Members of the PYHIN protein family have recently come to prominence as receptors mediating the detection of foreign DNA and initiating innate immune responses. Absent in melanoma 2 (AIM2) binds DNA in the cytosol of macrophages and mediates activation of the inflammasome pathway [1–4]. A second family member, p202, binds to cytosolic DNA and antagonises this pathway . The inflammasome is a protein complex initiating activation of the protease precursor procaspase 1. Active caspase 1 cleaves proIL-1β and proIL-18 prior to their secretion as active inflammatory cytokines, and also leads to rapid lytic cell death termed “pyroptosis” . AIM2-mediated responses are elicited by viruses such as mouse cytomegalovirus (MCMV) and vaccinia, the cytosolic bacteria Francisella tularensis and Listeria monocytogenes, and even extracellular bacteria such as Streptococcus pneumoniae[6–11]. AIM2 was necessary for effective control of Francisella tularensis and MCMV infection of mice [6, 7, 11].
Another PYHIN family member, human IFI16, was shown to mediate inflammasome responses to Kaposi’s sarcoma virus DNA in the nucleus . Infection led to increased nuclear colocalisation of IFI16 with ASC, followed by emigration of both factors into the perinuclear region. IFI16 but not AIM2 knockdown decreased procaspase-1 cleavage in response to viral infection. On the other hand, IFI16 and mouse PYHIN protein p204 were found to play a role in the recognition of foreign DNA leading to induction of interferon-β (IFN-β), which is a pathway distinct from the inflammasome . Induction of IFN-β by cytosolic DNA requires the adapter protein STING (stimulator of interferon genes), and subsequent activation of TANK-binding kinase 1 (TBK1) leading to phosphorylation and nuclear translocation of the transcription factor interferon regulatory factor-3 (IRF-3) [14–18]. IFI16 is not the only contender for such a role in DNA recognition, as recent work suggests that the unrelated helicase protein DDX41 is the DNA-binding protein primarily required for the early induction of signalling leading to IFN-β production whilst IFI16-mediated responses to DNA may prolong the induction of IFN-β later in the response . Both IFI16 and DDX41 were reported to bind to STING [13, 19]. Overall, study of the PYHIN gene family has been hampered by the complexity of the family in mouse, and a lack of understanding of orthology between mouse and human genes.
The PYHIN genes were identified as a cluster on mouse and human chromosome 1 and were named mouse Ifi200 (“interferon inducible”) [20, 21] and human HIN-200 (“hematopoietic, interferon-inducible nuclear proteins with a 200 amino acid repeat”) . They have more recently been annotated as the “PYHIN” family, acknowledging the defining features of an N-terminal pyrin domain and C-terminal HIN domain. There are four human PYHIN proteins: IFI16 (interferon inducible protein 16) , MNDA (myeloid nuclear differentiation antigen) , AIM2  and IFIX (interferon inducible protein X) . Publications have so far detailed seven mouse proteins p202(a/b), p203, p204, p205, p206, Aim2/p210, and Mndal (MNDA-like) as well as a number of predicted proteins [22, 27–30]. Family members are predominantly nuclear proteins, some with defined nuclear localisation signals . There is potential for regulated localisation, since acetylation of the nuclear localisation signal of IFI16 led to its cytosolic accumulation . Some family members have characterised nuclear export sequences , suggesting they may shuttle in and out of the nucleus. In contrast, p202 and AIM2 lack nuclear localisation signals and reside in the cytoplasm of untreated cells [2, 4], consistent with the role of AIM2 and p202 in the recognition of cytosolic DNA. p206 is also reported to have cytoplasmic location .
Prior to the finding that members of the family function in pathogen recognition, publications focused on roles in cell growth and cell cycle control, tumour suppression, apoptosis, DNA damage response, senescence, muscle and myeloid differentiation and autoimmunity [33–38]. These functions are comprehensively reviewed elsewhere [22, 39–42]. There is as yet limited insight into the specific molecular roles of the proteins in these diverse functions. Various family members have been found to bind tumour suppressors such as p53, BRCA1 (breast cancer 1, early onset) and retinoblastoma protein [34, 43–45], supportive of roles in cell cycle regulation, DNA repair and apoptosis. Interactions with a range of transcription factors and signalling molecules are also reported [46–50]. The novel roles being uncovered for PYHIN proteins in host defence now provide relevance for the long-established interferon-inducibility of these genes. Consistent with viral need to evade detection, several viral proteins are characterised to bind PYHIN family members [51–53].
The PYHIN proteins are defined by the possession of one or two 200-amino acid HIN domains at the C terminus, and a pyrin domain at the N terminus [22, 40]. The roles of the HIN and pyrin domains are well established for AIM2-mediated inflammasome responses [1, 2]. AIM2 recognises DNA via its HIN domain, and then recruits the inflammasome adapter protein ASC (apoptosis-associated speck-like protein containing a CARD) via homotypic interaction of pyrin domains. ASC itself recruits procaspase 1 via its caspase recruitment domain (CARD), resulting in intermolecular cleavage to give active caspase 1. The pyrin domain of AIM2 can therefore be considered the effector domain eliciting inflammasome formation. Pyrin domains (also known as PYD, PAAD or DAPIN) are also found in other proteins involved in inflammasome formation, such as NOD-like receptors. They are part of the death domain superfamily which also includes the death domain, death effector domain, and CARD . Death domains form six-helix bundles and are frequently involved in recruitment of proteins in apoptotic and inflammatory responses through homotypic interactions.
The HIN domain is unique to the PYHIN family, and three distinct sequence classes, HIN-A, -B, and -C, have been defined . The HIN domain was predicted to combine two oligonucleotide/oligosaccharide binding (OB)-folds . OB-folds are five-stranded β-barrel structures found in a number of single stranded DNA (ssDNA)-binding proteins such as replication protein A and breast cancer 2, early onset (BRCA2). The OB-fold prediction is now supported by the crystal structures of the HIN domains of IFI16 and AIM2 [56, 57]. The structure of double stranded (ds) DNA-bound proteins  showed that interaction between the HIN domains and DNA was primarily by electrostatic interaction with the sugar-phosphate backbone, explaining the DNA sequence-independent responses to cytosolic DNA. This work also suggested that in the absence of DNA, the pyrin domain is bound to the HIN domain in an autoinhibited state. Unterholzner et al. showed that the tandem HIN domains of IFI16 were more effective in DNA binding than its single HIN-B domain, with the HIN-A domain alone being ineffective . Interestingly, recent work showed that IFI16 had a preference for binding cruciform structure DNA . Native mouse p202, which also has two HIN domains, strictly bound to dsDNA and not ssDNA , and biological responses mediated via AIM2 are dependent on dsDNA, and are not elicited by ssDNA . Beyond this, whether the HIN domains of different PYHIN family members have any specificity for particular DNA sequences or structures remains to be established.
The presence of four PYHIN family members in human has been known for a number of years, but the number of predicted mouse genes has increased with each new release of the mouse genome. In this paper, we describe the mouse, human and rat gene loci and proteins, address the issue of orthology between mouse and human genes and expression of the many mouse genes, and examine the evolution of the gene family within mammals. This provides a picture of a rapidly evolving locus with vastly different gene repertoires in different mammalian species and even within mouse strains. Phylogenetic analysis shows a clear distinction between AIM2 and other family members, suggesting divergence in function. Surprisingly, given the important role for AIM2 in host defence in mouse, AIM2 appears only as a pseudogene in a number of different lineages, and appears to have been lost from genomes on several independent occasions during evolution.