software.

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deposited with GenBank (Accession Nos. AY190044 for mouse Ifne1 and AY190045 for human IFNE1). Southern blot analysis of restriction endonuclease-digested human and mouse genomic DNA showed that the human IFNE1 and mouse Ifne1 genes occur as single copies in the two genomes and confirmed the predicted restriction patterns determined from the genomic sequence (data not shown). Expression analyses of human IFNE1 and mouse Ifne1 To obtain information that would contribute to our understanding of the biological functions of IFN-q1 in both humans and mice, we determined their mRNA expression patterns by both RT-PCR and Northern blot analysis on a number of human and mouse tissues and/or cell lines. RTPCR analysis showed that human IFNE1 was expressed in the human prostate cancer cell line PC-3, amnion-derived WISH cells, SK-MEL28 melanoma cells, and Daudi cells and very weakly in MCF-7 human breast cancer cells (Fig. 4A). Human IFNE1 mRNA was not detected in Namalwa cells (Burkitt’s lymphoma) or in Jurkat (T) cells (Fig. 4A). Mouse Ifne1 mRNA was expressed in ovaries, uterus, fibroblasts, lung, brain, adrenal glands, and heart but was not detected in thymus, spleen, testis, liver, and kidney (Fig. 4B). Although this RT-PCR analysis was nonquantitative, the ovary and uterus appeared to express relatively higher levels of mouse Ifne1 mRNA than other mouse tissues. Northern blot analysis of mouse Ifne1 mRNA expression revealed a single mRNA transcript of approximately 1.5 kb in L929 cells, MEFs, and M1 cells (Fig. 4C). A 1.5-kb transcript encoding mouse Ifne1 was just detectable at all stages of embryonic development from E14 in the developing brain, liver, lung, kidney, large intestine, and thymus (data not shown). Interestingly, a 0.9-kb transcript in addition to the 1.5-kb mRNA transcript (Fig. 4D) was present in total mouse embryos from E8 to E12. The 0.9-kb mRNA transcript was also present in the placentas of gestating mice at various stages of pregnancy, with the highest expression occurring at E10 (Fig. 4D). The expression of mouse IFN-q1 in the placenta is consistent with this protein having a role in embryonic development. Induction of mouse Ifne1 mRNA by virus With the exception of the ruminant IFN-H s the majority of type I IFNs have the ability to be induced by viral infection. To determine whether the expression levels of Ifne1 were also able to be up-regulated upon viral infection, mouse L929 cells were treated with Semliki Forest virus (SFV). Northern blot analysis was then performed on poly(A)+ mRNA isolated from these cells using an Ifne1 cDNA probe. Ifne1 mRNA was constitutively expressed and when the levels of the mRNA transcripts encoding Ifne1 were quantified relative to Gapdh the levels increased only threefold 12 h after infection by SFV (Fig. 4E).

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Comparative analysis of human IFNE1 and mouse Ifne1 loci Elements regulating gene expression have previously been identified by comparing human and mouse coding and noncoding genomic sequences in regions of conserved synteny [23]. We therefore aligned part of the human genomic locus containing IFNE1 and IFNA1 (GenBank Accession No. NT_023974.11) with the segment of the mouse genomic locus containing Ifne1 and Ifna1 (Contig GA_x5J8B7W6711) using the program AVID [24] and visualized the results using the program VISTA [25]. Fig. 5A shows that the IFNE1 and IFNA1 genes were highly conserved between human and mouse. In addition, f1 kb of the 5V flanking sequence of human IFNE1 had over 70% identity with f1 kb of the mouse Ifne1 5V flanking region. This suggests that the proximal promoter of both human IFNE1 and mouse Ifne1 may be contained within this genomic region. A more detailed analysis of this region was then performed using ClustalW (Fig. 5B). Putative transcription factor binding sites were conserved between the human and the mouse sequence. These conserved motifs include sites for the signal transducers and activators of transcription (STATs) [26], progesterone receptor response element (PRE) [27], and CCAAT/enhancing protein h (CEBPh) (Fig. 5B). Comparative analysis of IFN family proteins The phylogenetic analysis of the IFN peptides identified a single most likely tree (Fig. 6) and 3736 near-optimal trees, none of which differed significantly from the former tree. Based on these 3,737 trees, a majority-rule consensus tree was inferred (Fig. 6); this tree is not as likely (lnL = 9733.76) as the most likely tree (lnL = 9724.15) but the difference is insignificant ( p c 0.71). Importantly, the consensus tree differed from each of the 3736 near-optimal trees, implying that topological model uncertainty may be more widespread than this analysis suggests. The second phylogenetic analysis produced yet another near-optimal tree; hence, we conclude that there is evidence of topological model uncertainty and that its size is underestimated, which, in turn, means that the relative likelihood scores may be somewhat biased. The length of the tree is 14.4, implying that every site in the alignment has changed on average 14.4 times, which, in turn, implies that interpretation of the phylogenetic tree must be done with caution; this is also reflected in the many low relative-likelihood score (RLS) values (Fig. 6) and the many low local bootstrap probability (LBP) values (data not shown). Rooted using the fish IFN sequences, the phylogenetic tree shows that there is a well-defined separation among most of the subtypes of IFN. All the IFN-a peptides group together, except the chicken and duck IFN-a peptides, which, in light of this phylogenetic tree, may need to be

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reclassified as a new subtype of IFN. Among the IFN-a subtype, there is a clear separation between those found in the mouse and those in the human genome. This implies that many gene duplications must have occurred independently within these two genomes since their divergence f120 Mya [28] and raises the question of whether the products of the gene duplicates have maintained the same function or acquired different functions. The IFN-N and IFN-H subtypes form a strongly supported sister group to the IFN-a subtype, and it appears that the IFN-H subtype has arisen from the IFN-N subtype. Whether this is an analytic artifact or a reflection of the evolutionary history is uncertain because there is strong evidence of lineage-specific differences in the evolutionary rate (IFN-H appears to have evolved much faster than IFNN). A more rigorous phylogenetic analysis of a larger alignment comprising the IFN-N and IFN-H subtypes is required to shed light on this particular part of the phylogenetic tree. The IFN-a, IFN-N, and IFN-H subtypes form another well-supported sister group to the Limitin and IFN-y subtypes. It is, however, uncertain whether these two subtypes should be remain defined as different subtypes or should be united into a single subtype. The edges leading to pig IFN-y and mouse Limitin sequences are very long (Fig. 6), lending support to the notion that the two subtypes should remain as

currently defined but more sequence data are really needed to address this question in more depth. The IFN-h and IFN-q subtypes form strongly supported sister groups to each other but the relationship between those groups, the IFN-n subtype, and the clade comprising the IFN-a, IFN-y, IFN-N, IFN-H , and Limitin subtypes is clearly uncertain. Part of the problem may be due to the small number of peptides representing the IFNh, IFN-y, IFN-q, IFN-n, and Limitin subtypes, but a more likely reason is the loss of clear phylogenetic signal, which is implied by the long edges in the tree (Fig. 6). A more rigorous phylogenetic analysis of an expanded alignment comprising these subtypes may resolve the uncertainty. The chicken and duck IFN-a peptides form a strongly supported sister group to all the other tetrapod IFN peptides. If the former two peptides indeed belong to the IFN-a subtype, then we can conclude that the other subtypes must have arisen from within the IFN-a subtype. However, this seems unlikely because we would have expected to find them also in many of the other mammalian genomes that have already been surveyed. A more probable explanation is that the chicken and duck IFN peptides belong to an as yet undefined subtype. More rigorous phylogenetic research of a data set comprising many more IFN peptides is needed to address this important issue.

Fig. 5. Alignments of mouse and human IFNE1 genomic loci. (A) Global alignment of the mouse and human region encompassing the IFNE1 and IFNA1 genes using the program AVID. Visualization of the graphical output was performed using the program VISTA. Numbers on the y axis represent the percentage identity of nucleotides in a 100-bp window with a cut-off of 50%. Numbers on the x axis indicate the nucleotide position from f10 kb 5V of IFNE1 to a position f2 kb 3V of IFNA1. Conserved coding regions where the average identity is >70% are shaded in blue; conserved noncoding sequences are shaded in pink. (B) Alignment of approximately 1 kb of 5V flanking region of human IFNE1 and mouse Ifne1. Asterisks between sequences indicate nucleotide identity. Conserved transcription factor binding sites are shaded (dark gray) and labeled; other, nonconserved transcription factor binding sites are also shaded (light gray); conserved core ETS-binding motifs are underlined. Known 5V untranslated sequences are shown in bold and the ATG is underlined.

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Fig. 5 (continued).

Structural analysis of human and mouse IFN-e1 The primary, secondary, and tertiary structure predictions are consistent with mouse and human IFN-q1 having overall similarity to the type I IFN class of cytokines. According to the above phylogenetic relationships the new IFN-q’s are more closely related to IFN-h and IFN-n and more distantly related to IFNs H , N, and y. Hence they have been designated IFN-q, i.e., a new subtype rather than an existing one since there is so far insufficient evidence to support the

designation of these IFNs as orthologues of any existing type I IFN. The type I IFNs have a characteristic barrel-shaped tertiary structure formed by the up –down arrangement of five a helices [29]. The mature 185-amino-acid human IFNq1 was 15 residues longer than mouse IFN-q1, otherwise they shared 54% amino acid identity plus 15% amino acid similarity. Secondary structure analysis predicted that human IFN-q1 and mouse IFN-q1 have six and five putative a helices, respectively (Fig. 7A). Helix F on human IFN-q1

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Fig. 6. Phylogeny showing the inferred evolutionary history of the IFN family. The majority-rule consensus tree was inferred using topological model averaging based on the most likely tree and the 3736 near-optimal trees found during the maximum-likelihood analysis. The relative-likelihood scores (0 – 100%), which are associated with the internal edges, represent the level of confidence that one can have in these edges. All the edges are drawn to scale (the scale bar represents 100 substitutions per 100 sites).

was located on its C-terminal extension and is not present on mouse IFN-q1 (Fig. 7A). The amino acids within helices A – E and the putative A – B loop are well conserved between human and mouse IFN-q1 (Fig. 7A), but only helices B, D, and E and the A – B loop of human IFN-q1 (and, to a lesser extent, mouse IFN-q1) show any degree of conservation

with other type I IFNs (Fig. 7B). A small a helix was predicted in both human and mouse IFN-q1 in the A – B loop (Fig. 7A), although not all programs used predicted this. The structure of several type I IFNs has been solved by X-ray crystallography or nuclear magnetic resonance [30 –

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Fig. 7. Analysis of mouse and human IFN-q1 amino acid sequences. (A) Alignment of the human (Hu) and mouse (Mu) IFN-q1 amino acid sequences. Leader sequences are italicized. Identical amino acids are shaded dark gray and similar amino acids are shaded light gray. Putative a helices are boxed and labeled underneath. (B) Alignment of human IFN-q1 with other type I IFNs. Putative a helices are boxed and labeled underneath. (C) Predicted three-dimensional ribbon structures of human and mouse IFN-q. The positions of helices A to E are labeled.

33]. The relatively high sequence homology between the amino acid sequences of the known IFN structures and that of IFN-q1 has made it possible to use these three-dimensional structures as templates for homology modeling and thereby predict the structures for human IFN-q1 and mouse IFN-q1. Using this approach, we showed that both human and mouse IFN-q1 have putative tertiary structures very similar to each other (Fig. 7C) and to other type I IFNs. Helix F of human IFN-q1 was unable to be modeled due to poor homology with other type I IFNs and a small helix within the C – D loop of mouse IFN-q1 not determined by secondary structure prediction is also shown (Fig. 7C). These data suggest that the predicted structures of both human and mouse IFN-q1 have the general characteristics of type I IFNs.

Discussion The characterization of gene clusters has provided important insights into the physiological and pathological functions of related genes. Functionally related genes are often clustered together: see, for example, the major

histocompatibility genes [34], the antimicrobial h-defensin genes [35], the class II cytokine receptor genes [36], and the genes encoding cytokines, such as interleukins 4, 13, and 5. This may give efficiency for coregulation, for example, via locus control regions [23]. Functional clusters of genes may have important pathological consequences such as the chromosomal translocations or deletions that occur in cancer. The type I IFNs are an important class of genes and their coordinate expression is crucial in homeostasis and host defense. Furthermore, deletions of HSA chromosome 9 may be important in carcinogenesis [12]. We therefore undertook a comprehensive annotation of the type I Ifn cluster at 42.6 cM on MMU chromosome 4. Table 1 outlines all the known mouse and human IFNs and specifies which are unique in humans and which are unique in mice. We discovered the presence of 13 Ifna genes, Ifnb, at least 2 Limitin/Ifnl (Ifnl2 and Ifnl3) genes, 6 Ifnp pseudogenes, and 4 putative novel genes in a 430-kb region of genomic DNA. Six of these Ifnas at the centromeric end of the cluster have previously been physically mapped together in tandem array [16,18,19]. The two novel Ifns have been designated Ifna12 and Ifna13. Both genes encode putative peptides with

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sequences that differ from the consensus IFN-a sequence by only a few amino acids; hence, they are assigned to the IFNa subtype. All mouse Ifn genes found on the 5V genomic contig were arranged in tandem with the direction of transcription towards the 5V telomere. Given the high nucleotide and amino acid similarity between the different mouse Ifnas, it is likely that these genes arose by gene duplication via unequal recombination following the divergence of Ifna and Ifnb 253 million years ago [12,37,38]. This is consistent with the high percentage of repetitive sequences within the mouse type I Ifn locus. At the 3V end of the mouse type I Ifn gene cluster, the Ifnas are arranged in tandem with the direction of transcription toward the 3V telomere. The notion that these genes arose by successive gene duplication is

Table 1 (continued)

Table 1 Human and murine type I interferon genes

Highlighted IFNs represent those unique to their species and those in bold denote novel IFNs.

much more apparent than elsewhere in the locus, and it is likely that these duplications are recent since there are no pseudogenes present between Ifna7 and Ifna1. The reasons for these duplications are as yet unknown, although it is tempting to speculate that the production of IFNs for host defense required a greater range of genes for survival. While it has been assumed that there is no functional mouse equivalent of human IFNW1, the Ifnp4 pseudogene is similar to human IFNW1, so it is possible that they have a common ancestor and that the mouse orthologue was rendered nonfunctional. Mouse Kiaa1354 contains a substantial open reading frame, is encoded by an expressed sequence tag, contains a CpG island at its 5V end and polyadenylation signal sequences, and has a human orthologue in a position of conserved synteny in the human type I IFN locus on HSA 9. There is another transcriptional unit between Ifna13 and Ifna12 but there is insufficient sequence information to determine its status as a real gene or pseudogene. A mitochondrial ribosomal protein pseudogene is located adjacent to Ifna12. Future experiments will clarify the identities and functions of these genes. The existence of these genes would make deletion of the type I Ifn locus impractical for studying the function of the type I IFNs. At the 3V ends of both the mouse and the human type I IFN loci are genes encoding a novel type I IFN, designated

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IFNE1. These putative IFNs, like mouse Limitin/Ifnl1, share limited homology to other type I IFNs but their presence within the type I Ifn locus and their structural similarity, despite subtle variations compared with other type I IFNs (as determined by computer modeling), suggest that they are bona fide type I IFNs. Their ability to bind the type I IFN receptor complex is unknown as yet, but would confirm them as type I IFNs. Both mouse Ifne1 and human IFNE1 are expressed in cells and tissues with a reproductive function, such as the uterus and ovary, and in the placentas of gestating mice. No human or mouse equivalent of IFN-H , the pregnancy recognition IFN in ruminants, such as sheep and cattle [9], has yet been found. The analysis described here indicates, based on coding sequence, that the IFN-q1 are most closely related to IFN-hs from an evolutionary perspective. However, these observations, together with the proposed developmental functions of the type I IFNs [39], suggest that mouse and human IFN-q1 may have a role in reproduction and/or development. This is consistent with the conserved progesterone receptor binding sites [27] in the proximal promoters of both human IFNE1 and mouse Ifne1 since progesterone is essential for establishing and maintaining pregnancy in mammals [40]. Consequently, type I IFNs may have an important role in reproduction in mice and humans. Their true function will require further experimentation.

Materials and methods Analysis of the type I IFN locus Approximately 300 kb of mouse genomic DNA from contig GA_x5J8B7W6GG0 (encompassing the centromeric end of the type I Ifn locus) and f120 kb of mouse genomic DNA from contig GA_x5J8B7W6711 (encompassing the 3V end of the type I Ifn locus on MMU chromosome 4) was obtained from Celera (www.celera.com). Whenever possible, ambiguous bases in these data were identified by comparison with Contig NW _ 000209 from NCBI (www.ncbi.nlm.nih.gov). Approximately 150 kb of human genomic DNA from contig NT_023974.11 (encompassing the telomeric end of the type I IFN locus on HSA chromosome 9p21) was also obtained from NCBI. Identification of transcribed nucleotide sequences and repeat sequences in the genomic DNA was done by using NIX from the Human Genome Mapping Project’s Resource Centre in UK application (http://www.menu.hgmp.mrc.ac.uk/menu-bin/Nix/ Nix.pl). Sequences obtained from these contigs were compared with those of known human genes (IFNA1, NM_024013; IFNA8, NM_002170; IFNA2, NM_000605; IFNA13, NM_006900; IFNA6, NM_021002), human pseudogenes (IFNWP19, LOC169548; IFNWP2, LOC138685; IFNWP12, LOC158011), predicted human genes (human KIAA1354, XM_027604; LOC195555; and AK007653), mouse genes (Limitin/Ifnl1, AB024521; Ifna1,

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NM_010502; Ifna2, NM_010503; Ifna3 (IfnaA), M28587; If na4 , N M _ 01 050 4; Ifna5 , N M _ 010 50 5; Ifna6 , NM _ 008335; Ifna7, NM _ 008334; Ifna8 (IfnaB), NM_008336; Ifna9, M13660; Ifna11, NM_008333; Ifnb, NM_010510; Ifnp2, M10076), predicted mouse genes (Kiaa1354, LOC242521; Mrpl48, LOC269553; Ifnl2, LOC230400; and LOC242520), and predicted fish genes (Tetraodon, CAD67762; Takifugu, CAE47314; Dario_a, CAD67753; Dario_b, CAD67754; Dario_1, AMM95448; Dario_2, CAD67752) obtained from NCBI’s Web site. Alignment and phylogenetic analysis of the interferon proteins The amino acid sequences of 48 IFNs from human, mouse, chicken, sheep, goat, musk ox, giraffe, cow, pig, horse, rabbit, duck, zebrafish, Takifugu rubripens, and Tetraodon negroviridis (all obtained from GenBank or sequenced as part of this project) were aligned using ClustalW [41], and the result was subsequently refined using GDE [42] to yield an alignment that is available as supplementary material via the World Wide Web (http:// www.bio.usyd.edu.au/fjermiin/alignment_37.pdf). The alignment was analyzed using ProtML [43] with the JTT-F model of amino acid substitution [44]. Initially, we surveyed the tree space with the aim of producing a set of 5000 near-optimal trees (using the –jfqn 5000 option). This set of trees was then analyzed more rigorously (using the – jfu options) and later compared statistically using the Kishino –Hasegawa test [45]. Topological model uncertainty [46] was considered using model averaging [47]; when considered, we used the Class V weighting scheme with a = 0.05 to produce (i) a majority-rule consensus tree and (ii) RLSs for internal edges in the inferred phylogeny. Subsequently, a second phylogenetic analysis was done, but this time we first generated a neighbor-joining tree using NJDist [43] and a distance matrix obtained using ProtML with the –jfD option; this tree was then changed using the nearest-neighbor interchange algorithm (using ProtML with the –jfRu option) with the aim of finding the most likely tree. LBPs were estimated using the RELL method for internal edges in the trees examined. Other computational analysis Putative open reading frames were inferred using MacVector (MacVector v7.1; Oxford Molecular Ltd.). Transcription factor binding sites were identified using MatInspector Release Professional [48]. Secondary structure elements were predicted using the algorithms available from NPS [49] (http://www.npsa-pbil.ibcp.fr.) and with the algorithms available from SwissProt [50]. Global alignment of the human and mouse genomic sequence of conserved synteny was performed as previously described [25,51]. A higher resolution comparison of the conserved noncoding sequences upstream of human IFNE1 and mouse Ifne1 was carried

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out using ClustalW v1.8 (http://www.searchlauncher. bcm.tmc.edu). Cell culture and isolation and Northern analysis of poly(A)+ mRNA All cell lines used, except MEFs, were cultured essentially as previously described [52]. MEFs were isolated from mice of mixed genetic background and maintained in DMEM (Invitrogen, Ontario, Canada). Poly(A)+ mRNA was prepared as previously described [53] from cells grown to midlog phase or from tissues dissected from C57BL/6 mice and snap-frozen in liquid nitrogen. Northern blots of poly(A)+ mRNA were prepared and hybridized with a 32Plabeled 664-bp mouse Ifne1 cDNA or Gapdh cDNA probe, as previously described [52]. Reverse-transcriptase polymerase chain reaction Preparation of cDNA was carried out using Omniscript reverse transcriptase (Qiagen, Chatsworth, CA, USA) and a cDNA synthesis primer (5V-TTCTAGAATTCAGCGGCCGC(T)30N.1N-3V) according to the manufacturer’s recommendations. Minus reverse transcriptase samples were prepared as above, but without the addition of Omniscript. Amplification of Ifna12 cDNA was carried out on a Perkin –Elmer 2400 thermocycler using the following conditions: 2.5 Al 10 Taq buffer (Promega, Madison, WI, USA), 0.5 Al 10 mM dNTPs, 3.0 Al 25 mM MgCl2 (Promega), 0.175 Al Taq (5 U/Al; Promega), 2.5 ng cDNA, 1.0 Al 5V primer (10 AM; 5V-AGAGAACCTAGAGGGGAAGGTTCAGGA-3V), 1.0 Al 3V primer (10 AM; 5V-CCTTTGCAGAGAGACATGACATTGCTA-3V), 15.825 Al H 2 O. PCR cycling conditions were 94jC for 30 s, then 35 cycles of 94jC for 20 s, 60jC for 20 s, 72jC for 1 min. Amplification of Ifna13 cDNA was performed as above with the exception of the 5V and 3V primers (5V-AGAGAACTTAAAAGGGAAGACTCAGAACTA-3V and 5V-AAGACTGACAGACTGAAAATAAATACATGC-3V, respectively) and the annealing temperature was 53jC. Amplification of human IFNE1 cDNA was performed as above with the exception of the 5V and 3V primers (5V-TTCACCATGATTATCAAGC A C T T C T T T G G A - 3 V a n d 5 V- T C C C T C C A C C TACCTCGGGCTTCT-3V, respectively) and the annealing temperature was 62jC. Amplification of mouse Ifne1 cDNA was performed as above with the exception of the 5V and 3V primers (5V-AGATCCCTGTGTGCCCTCCACCAT-3V and 5V-TCCTCCCACTCCGCCACCAA-3V, respectively). RT-PCR of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA was also carried out as above using specific primers (5V-GAACGGGAAGCTTGTCATCAA-3V and 5VCTAAGCAGTTGGTGGTGCAG-3V) and cycling parameters of 25 cycles of 94jC for 10 s, 55jC for 10 s, 72jC for 30 s. PCR products were then electrophoresed on 1.5% agarose gels and, when necessary, transferred onto Genescreen Plus membranes (Dupont) and their specificity was determined by

hybridization with internal oligonucleotides (human IFNE1, 5V-ACTGTGTTGGTGCTGCTGGCCTCT-3V; mouse Ifne1, 5V-CAGCTCCCTGAAACGGTGTTGCTG-3V; Gapdh, 5VTCTTCCAGGAGCGAGATCCC-3V). PCR products were electrophoresed on a 1.5% agarose gel, excised, gel-purified, and ligated into pGEM-T (Promega). Automated sequencing was carried out on plasmid templates on a Prism Ready Reaction Dye Termination mix on an ABI automated sequencer (PE Applied Biosystems).

Acknowledgments Xiang-ming Liu (Center for Functional Genomics and Human Disease) and Mireille Lahoud (Walter and Eliza Hall Institute, Parkville, Australia) are thanked for their generous gifts of cDNA. This is Research Paper 005 from SUBIT.

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