The immune system defends us from attacks by viruses, bacteria and other microbes. One way that the immune system can identify these invaders is by detecting genetic material from the microbes. Like us, many of these microbes have genetic material made of a two-stranded molecule called DNA. A protein called IFI16 is an important sensor in immune cells that can bind to foreign DNA, but not to the DNA of the host. If the ability to discriminate between host and foreign DNA is lost, then immune cells start to attack the body’s own tissues, which can lead to severe “autoimmune” diseases. However, it is not clear how IFI16 and other sensor proteins are able to tell foreign and host DNA apart.

DNA in host cells is packaged in particular proteins to form structures called nucleosomes that can make it difficult for other proteins to bind to the DNA. When foreign DNA enters the cell, IFI16 promotes the formation of nucleosomes on these molecules, but the nucleosomes are further apart than those in host DNA, which leaves longer stretches of “exposed” DNA between the nucleosomes. In 2014, a group of researchers reported that multiple molecules of IFI16 associate with each other and form clusters on exposed foreign DNA. Here, Stratmann, Morrone et al. – including some of the researchers from the earlier work – investigated how IFI16 selectively binds to foreign DNA using a combination of biochemical and single-molecule techniques.

The experiments show that to start with, a few molecules of IFI16 bind to stretches of exposed foreign DNA and then move along the DNA strands. As more molecules of IFI16 bind to this DNA, they bump into each other and start to form clusters that eventually become immobile. Further experiments show that the more tightly packed nucleosomes in host DNA molecules act as a barrier to IFI16 cluster formation because they interfere with the ability of the protein to move along the DNA.

Stratmann, Morrone et al.’s findings show that IFI16 can only trigger immune responses when it binds to stretches of exposed DNA that are long enough to allow the assembly of IFI16 clusters. The next challenge will be to see whether other DNA sensors employ similar strategies to detect foreign DNA.

The host innate immune system detects infection by directly recognizing molecular signatures associated with pathogens (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2000). Remarkably, such signatures include universal building blocks of all life, such as DNA and RNA (Janeway and Medzhitov, 2002; Orzalli and Knipe, 2014; Paludan and Bowie, 2013). In the cytoplasm, the immune system relies on the absence of endogenous DNA, and thus marks all detected DNA as 'foreign' (nonself) (Orzalli and Knipe, 2014; Paludan and Bowie, 2013). However, DNA viruses often evade the cytosolic detection machineries, as their genomes are not exposed until reaching the nucleus (Orzalli and Knipe, 2014; Paludan and Bowie, 2013). The host counters this infection strategy in the nucleus by directly assembling supramolecular signaling platforms that trigger inflammatory responses on invading foreign DNA, but not on its own genomic material (Johnson et al., 2013; Li et al., 2012; Kerur et al., 2011). Although key players that target foreign dsDNA in the host nucleus have been identified (Orzalli and Knipe, 2014; Paludan and Bowie, 2013), the molecular mechanisms by which these sensors distinguish self from nonself dsDNA remain unknown.

The interferon-inducible protein 16 (IFI16) is a key innate immune sensor that detects foreign dsDNA and uses it as a scaffold to assemble supramolecular signaling platforms in both the host nucleus and cytoplasm (Unterholzner et al., 2010; Johnson et al., 2013; Li et al., 2012; Kerur et al., 2011; Orzalli et al., 2012) (Figure 1A). IFI16 plays a central role in defense against a number of pathogens (e.g herpes simplex virus-1) (Unterholzner et al., 2010; Johnson et al., 2013; Li et al., 2012; Kerur et al., 2011; Orzalli et al., 2012). On the other hand, persistent IFI16 signaling is associated with autoimmunity (e.g. Sjögren’s syndrome) (Mondini et al., 2007; Choubey et al., 2010; Mondini et al., 2010; Gugliesi et al., 2013; Smith and Jefferies, 2014). The molecular mechanisms by which IFI16 selectively targets foreign dsDNA remain unknown. To establish a functional signaling platform, IFI16 must overcome two challenges. First, individual IFI16 molecules must be able to locate one another on large pathogen genomes with sizes ranging from 10⁵ to 10⁶ base pairs (bps). Second and more importantly, this assembly mechanism can only take place on foreign dsDNA and must be inhibited on host dsDNA (Figure 1A). Here, we report the observation of a unifying molecular mechanism that explains how IFI16 resolves these central issues in initiating its foreign-dsDNA sensing pathways.

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To identify the mechanisms underlying assembly of IFI16 signaling platforms on DNA, we monitored the oligomerization kinetics of FRET donor and acceptor labeled IFI16 on naked dsDNA (FRET: fluorescence resonance energy transfer; Figure 1B and Figure 1—figure supplement 1). Previous work demonstrated the existence of such oligomers and reported on their equilibrium binding properties but did not provide insights into the assembly mechanisms (Morrone et al., 2014). Using various dsDNA fragment sizes present in excess, we observe that the assembly rate increased non-linearly and by 50-fold from 60 to 200 bps dsDNA, above which it stayed constant (up to 600 bps; Figure 1B,C). With a dsDNA-binding footprint of ~15 bp for one IFI16 (Morrone et al., 2014), our results indicate that about 4 copies are required to initiate assembly, and about 10 IFI16 molecules are required for optimal oligomeric assembly (Figure 1C). Further, the assembly rate constants scaled linearly with the IFI16 concentration for all measured DNA lengths (Figure 1—figure supplement 1 and Supplementary file 1A), indicating that a purely cooperative assembly mechanism is unlikely. In line with this observation, previous work reported relatively small contributions of cooperativity in oligomerization with Hill constants near 2 for DNA substrates up to 2000 bp (Morrone et al., 2014).

Our ensemble-averaged, solution-phase observations of the faster assembly on longer dsDNA suggest a model in which IFI16 scans along dsDNA to increase the probability of encountering other IFI16 molecules (Figure 1D). To directly test such a mechanism, we used single-molecule fluorescence imaging to track the movements of individual Cy5-labeled IFI16 molecules on stretched, double-sided attached λ-phage dsDNA (λdsDNA; 48.5 kbps) (Figure 2A and Video 1). Figure 2B shows that individual IFI16 molecules one-dimensionally (1D) diffuse on λdsDNA while bound for several seconds. The diffusion coefficient of IFI16 increased with ionic strength, indicating that IFI16 does not maintain a continuous electrostatic interaction with the dsDNA backbone, but instead moves along the λdsDNA scaffold by executing microscopically small steps (Blainey et al., 2006) (Figure 2B and Figure 2—figure supplement 1). An IFI16 construct lacking the oligomerizing pyrin domain (PYD) (IFI16^HinAB; see also Figure 1A) showed similar diffusional properties, suggesting that the dsDNA-binding HIN200 domains are responsible for 1D diffusion. Upon applying higher concentrations of IFI16 with a constant supply of proteins into our flow cell, we observed a gradual formation of distinct, immobile clusters along λdsDNA (Figure 2C, Figure 2—figure supplement 2, and Video 2). Over time, we also observed an increase in the number of molecules per cluster and a concomitant decrease in the diffusion coefficient (Figures 2C,D). We analyzed the impact of flow on the diffusion coefficient and diffusion bias and found it to be not significant for cluster formation (Figure 2—figure supplement 4). Single-molecule intensity analysis revealed that the lower limit of the number of IFI16 molecules found in immobile clusters is equivalent to eight protomers (Figure 2D and Figure 2—figure supplement 3), which also corroborate the optimal complex (ten protomers) suggested from Figure 1C. Furthermore, individual IFI16 molecules either formed new clusters or joined other clusters in a stochastic manner, and the immobile clusters formed faster with higher IFI16 concentrations (Figure 2E). The rate of addition of IFI16 molecules to clusters is independent on the size of the existing cluster, confirming the absence of strong cooperativity in assembly (Figure 2C; bottom panel).

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The 1D diffusion of IFI16 on dsDNA explains why the assembly rates increase with the DNA length in the bulk experiments (Figure 1C). With the longer dsDNA acting as an antenna, it allows binding of more IFI16 while 1D diffusion facilitates dynamic association (Figure 1D). The saturation of the assembly rate (Figure 1C) can be explained by the square dependence of the diffusional search time on length: at a sufficiently long dsDNA length, the dissociation rate of an individual IFI16 will be faster than the time needed to scan along the entire length of the DNA. In addition, longer DNA substrates work no longer as antennae, but as traps, since individual IFI16 molecules are farther apart and thus less likely to encounter one another (Hu et al., 2006; Turkin et al., 2015; 2016). Overall, the results of our bulk and single-molecule experiments are consistent with the dsDNA-size dependent binding in vitro(Morrone et al., 2014), which also correlates with the IFI16-induced inflammatory responses in vivo(Unterholzner et al., 2010). Thus, we propose that the 1D-diffusion mediated assembly plays a key role in regulating the overall IFI16-mediated immune responses.

It has long been speculated that chromatinization acts as the key feature that allows IFI16 to distinguish host from foreign DNA in the nucleus (Kerur et al., 2011; Unterholzner and Bowie, 2011; Li et al., 2012; Orzalli et al., 2012; 2013; Johnson et at., 2014); IFI16 oligomerizes on exposed invading foreign-dsDNA before it becomes hetero-chromatinized. Previous in vivo work demonstrated that transfected chromatinized SV40 DNA is able to evade IFI16 oligomerization and downstream responses (Orzalli et al., 2013). Nevertheless, the molecular mechanism by which IFI16 could use chromatinization to distinguish self from nonself has yet to be identified. To directly address this issue, we first used a competition-binding assay to investigate how IFI16 interacts with dsDNA fragments containing two nucleosomes with varying spacer sizes (6, 30, 50, and 70 bps; Figure 3A and Figure 3—figure supplement 1). Here, di-nucleosomes with 6-, 30-, and 50-bp spacer failed to compete against IFI16-bound FAM-labeled 70-bp dsDNA, (Figure 3B). On the other hand, the di-nucleosome with 70-bp spacer competed similarly as 70-bp naked dsDNA, but significantly more weakly than naked 300-bp dsDNA (Figure 3B). In FRET assembly assays, di-nucleosomes with spacers shorter than 70-bp failed to support assembly (Figure 3C), consistent with our FRET kinetics assays using naked dsDNA (Figure 1B). The 70-bp spacer di-nucleosome supported oligomerization of IFI16; however, the assembly kinetics was again similar to that of naked 70-bp dsDNA, but not that of naked 300-bp dsDNA (Figure 3C). Taken together, these results show that efficient IFI16 cluster formation requires a minimal length of 50-70 base pairs of exposed dsDNA. Considering that the size of dsDNA linker between two nucleosomes is about 20 to 30 bps in mammals (McGhee et al., 1983), these results directly support the hypothesis that chromatinization is a key deterrent for preventing the assembly of IFI16 signaling platforms on self-dsDNA.

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To test whether the inhibitory effect of chromatin directly arises by interfering with the 1D diffusion of IFI16, we visualized the movement of individual IFI16 molecules on DNA with nucleosomes. We introduced randomly localized nucleosomes in λdsDNA using recombinant human histone octamers and tagged nucleosome positions with fluorescent antibodies against the N-terminal tail of histone H4 (Figure 4—figure supplement 1). The application of hydrodynamic flow resulted in the single IFI16 molecules being pushed to one direction and clustering at nucleosomal sites on λdsDNA, unable to overcome the octamers by diffusion (Figure 4A, Figure 4—figure supplement 2, and Video 3). Without the antibody, the motion of IFI16 was still confined, whereas for bare λdsDNA, IFI16 moved with a high processivity along the entire strand (Figures 4B,C, and Figure 4—figure supplement 3). These observations are consistent with the bulk experiments (Figure 3), and confirm that nucleosomes directly restrict the 1D diffusion of IFI16 and consequently limit the assembly of IFI16 signaling platforms on dsDNA.

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The molecular mechanism by which innate immune sensors distinguish self from foreign dsDNA in the host nucleus has been a major unresolved question in innate immunology (Kerur et al., 2011; Unterholzner and Bowie, 2011; Li et al., 2012; 2013; Orzalli et al., 2012; 2013; Johnson et al., 2014). The oligomerization of IFI16 on under-chromatinized foreign DNA plays a key role not only in initiating inflammatory and antiviral responses, (Monroe et al., 2014; Orzalli et al., 2012; Kerur et al., 2011), but also in regulating the hetero-chromatinization and silencing of viral dsDNA (Johnson et al., 2014; Orzalli et al., 2013). By using time-resolved bulk and single-molecule fluorescence assays, we demonstrate here that IFI16 ID scans along exposed dsDNA to assemble into distinct clusters and that chromatinization is sufficient to inhibit IFI16 from targeting host dsDNA for assembly. In vivo, this 1D scanning mechanism allows a limited number of IFI16 molecules to allocate each other on large genomes of invading pathogens. In combination with 3D sampling of binding sites on a collapsed DNA molecule, this process optimizes the oligomerization and downstream signaling time. While the clustering on dsDNA presents a tempting explanation for the role of IFI16 in viral gene silencing, future in vivo experiments await to test this. IFI16 belongs to the family of AIM2-like receptors, which include other nuclear and cytosolic foreign dsDNA-sensors. It will be interesting to determine whether and how these other related sensors use exposed dsDNA as a 1D 'digital ruler' to regulate their signaling platform assembly. This family of sensors is implicated in a number of autoimmune disorders (Mondini et al., 2007; 2010; Choubey et al., 2010; Gugliesi et al., 2013; Smith and Jefferies, 2014); how regulation of assembly is disrupted may provide insights into these afflictions.

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Author details

1. Sarah A Stratmann

   University of Groningen, Groningen, Netherlands

   Contribution

   SAS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Seamus R Morrone

   Competing interests

   No competing interests declared.

2. Seamus R Morrone

   Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   SRM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Sarah A Stratmann

   Competing interests

   No competing interests declared.

3. Antoine M van Oijen

   1. University of Groningen, Groningen, Netherlands
   2. University of Wollongong, Wollongong, Australia

   Contribution

   AMvO, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   vanoijen@uow.edu.au

   Competing interests

   AMvO: Reviewing editor, eLife.

4. Jungsan Sohn

   Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   JS, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   jsohn@jhmi.edu

   Competing interests

   No competing interests declared.

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