Identification of a novel gene family that includes the interferon-inducible human genes 6-16 and ISG12

Abstract

Background: The human 6-16 and ISG12 genes are transcriptionally upregulated in a variety of cell types in response to type I interferon (IFN). The predicted products of these genes are small (12.9 and 11.5 kDa respectively), hydrophobic proteins that share 36% overall amino acid identity. Gene disruption and over-expression studies have so far failed to reveal any biochemical or cellular roles for these proteins.

Results: We have used in silico analyses to identify a novel family of genes (the ISG12 gene family) related to both the human 6-16 and ISG12 genes. Each ISG12 family member codes for a small hydrophobic protein containing a conserved ~80 amino-acid motif (the ISG12 motif). So far we have detected 46 family members in 25 organisms, ranging from unicellular eukaryotes to humans. Humans have four ISG12 genes: the 6-16 gene at chromosome 1p35 and three genes (ISG12(a), ISG12(b) and ISG12(c)) clustered at chromosome 14q32. Mice have three family members (ISG12(a), ISG12(b1) and ISG12(b2)) clustered at chromosome 12F1 (syntenic with human chromosome 14q32). There does not appear to be a murine 6-16 gene. On the basis of phylogenetic analyses, genomic organisation and intron-alignments we suggest that this family has arisen through divergent inter- and intra-chromosomal gene duplication events. The transcripts from human and mouse genes are detectable, all but two (human ISG12(b) and ISG12(c)) being upregulated in response to type I IFN in the cell lines tested.

Conclusions: Members of the eukaryotic ISG12 gene family encode a small hydrophobic protein with at least one copy of a newly defined motif of approximately 80 amino-acids (the ISG12 motif). In higher eukaryotes, many of the genes have acquired a responsiveness to type I IFN during evolution suggesting that a role in resisting cellular or environmental stress may be a unifying property of all family members. Analysis of gene-function in higher eukaryotes is complicated by the possibility of functional redundancy between family-members. Genetic studies in organisms (e.g. Dictyostelium discoideum) with just one family member so far identified may be particularly helpful in this respect.

Figure 1

Alignment of putative protein sequences identifies a ISG12 protein motif. Putative protein product of the genes in Table 1 were aligned using Clustal W [26] and annotated using Boxshade . The section of this alignment where the predicted proteins share greatest amino-acid identity is shown. The consensus sequence for this region represents residues that are conserved in 50% or more of the sequences and defines the ISG12 motif (Pfam accession number: PF06140). Black squares represent sequence identity and grey squares represent sequence similarity. Numbers flanking the sequence represent amino-acid numbers in the putative proteins.

Figure 2

Predicted protein hydrophobicity. Kyte-Doolittle schematics were formulated using GREASE software to show the hydrophobicity of predicted protein sequences for mouse ISG12(a), human ISG12(a) and human 6–16. ISG12 motifs are highlighted in yellow.

Figure 3

Phylogenetic analysis of the ISG12 family. Maximum parsimony tree with bootstrap confidence levels based on putative protein coding sequence of higher mammals using D. discoideum as an out-group (see materials and methods). Two stars represent a bootstrap confidence level >85%, one star >60%.

Figure 4

Genomic organisation of ISG12 genes in humans and mice showing ISRE positions. Regions of human chromosomes 1p35 and 14q32, and mouse chromosome 12F1, carrying ISG12 genes are shown. Transcribed regions are coloured (thickened lines, introns; boxes, exons) and arrows represent direction of transcription. Translational initiation and termination sites are indicated by green and red circles, respectively. Exons encoding an ISG12 motif are starred. The positions of numbered ISRE sequences (as defined in Table 2) are indicated. Orientations relative to Telomeres (TEL) and Centromeres (CEN) are indicated. The regions shown can be accessed from the Ensembl website using the following addresses: .

Figure 5

Alignment of introns in ISG12 amino-acid sequences. Predicted ISG12 protein sequences, for genes whose intron/exon structures are available, are aligned as in Figure 1, with the ISG12 motifs highlighted in yellow. Positions of introns lie between the amino acids that have been marked (⇓).

Figure 6

Expression of human and mouse ISG12 genes in cell lines. Transcripts for the indicated human and mouse ISG12 genes were detected by RT-PCR. RNA was isolated from the human HT1080 cells (a) or mouse L-929 cells (b) treated with (left hand panels) or without (right hand panels) type I IFN for 24 h. The indicated serial five-fold dilutions of reverse transcripts were analysed by PCR. Most PCR assays included, as an internal control, primers for beta-actin (β-Ac). Diagnostic products for each transcript are arrowed. M = size markers.