Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16

Abstract

Detection of pathogenic nucleic acids is essential for mammalian innate immunity. IFN-inducible protein IFI16 has emerged as a critical sensor for detecting pathogenic DNA, stimulating both type I IFN and proinflammatory responses. Despite being predominantly nuclear, IFI16 can unexpectedly sense pathogenic DNA in both the cytoplasm and the nucleus. However, the mechanisms regulating its localization and sensing ability remain uncharacterized. Here, we propose a two-signal model for IFI16 sensing. We first identify an evolutionarily conserved multipartite nuclear localization signal (NLS). Next, using FISH and immunopurification, we demonstrate that IFI16 detects HSV-1 DNA primarily in the nucleus, requiring a functional NLS. Furthermore, we establish a localization-dependent IFN-β induction mediated by IFI16 in response to HSV-1 infection or viral DNA transfection. To identify mechanisms regulating the secondary cytoplasmic localization, we explored IFI16 posttranslation modifications. Combinatorial MS analyses identified numerous acetylations and phosphorylations on endogenous IFI16 in lymphocytes, in which we demonstrate an IFI16-mediated IFN-β response. Importantly, the IFI16 NLS was acetylated in lymphocytes, as well as in macrophages. Mutagenesis and nuclear import assays showed that NLS acetylations promote cytoplasmic localization by inhibiting nuclear import. Additionally, broad-spectrum deacetylase inhibition triggered accumulation of cytoplasmic IFI16, and we identify the acetyltransferase p300 as a regulator of IFI16 localization. Collectively, these studies establish acetylation as a molecular toggle of IFI16 distribution, providing a simple and elegant mechanism by which this versatile sensor detects pathogenic DNA in a localization-dependent manner.

The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

“…In contrast to AIM2, DNA recognition by IFI16 can occur in both the cytosolic and nuclear compartments. For example DNA which has been transfected, lentiviral DNA that has accumulated or herpesvirus DNA which has been exposed in the cytosol can be detected by cytosolic IFI16 [2,46,48] while DNA from nuclear viruses such as HCMV is detected in the nucleus [10,49]. As mentioned earlier, the NLS of IFI16 is required for the nuclear localisation of IFI16.…”

“…Viral DNA sensing by IFI16 leading to downstream signalling IFI16 is the second member of the human PYHIN family shown to be an innate immune sensor for dsDNA. Unlike AIM2, IFI16 is most abundant in the nucleus as a result of a NLS present in its Nterminal region [10]. IFI16 was initially identified as a novel DNA sensor using a 70 base pair IFN-b-inducing vaccinia virus dsDNA motif to purify DNA-binding proteins from cytosolic human monocytic extracts [2].…”

“…3). The acetylation status of IFI16 is thought to be regulated by the acetyltransferase p300 and the histone deacetylase HDAC and occurs in various cell types [10]. Thus an important factor in the regulation of IFI16 localisation is its acetylation status.…”

“…AIM2 and POP3 are predominantly cytosolic proteins, while IFI16, MNDA and PYHIN1 are predominantly found in the nucleus owing to the presence of an N-terminal nuclear localisation sequence (NLS) [3,9]. However, other factors such as acetylation or the presence of stimulatory DNA (in the case of IFI16), or the onset of apoptosis (in the case of MNDA), can also influence their cellular localisation [10][11][12].…”

Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy

“…First, as observed from several other DNAsensing pathways in mammals, it is not known why long dsDNA (>60 bp) is required to induce IFI16 activity (3, 9, 10). Second, why IFI16 does not bind self-DNA in the nucleus is not known (11)(12)(13)(14). Third, it is not known how IFI16 can selectively assemble into large signaling foci (e.g., inflammasome) with nonself-DNA (11)(12)(13)(14).…”