Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors. In particular, DNA is a potent activator of immune responses during infection or tissue damage, and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses. Here we report on a candidate DNA sensor, previously named DLM-1 (also called Z-DNA binding protein 1 (ZBP1)), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA-associated antimicrobial immunity and autoimmune disorders.

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

DNA, whether it is microbe-derived or host-derived, evokes immune responses when exposed to the cytosol of a cell. We previously reported that DNA-dependent activator of IFN regulatory factors (DAI), also referred to as DLM-1/ZBP1, functions as a DNA sensor that activates the innate immune system. In the present study, we examined the regulation of the complex DNAsensing system by DAI and other molecules. We first show that DAI directly interacts with DNA in vitro and that it requires three DNA-binding domains for full activation in vivo. We also show that the artificially induced dimerization of DAI results in the DNAindependent activation of type I IFN genes, thereby providing a better understanding for the molecular basis of DAI activation. Furthermore, we provide evidence for the presence of additional DNA sensors, either positively or negatively regulating cytosolic DNA-mediated innate immune responses. These results in toto provide insights into the mechanism of DAI activation and reveal the complex regulatory mechanisms underlying DNA-mediated protective and pathologic immune responses.ADAR1 ͉ E3L ͉ interferon ͉ IFN regulatory factor N on-self-nucleic acids from invading microbes or self-nucleic acids exposed in a cell by infection or incomplete clearance during cell damage commonly evoke immune responses (1-5). In addition to membrane-type Toll-like receptors (TLRs), such as TLR3, TLR7, and TLR9, that are activated by RNA or DNA (6, 7), there are cytosolic receptors that also evoke the nucleic acid-mediated activation of innate immune responses, the hallmark of which is the induction of type I IFN genes (8-11). Indeed, RIG-I and MDA5 molecules function as cytosolic RNA sensors and activators, whereas LGP2 acts as a negative regulator, probably by competing with RIG-I and MDA5 for RNA binding (8,10,12). Recent attention has focused on the regulation of DNA-sensing systems, because they also relate to protective and pathologic immune responses. In this context, we identified DAI [also referred to as DLM-1 and ZBP1 (13); we refer to it as DAI hereafter for convenience] as the first cytosolic DNA sensor and activator of innate immune responses activated by cytosolic DNAs (14).We have shown that the artificial expression of otherwise IFN-inducible DAI selectively enhances the DNA-mediated induction of type I IFN genes and other genes involved in innate immunity and that the RNA interference of DAI mRNA in cells, on the other hand, strongly inhibits this gene induction program in mouse fibroblast L929 cells (14). In addition to two binding domains for left-handed Z-form DNA (Z-DNA), termed Z␣ and Z␤ (15, 16), DAI also contains an additional candidate DNAbinding region/domain, termed the D3 region, that may also interact with DNA (Fig. 1a) (14). Furthermore, we adduced evidence that DAI recruits TBK1 serine/threonine kinase and IFN regulatory factor 3 (IRF3) transcription factor, both of which play critical roles in the induction of type I IFN genes and other genes (14). A DAI mutant lacking the DNA-binding reg...

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The activation of the innate immune responses by DNA exposed within the cytosol has gained much attention and, in this context, several cytosolic DNA sensors have been identified. However, previous studies revealed the operation of redundant and complex mechanisms and it still remains to be clarified how the DNAmediated evocation of diverse innate immune responses can be achieved. Here we show that two RIG-I-like receptors (RLRs), RIG-I and MDA5, known as cytosolic RNA receptors, nonredundantly function as cytosolic DNA receptors that lead to the selective activation of type I IFN genes. We demonstrate that overexpression of otherwise IFN-inducible RIG-I or MDA5 in IFN signal-deficient cells results in a marked enhancement of type I IFN gene induction upon cytosolic DNA stimulation, while in their absence the induction is impaired. Interestingly, the DNA-mediated induction of other cytokine genes was barely affected by the absence of RLRs. Indeed, unlike the RNA-RLR pathway that activates the transcription factors IRF3 and NF-B, the DNA-RLR pathway is primarily responsible for the IRF3 activation critical for type I IFN gene transcription, illustrating a deliberate divergence of the DNA signaling pathways. Expectedly, the RLR pathway also contributes to intricate innate immune responses against infection by a DNA virus. Our study may provide insights into the complexity of host defense mechanisms that thwart immune evasion by DNA-containing pathogens.DNA sensor ͉ innate immunity ͉ IRF3 ͉ NF-B