Entry of foreign DNA into the nucleus of a eukaryotic cell triggers numerous innate immune responses such as the induction of the inflammasome, type 1 interferon and DNA damage responses, as well as the recruitment of the ND10 or PML body components. In addition to the secretion of pro-inflammatory cytokines as a defense, the cell also maintains its homeostasis by limiting the expression of the invading foreign genes. Transcription factors like Daxx, Sp1 and PML (Poleshko et al., 2008; Merkl et al., 2018), and heterochromatin inducing factors like HDAC1 and CBX3 (Poleshko et al., 2008) have been shown to be instrumental in silencing foreign viral DNA. Interestingly, a Pyrin-Hin200 family nuclear innate immune DNA sensor, IFI16 (Interferon-γ-inducible protein 16) has emerged as a preeminent viral transcription restriction factor against a number of DNA viruses (Gariano et al., 2012; Orzalli et al., 2013; Merkl and Knipe, 2019; Johnson et al., 2014; Lo Cigno et al., 2015). Although the innate immune DNA sensor role of IFI16 where it senses foreign (Kerur et al., 2011; Orzalli et al., 2013) or damaged self DNA (Ouchi and Ouchi, 2008) and induces the ASC-dependent inflammasome pathway (Kerur et al., 2011; Ansari et al., 2013; Dutta et al., 2015) and the IFN-β pathway through the STING-TBK1-IRF3 axis (Unterholzner et al., 2010; Iqbal et al., 2016) is well understood, its transcription restriction role is not fully understood. Gariano et al. (2012) observed that IFI16 restricted human cytomegalovirus (HCMV) transcription and replication by displacing the Sp1 transcription factor. Orzalli et al. (2013), and Merkl and Knipe (2019) showed that IFI16 promotes silencing of human herpes simplex virus type-1 (HSV-1) gene expression and replication by adding the repressive chromatin mark, H3K9me3. Our studies showed that IFI16 restricts HSV-1 replication by modulating the binding of H3K4me3 and H3K9me3 histone marks and the RNA Pol II, TATA-binding protein (TBP) and Oct1 transcription factors (Johnson et al., 2014). Similar observations were made by Lo Cigno et al. (2015) for human papillomavirus 18 (HPV18).

Recently, we observed that IFI16 is essential for the repression of Kaposi’s sarcoma associated herpesvirus (KSHV) lytic gene transcription during latency and thus facilitates latency maintenance (Roy et al., 2016). IFI16 knockdown (KD) in the latently KSHV-infected primary effusion B-lymphoma (PEL) BCBL-1 and BC-3 cell lines resulted in a global increase of KSHV lytic transcripts, proteins, and viral genome replication but not latent genes. We also demonstrated these results during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by a chemical agent TPA. Intracellular viral genome copy number and virion particle associated KSHV DNA copy numbers were also elevated as a result of this KD. IFI16 chromatin immunoprecipitation assays (ChIP) showed that IFI16 binds to the promoters of all temporal KSHV gene classes. In addition, IFI16 repressed the transcription of KSHV luciferase promoter constructs in the uninfected epithelial SLK and osteosarcoma U2OS cells. Furthermore, during lytic reactivation of KSHV, we observed that IFI16 was polyubiquitinated and degraded via the proteasomal pathway, and thus relieving the lytic promoters of the repressive action of IFI16 to create a conducive environment for lytic gene expression. Blocking of KSHV DNA replication and late lytic gene expression resulted in the absence of IFI16 degradation. Our studies suggested that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency by repressing the lytic gene transcription.

The transcription regulatory role of IFI16 has been also reported in other unrelated systems (Johnstone et al., 1998; Caposio et al., 2007; Kang et al., 2014). Johnstone et al. (1998) fused IFI16 to GAL4 DNA binding domain (DBD) and observed that it acts as a potent transcription repressor when positioned in proximity to a GAL4DBD binding sequence containing promoter. Caposio et al. (2007) reported that overexpressing IFI16 resulted in an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. IFI16 has been shown to interact with cellular transcription factors such as SP1 (Luu and Flores, 1997; Lo Cigno et al., 2015) and p53 (Johnstone et al., 2000; Kwak et al., 2003). Kang et al. (2014) found that IFI16 is associated with the promoter of the estrogen receptor α (ERα) gene ESR1 and plays a role in the transcriptional regulation of ESR1 gene in breast cancer (Kang et al., 2014). Thompson et al. (2014) described a regulatory role for IFI16 in the transcriptional regulation of IFN-α gene expression. Recently, a closely related murine PYHIN family member p205 has been shown to control ASC gene expression by regulating RNA polymerase II binding to its promoter (Ghosh et al., 2017).

In this study, we used KSHV latency establishment and maintenance as a model system to study the transcription and possible epigenetic modulatory roles of IFI16. KSHV, an oncogenic human herpesvirus, is etiologically associated with endothelial Kaposi's sarcoma (KS), primary effusion B-cell lymphoma (PEL) and B-lymphoproliferative multicentric Castleman’s disease (MCD) (Chang et al., 1994; Cesarman et al., 1995; Soulier et al., 1995). Like all other herpesviruses, KSHV establishes a lifelong latency in humans with periodic lytic reactivation and reinfection (Boshoff and Chang, 2001). Based on the temporal regulation of expression, the lytic genes are classified as immediate early (IE), early (E) and late (L) genes. After primary infection of permissive cells, the chromatin-free input KSHV genome enters the nucleus and undergoes rapid chromatinization which is mediated by cellular epigenetic factors and viral chromatin regulatory elements (Günther and Grundhoff, 2010; Toth et al., 2013b; Günther et al., 2014). This chromatinized viral genome is maintained as an extrachromosomal episome. During latency, lytic genes are under tight transcriptional repression by epigenetic factors and transcriptional repressors (Krishnan et al., 2004). Upon conducive conditions such as hypoxia, infection by other pathogens, inflammatory cytokines, or immune suppression, KSHV transitions from latency to lytic reactivation leading to progeny virion formation (Toth et al., 2013b). KSHV lytic reactivation can be induced by treating latently infected cells in culture with small molecule inhibitors of epigenetic modulators such as histone methyltransferases (HMTases), histone deacetylases (HDACs), histone acetyltransferases (HATs) and DNA methyltransferases (DNMTs) (Hopcraft et al., 2018).

The mechanisms of KSHV genome maintenance during latency are under intense investigations. Toth et al. (2010) reported that activating histone marks AcH3 and H3K4me3 colocalized on the KSHV genome and predominantly occupied the latency and the early-lytic regions during latency. On the other hand, the repressive H3K27me3 mark was widely distributed throughout the KSHV genome, while H3K9me3 was restricted mainly to two regions spanning 30–60 kb and 95–115 kb encoding several late lytic genes (ORF16–40 and ORF58–68). In agreement with Toth et al.’s results, Günther and Grundhoff (2010) also found H3K9me3 to be mainly restricted to two consecutive KSHV genomic regions, ~33–46 kb and 100–114 kb in latently infected cells. Both these authors found that many lytic loci which are transcriptionally inactive during latency are occupied by both activating as well as repressive histone marks, instigating the hypothesis that these promoters are ‘bivalent’ and are suspended in a transcriptionally poised state capable of reactivating rapidly when needed (Toth et al., 2013a; Ter Horst and Luiten, 1987; Günther and Grundhoff, 2010). More recently, Sun et al. (2017) performed ChIP-Seq experiments on classic KS tissues and found that the activating acetylated H3 (H3Ac) marks were restricted to the latency locus, while the repressive H3K27me3 was widespread on KSHV genome.

Although the transcription repression and the possible epigenetic histone mark modification roles of IFI16 are known, no definitive mechanism has been identified till date linking IFI16 to epigenetic chromatin remodeling. Here, we used KSHV latency as a model and asked the question - ‘how does IFI16 modulate epigenetic histone marks leading to its transcription regulatory activity?'. Our studies show that KD of IFI16 most significantly reduces H3K9me3 and increases H3K4me3 on the KSHV gene promoters, and that IFI16 interacts with H3K9 histone methyltransferase (H3K9-MTase) Suppressor of variegation 3–9 homolog 1 (SUV39H1) and G9a-like protein (GLP) both in uninfected and infected cells. IFI16 KD and knockout (KO) drastically reduces the recruitment of these two H3K9-MTase onto the KSHV genome which was confirmed by the IFI16 rescue experiments in KO cells. Our studies suggest that IFI16 dependent recruitment of GLP mono and di-methylates (me1/me2) at H3K9 on the KSHV genome, while SUV39H1 further establishes tri-methylated H3K9me3 marks. This consequently regulates the recruitment of heterochromatin protein 1-α (HP1α) protein which functions downstream of H3K9me3 resulting in DNA compaction and heterochromatization of the KSHV genome and silencing of the KSHV genes, especially the late lytic genes. Our findings unravel a previously unknown function of IFI16 namely the interactions with SUV39H1 and GLP H3K9-MTases that facilitate the epigenetic silencing of foreign viral DNA.

The transcriptional repressor function of IFI16 has been identified in diverse experimental models (Caposio et al., 2007; Johnstone et al., 1998; Kang et al., 2014; Thompson et al., 2014; Roy et al., 2016). In addition, IFI16’s ability to restrict transcription and replication of episomal viral DNA of KSHV, HSV-1, HCMV and HPV18 is also well established (Roy et al., 2016; Gariano et al., 2012; Orzalli et al., 2013; Merkl and Knipe, 2019; Johnson et al., 2014; Lo Cigno et al., 2015). Although these studies suggested that IFI16 may have the ability to promote epigenetic modification of foreign/viral DNA leading to transcriptional silencing (Orzalli et al., 2013; Merkl and Knipe, 2019; Johnson et al., 2014; Lo Cigno et al., 2015), till date, no precise epigenetic function of IFI16 has been identified that can explain its ability to inhibit transcription. Our comprehensive studies here demonstrate that under physiological conditions, IFI16 is associated with the H3K9 methyltransferases SUV39H1 and GLP which mediates the deposition of H3K9me2/me3, and thus unravels one of the potential mechanisms by which IFI16 mediates epigenetic modifications and transcriptional regulation.

Lysines 4, 9, 27, 36 and 79 of histone H3, and 20 of histone H4 can be methylated, but, no attempt has been made to comprehensively study the possible role of IFI16 in recruiting these H3 lysine tri-methylations. Hence, we undertook a systamatic approach toward identifying the H3 lysine tri-methylations that can be modulated by IFI16 on the KSHV epigenome and tested all five identified lysine tri-methylations of H3. Our studies demonstrate that depletion of IFI16 results in increased recruitment of H3K4me3 and decreased recruitment of H3K9me3 on the KSHV promoters in both latent BCBL1 cells and in de novo infected TIME cells (Figure 1C and F). Both these epigenetic modifications are conducive of increased transcriptional activity which is in agreement with our previous observation that KD of IFI16 results in increased transcription of lytic genes (Roy et al., 2016). As a further confirmation of this, we found that KD of IFI16 resulted in increased recruitment of RNA Pol II on all the lytic promoters tested (Figure 1C and F). It is interesting to note that Toth et al. (2010) and Günther and Grundhoff (2010) found that abundant levels of H3K9me3 recruitment was restricted mainly to two regions of KSHV genome (30–60 kb and 95–115 kb) encoding a number of late genes (ORF16–40 and ORF58–68) in TRExBCBL1-Rta cells. However, lesser basal levels were detectable throughout the KSHV genome. Interestingly, Günther and Grundhoff (2010) did not observe significant deposition of H3K9me3 on the KSHV genome during de novo infection of SLK cells. However, it must be considered that SLK cells are of epithelial origin and have been shown to be a contaminant from the renal-cell carcinoma cell line Caki-1 (Stürzl et al., 2013). In contrast, we used the hTERT immortalized human dermal microvascular endothelial cell line, TIME, which we believe is a more suitable cell model for KSHV de novo infection.

After observing that IFI16 regulates the recruitment of H3K9me3, we first asked the question ‘Is the H3K9me3 mark important for the KSHV life cycle?”. Our studies demonstrate that after lytic induction of TRExBCBL1-Rta cells, levels of H3K9me3 reduced by half on the late ORF63 promoter as early as day 1 (Figure 1I). The location of the ORF63 promoter on the KSHV genome is between 102 and 103 kb which is within the high abundance H3K9me3 distribution region described previously (Toth et al., 2010; Günther and Grundhoff, 2010). In contrast, abundance of H3K27me3 on the ORF63 promoter decreased more gradually between days 2 and 4 post-induction. This strongly suggested that H3K9me3 does play a role in the KSHV lifecycle. Further evidence of the role of H3K9me3 in KSHV lifecycle became evident when we treated BCBL1 cells with A366, a specific inhibitor of H3K9me2 and H3K9me3 methyltransferase (Kaniskan et al., 2018). This caused significantly higher transcription of KSHV lytic genes, while expression of the latent genes and the control GAPDH and IFI16 genes did not alter significantly (Figure 2B). This is a hallmark of successful lytic reactivation of the latent KSHV genome. Thus, H3K9me2/3 recruitment and maintenance are indispensable for the maintenance of latency. This experiment also suggests that the H3K9me2/3 mark and possibly all the other epigenetic marks on the KSHV genome is dynamic in nature and must be continuously recruited to maintain latency. This may be solely or partly due to the fact that as the BCBL1 cell divides, the newly replicated latent epigenetically naive KSHV genomes must recruit the proper epigenetic marks to be effectively silenced in order to maintain latency.

It is generally considered that H3K9me3 is a hallmark of constitutive heterochromatin while H3K27me3 is typically seen in facultative heterochromatin. Constitutive heterochromatin are often considered to be irreversible such that they will never revert to euchromatin, as those seen at the telomeres and centromeres in mammalian cells. Alternatively, facultative heterochromatin is considered to be reversible and capable of undergoing active gene expression. However, resent advances disprove this notion and H3K9me3’s role in cell-type-specific regulation of facultative heterochromatin has been established (Becker et al., 2016). Moreover, numerous reports have demonstrated that herpes viral and retroviral genomes are associated with H3K9me3 during latency and almost all these genes undergo active transcription during lytic reactivation (Günther and Grundhoff, 2010; Toth et al., 2010; Orzalli et al., 2013; Lo Cigno et al., 2015; Merkl and Knipe, 2019; Bloom et al., 2010; Cliffe et al., 2009).

Our observations confirm that IFI16 recruits the H3K9MTases SUV39H1 and GLP on to the KSHV genome resulting in the enrichment of H3K9me2/me3 marks (Figures 3–7). GLP and its partner G9A are known to be responsible for mono and di-methylation (me1/me2) of H3K9 (Tachibana et al., 2005). On the other hand, SUV39H1 has been established to use mono and di-methylated H3K9 as a primary substrate and establish tri-methyl H3K9 (Rea et al., 2000; Peters et al., 2001). Therefore, we believe, that the establishment of H3K9me3 heterochromatic locus on the KSHV genome requires the concerted action of GLP and SUV39H1. The fact that IFI16 interacts with and recruits both these H3K9MTases explains the functional importance of this mechanism and that IFI16 has evolved to specifically interact/recruit these MTases to establish heterochromatin. Although KD of SUV39H1 and GLP resulted in the induction of KSHV lytic transcripts in BCBL-1 cells, KD of GLP only caused a marginal twofold increase in KSHV genome DNA replication, while KD of SUV39H1 alone had no effect. This may signify that IFI16-mediated recruitment of H3K9me3 is one among the multiple barriers that herpesviruses have to overcome before the successful induction of lytic replication. Other important barriers including H3K27me3 exists and a concerted action of all these viral restriction factors help to establish and maintain latency.

The above suggestion is also supported by the observation that H3K4me3 modification on ORF73 promoter after IFI16 KD is significant (Figure 1.F, p-value **). We have previously reported ORF73 mRNA expression under similar conditions after IFI16 KD (Roy et al., 2016), where we observed that KD of IFI16 lead to a marginal increase (2-fold) in ORF73 expression in contrast to lytic genes which increased by several folds. This observation can be explained by the fact that the ORF73 promoter is under the concerted regulation of numerous transcription and epigenetic factors including Oct1, GATA-1 Ap1, and RBP-Jκ (Lan et al., 2005). In addition, the ORF73 promoter consists of two mRNA start sites – the LANA constitutive (LTc) and the RTA-inducible (LTi) mRNA start sites (Veeranna et al., 2012). This makes the ORF73 promoter particularly complex and it is possible that additional factors are involved in keeping the expression of LANA in check after IFI16 KD despite the fact that H3K4me3 is significantly enriched on the ORF73 promoter.

Chromatin marked by H3K9me3 are recognized by the chromo domain protein, HP1α which facilitate heterochromatin formation (Bannister et al., 2001; Lachner et al., 2001). HP1 proteins serves as a scaffold to recruit other chromatin modifying proteins, including more H3K9 MTases and histone deacetylases (Bannister et al., 2001; Nakayama et al., 2001; Lachner et al., 2001). We therefore reasoned that if IFI16 recruits SUV39H1 and GLP to establish H3k9me3 marks on the KSHV genome, the subsequent recruitment of HP1α should also be dependent on IFI16. Our data showed that depletion of IFI16 resulted in reduced recruitment of HP1α on the KSHV genome (Figure 9E–G) and KD of HP1α caused robust induction of KSHV lytic gene transcription. Together, these confirmed that HP1α is important for maintenance of latency and IFI16 is indispensable for its recruitment. Studies of HP1 dynamics have revealed that HP1α is not a stable component of heterochromatin but is highly mobile (Krouwels et al., 2005). This dynamicity of HP1α together with the need to epigenetically silence nascent KSHV genomes synthesized as a result of multiplication of latently infected cells helps explain the indispensable nature of IFI16 in HP1α recruitment.

One common factor binding all the identified functions of IFI16 is its DNA binding ability. However, crystal structure of IFI16 HIN domains in complex with B-form dsDNA revealed that it binds dsDNA in a non-sequence specific manner (Jin et al., 2012). Therefore, how IFI16 distinguishes between self and non-self DNA and if there is any specific signal that dictates both innate sensing and epigenetic silencing by IFI16 remains unanswered. Further studies are required to determine the possible existence of nuclear macromolecular complex(es) detecting and epigenetically silencing foreign invading DNA. Multiple observations point such a possibility. We have observed that silencing of IFI16 also effects the recruitment of H3K4me3 (Figure 1C and F). Therefore, it is possible that a H3K4-specific demethylase is also recruited by IFI16. Fritsch et al. (2010) reported that Suv39H1, G9a, GLP, and SETDB1 participate in a multimeric complex. Suv39H1 has been found to interact with the DNA methyltransferase DNMT3B (Lehnertz et al., 2003) and HP1α has been reported to recruit DNMT3B. Previous reports have also shown that IFI16 is instrumental in recruiting transcription factors like Sp1 and Oct1 (Gariano et al., 2012; Johnson et al., 2014). Therefore, the possibility of all these chromatin silencing factors acting in a concerted fashion is plausible.

Our earlier studies have demonstrated that IFI16 is in association with different proteins in the nucleus and mediates the innate immune responses (Dutta et al., 2015; Iqbal et al., 2016). We identified two IFI16 complexes, namely IFI16-BRCA1 and IFI16-BRCA1-H2B. These complexes recognized the KSHV and HSV-1 genomes soon after their entry into the nucleus, leading into BRCA1-mediated p300 recruitment and acetylation of IFI16 and H2B by p300. IFI16 acetylation resulted in the formation of BRCA1-IFI16-ASC-procaspase-1 inflammasome formation in the nucleus, transport to the cytoplasm, pro-IL-1β cleavage and IL-1β formation. Cytoplasmic transport of acetylated IFI16-H2B-BRCA1 results in the association with cGAS and STING leading into phosphorylation of TBK1 and IRF3, nuclear translocation of p-IRF3 and IFN-β production. Our present study identified a novel epigenetic function of IFI16 and demonstrates that IFI16 is in association with H3K9MTases SUV39H1 and GLP in the nucleus and IFI16 recruits these MTases to the KSHV genome which sequentially methylates H3K9 to me1/me2 and me3. This serves as a docking site for HP1α which recruits further chromatin compaction factors leading to heterochromatin formation and lytic gene silencing (Figure 9J). Thus, our studies uncovered an important paradigm and demonstrated that IFI16-mediated innate immune sensing of foreign viral DNA not only leads to the induction of innate interferon and inflammasome pathways, but also results in the epigenetic silencing of the foreign DNA.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Arunava Roy

   Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft

   For correspondence

   arunavaroy@usf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8486-0539

2. Anandita Ghosh

   Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, United States

   Contribution

   Data curation, Visualization, Methodology

   Competing interests

   No competing interests declared

3. Binod Kumar

   Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, United States

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

4. Bala Chandran

   Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   chandran@usf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5319-8714

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