ZNFX1 antisense RNA1 promotes antiviral innate immune responses via modulating ZNFX1 function.

Increasing evidence suggests that natural antisense transcriptional lncRNAs regulate their adjacent coding genes to mediate diverse aspects of biology. Bioinformatics analysis of the previously identified antiviral gene ZNFX1 revealed neighboring lncRNA ZFAS1 transcribed on the opposite strand from ZNFX1. Whether ZFAS1 exerts antiviral function via regulating the dsRNA sensor ZNFX1 is unknown. Here we found that ZFAS1 was upregulated by RNA and DNA viruses and type I IFNs (IFN-I) dependent on Jak-STAT signaling, similar to the transcription regulation of ZNFX1. Knockdown of endogenous ZFAS1 partially facilitated viral infection, while ZFAS1 overexpression showed opposite effects. In addition, mice were more resistant to VSV infection with the delivery of human ZFAS1. We further observed that ZFAS1 knockdown significantly inhibited IFNB1 expression and IFR3 dimerization, whereas ZFAS1 overexpression positively regulated antiviral innate immune pathways. Mechanistically, ZFAS1 positively regulated ZNFX1 expression and antiviral function by enhancing the protein stability of ZNFX1, thereby establishing a positive feedback loop to enhance antiviral immune activation status. In short, ZFAS1 is a positive regulator of antiviral innate immune response via regulating its neighbor gene ZNFX1, adding new mechanistic insight into lncRNA-mediated regulation of signaling in innate immunity.

Deficiency of SECTM1 impairs corneal wound healing in aging.

The corneal epithelium is the outermost transparent barrier of the eyeball and undergoes continuous self-renewal by limbal stem cells (LSCs) during its lifetime; however, the impact of aging on LSCs remains largely unknown. Here, we showed that the healing ability of the cornea in elderly macaques (Macaca fascicularis) was significantly decreased compared to that of younger macaques. This delayed wound closure accompanied a disordered cell arrangement and corneal opacity. A novel cytokine, Secreted and Transmembrane 1 (SECTM1), was found to facilitate corneal healing and was upregulated in young macaques upon wounding. Mechanistically, SECTM1 is essential for LSC migration and proliferation, and may partially function through Cell Division Cycle Associated 7 (CDCA7). Notably, the topical application of SECTM1 to aged wounded corneas dramatically promoted re-epithelialization and improved corneal transparency in both mice and macaques. Our work suggests that aging may impair the expression of healing response factors and injury repair in non-human primate corneas, and that SECTM1 application could potentially benefit corneal wound healing in clinical treatment.

Single-cell transcriptomics implicates the FEZ1-DKK1 axis in the regulation of corneal epithelial cell proliferation and senescence.

Limbal stem/progenitor cells (LSC) represent the source of corneal epithelium renewal. LSC proliferation and differentiation are essential for corneal homeostasis, however, the regulatory mechanism remains largely unexplored. Here, we performed single-cell RNA sequencing and discovered proliferation heterogeneity as well as spontaneously differentiated and senescent cell subgroups in multiply passaged primary LSC. Fasciculation and elongation protein zeta 1 (FEZ1) and Dickkopf-1 (DKK1) were identified as two significant regulators of LSC proliferation and senescence. These two factors were mainly expressed in undifferentiated corneal epithelial cells (CECs). Knocking down the expression of either FEZ1 or DKK1 reduced cell division and caused cell cycle arrest. We observed that DKK1 acted as a downstream target of FEZ1 in LSC and that exogenous DKK1 protein partially prevented growth arrest and senescence upon FEZ1 suppression in vitro. In a mouse model of corneal injury, DKK1 also rescued the corneal epithelium after recovery was inhibited by FEZ1 suppression. Hence, the FEZ1-DKK1 axis was required for CEC proliferation and the juvenile state and can potentially be targeted as a therapeutic strategy for promoting recovery after corneal injury.

ETS1-HMGA2 Axis Promotes Human Limbal Epithelial Stem Cell Proliferation.

Purpose: This study aimed to investigate the role and molecular mechanism of ETS1 in the proliferation and differentiation of human limbal epithelial stem cells (LESCs). Methods: RNA-seq and quantitative real-time PCR were used to determine gene expression changes when ETS1 and HMGA2 was knocked down using short-hairpin RNAs or overexpressed by lentivirus. Immunofluorescence and flow cytometry experiments were performed to assess the roles of ETS1 and HMGA2 in LESC proliferation. ETS1-bound cis-regulatory elements and target genes in LESCs were identified using chromatin immunoprecipitation sequencing. The epigenetic features of ETS1-binding sites were assessed by the published histone modification and chromatin accessibility profiles. Results: ETS1 was robustly expressed in LESCs but dramatically reduced on differentiation into corneal epithelial cells (CECs). ETS1 knockdown in LESCs inhibited cellular proliferation and activated CEC markers (KRT3, KRT12, CLU, and ALDH3A1). When ETS1 was overexpressed during CEC differentiation, LESC-associated genes were upregulated while CEC-associated genes were downregulated. The genome-wide binding profile of ETS1 was identified in LESCs. ETS1 occupied H3K4me3-marked promoters and H3K27ac/H3K4me1-marked enhancers. ETS1-binding sites were also enriched for chromatin accessibility signal. HMGA2 showed a consistent expression pattern with ETS1. ETS1 activates HMAG2 by binding to its promoter. Knockdown and overexpression experiments suggested that HMGA2 can promote LESC proliferation and inhibits its differentiation. Conclusions: ETS1 promotes LESC proliferation and inhibits its differentiation via activating HMGA2.

dsRNA Induced IFNß-MMP13 Axis Drives Corneal Wound Healing.

Purpose: Cornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing. Methods: A corneal wound healing model was established via the surgical removal of half of the corneal surface and adjoining limbus. RNase III was then used to clarify the role of dsRNA in corneal wound closure and RNA-seq was performed to investigate the mechanism of dsRNA in the healing process. Related gene expression was assessed using immunofluorescence staining, qPCR, and Western blot. Flow cytometry and scratch assay were used to analyze the proliferation and migration of limbal stem/progenitor cells (LSCs) in vitro and functional analysis of the target genes was completed using the corneal wound healing model. Results: Corneal wound healing was delayed and impaired when the dsRNAs were removed or damaged following RNase III digestion. The dsRNAs released following corneal damage activate type I interferon (IFN-I) signaling, primarily IFNß, via the corneal epithelium and neutralizing IFNß or blocking IFN-I signaling delays corneal wound closure. Moreover, our data identified MMP13 as a downstream effector of IFNß where its expression promotes LSC proliferation and enhances corneal epithelial reconstruction in vivo. Conclusions: The dsRNA induced IFNß-MMP13 axis plays a key role in corneal wound healing.

Healing Ability of Central Corneal Epithelium in Rabbit Ocular Surface Injury Models.

Purpose: Wound healing of the corneal epithelium mainly involves two types of cells: limbal stem/progenitor cells (LSCs) and differentiated central corneal epithelial cells (CECs). The healing ability of CECs is still debatable, and its correlated transcriptomic alterations during wound healing are yet to be elucidated. This study aimed to determine the healing ability and mechanisms underlying the actions of CECs using rabbit ocular surface injury models. Methods: A central corneal ring-like residual epithelium model was used to investigate the healing ability of CECs. Uninjured and injury-stimulated LSCs and CECs were collected for transcriptomic analysis. The analysis results were verified by quantitative reverse transcriptase polymerase chain reaction, immunofluorescence staining, and two types of rabbit corneal injury models. Results: During wound healing, the upregulated genes in LSCs were mostly enriched in the mitotic cell cycle-related processes, but those in CECs were mostly enriched in cell adhesion and migration. CECs could repair the epithelial defects successfully at one-time injuries. However, after repetitive injuries, the CECs repaired notably slower and failed to completely heal the defect, but the LSCs repaired even faster than the one-time injury. Conclusions: Our results indicated rabbit CECs repair the epithelial defect mainly depending on migration and its proliferative ability is limited, and LSCs are the main source of regenerative epithelial cells. Translational Relevance: This study provides information on gene expression in the corneal epithelium during wound healing, indicating that regulation of the cell cycle, cell adhesion, and migration may be the basis for future treatment strategies for corneal wound healing.

Comprehensive 3D epigenomic maps define limbal stem/progenitor cell function and identity.

The insights into how genome topology couples with epigenetic states to govern the function and identity of the corneal epithelium are poorly understood. Here, we generate a high-resolution Hi-C interaction map of human limbal stem/progenitor cells (LSCs) and show that chromatin multi-hierarchical organisation is coupled to gene expression. By integrating Hi-C, epigenome and transcriptome data, we characterize the comprehensive 3D epigenomic landscapes of LSCs. We find that super-silencers mediate gene repression associated with corneal development, differentiation and disease via chromatin looping and/or proximity. Super-enhancer (SE) interaction analysis identified a set of SE interactive hubs that contribute to LSC-specific gene activation. These active and inactive element-anchored loop networks occur within the cohesin-occupied CTCF-CTCF loops. We further reveal a coordinated regulatory network of core transcription factors based on SE-promoter interactions. Our results provide detailed insights into the genome organization principle for epigenetic regulation of gene expression in stratified epithelia.

Generating an MEIS1 homozygous knockout human embryonic stem cell line using the CRISPR/Cas9 system.

Myeloid ecotropic viral integration site 1 (MEIS1) plays an essential role in the development of several embryonic organs, such as the central nervous system and eyes. To further investigate the role of MEIS1 in embryonic development, herein, we generated a MEIS1 homozygous knockout human embryonic stem cell (hESC) line using the CRISPR/Cas9 genome-editing technology. We believe that this cell line will be a good resource for exploring the function of the MEIS1 gene in embryonic development in vitro. Furthermore, the gene-knockout method reported in this study is efficient and labor-saving, which may provide an effective strategy for hESC gene deletion.

Mitochondria-localised ZNFX1 functions as a dsRNA sensor to initiate antiviral responses through MAVS.

In the past two decades, emerging studies have suggested that DExD/H box helicases belonging to helicase superfamily 2 (SF2) play essential roles in antiviral innate immunity. However, the antiviral functions of helicase SF1, which shares a conserved helicase core with SF2, are little understood. Here we demonstrate that zinc finger NFX1-type containing 1 (ZNFX1), a helicase SF1, is an interferon (IFN)-stimulated, mitochondrial-localised dsRNA sensor that specifically restricts the replication of RNA viruses. Upon virus infection, ZNFX1 immediately recognizes viral RNA through its Armadillo-type fold and P-loop domain and then interacts with mitochondrial antiviral signalling protein to initiate the type I IFN response without depending on retinoic acid-inducible gene I-like receptors (RLRs). In short, as is the case with interferon-stimulated genes (ISGs) alone, ZNFX1 can induce IFN and ISG expression at an early stage of RNA virus infection to form a positively regulated loop of the well-known RLR signalling. This provides another layer of understanding of the complexity of antiviral immunity.

The role of alternative polyadenylation in the antiviral innate immune response.

Alternative polyadenylation (APA) is an important regulatory mechanism of gene functions in many biological processes. However, the extent of 3' UTR variation and the function of APA during the innate antiviral immune response are unclear. Here, we show genome-wide poly(A) sites switch and average 3' UTR length shortens gradually in response to vesicular stomatitis virus (VSV) infection in macrophages. Genes with APA and mRNA abundance change are enriched in immune-related categories such as the Toll-like receptor, RIG-I-like receptor, JAK-STAT and apoptosis-related signalling pathways. The expression of 3' processing factors is down-regulated upon VSV infection. When the core 3' processing factors are knocked down, viral replication is affected. Thus, our study reports the annotation of genes with APA in antiviral immunity and highlights the roles of 3' processing factors on 3' UTR variation upon viral infection.