IGSF8 is an innate immune checkpoint and cancer immunotherapy target.

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival in vitro or in vivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells in vitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and T cell signaling in vivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Replication Timing Becomes Intertwined with 3D Genome Organization.

Through dissecting the link between spatial genome organization and DNA replication timing, Sima et al. (2018) discover early replicating control elements (ERCEs), a new type of cis-acting elements that regulate replication timing, transcription, and multiple layers of three-dimensional features of genome organization. The study has important implications for unraveling control elements of high-order genome structure and function.

Targeting LYPLAL1-mediated cGAS depalmitoylation enhances the response to anti-tumor immunotherapy.

Cyclic GMP-AMP synthase (cGAS) binds pathogenic and other cytoplasmic double-stranded DNA (dsDNA) to catalyze the synthesis of cyclic GMP-AMP (cGAMP), which serves as the secondary messenger to activate the STING pathway and innate immune responses. Emerging evidence suggests that activation of the cGAS pathway is crucial for anti-tumor immunity; however, no effective intervention method targeting cGAS is currently available. Here we report that cGAS is palmitoylated by ZDHHC9 at cysteines 404/405, which promotes the dimerization and activation of cGAS. We further identified that lysophospholipase-like 1 (LYPLAL1) depalmitoylates cGAS to compromise its normal function. As such, inhibition of LYPLAL1 significantly enhances cGAS-mediated innate immune response, elevates PD-L1 expression, and enhances anti-tumor response to PD-1 blockade. Our results therefore reveal that targeting LYPLAL1-mediated cGAS depalmitoylation contributes to cGAS activation, providing a potential strategy to augment the efficacy of anti-tumor immunotherapy.

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Computational methods for analysing multiscale 3D genome organization.

Recent progress in whole-genome mapping and imaging technologies has enabled the characterization of the spatial organization and folding of the genome in the nucleus. In parallel, advanced computational methods have been developed to leverage these mapping data to reveal multiscale three-dimensional (3D) genome features and to provide a more complete view of genome structure and its connections to genome functions such as transcription. Here, we discuss how recently developed computational tools, including machine-learning-based methods and integrative structure-modelling frameworks, have led to a systematic, multiscale delineation of the connections among different scales of 3D genome organization, genomic and epigenomic features, functional nuclear components and genome function. However, approaches that more comprehensively integrate a wide variety of genomic and imaging datasets are still needed to uncover the functional role of 3D genome structure in defining cellular phenotypes in health and disease.

A two-amino-acid substitution in the transcription factor RORγt disrupts its function in TH17 differentiation but not in thymocyte development.

The transcription factor RORγt regulates differentiation of the TH17 subset of helper T cells, thymic T cell development and lymph-node genesis. Although elimination of RORγt prevents TH17 cell-mediated experimental autoimmune encephalomyelitis (EAE), it also disrupts thymocyte development, which could lead to lethal thymic lymphoma. Here we identified a two-amino-acid substitution in RORγt (RORγtM) that 'preferentially' disrupted TH17 differentiation but not thymocyte development. Mice expressing RORγtM were resistant to EAE associated with defective TH17 differentiation but maintained normal thymocyte development and normal lymph-node genesis, except for Peyer's patches. RORγtM showed less ubiquitination at Lys69 that was selectively required for TH17 differentiation but not T cell development. This study will inform the development of treatments that selectively target TH17 cell-mediated autoimmunity but do not affect thymocyte development or induce lymphoma.

scGHOST: identifying single-cell 3D genome subcompartments.

Single-cell Hi-C (scHi-C) technologies allow for probing of genome-wide cell-to-cell variability in three-dimensional (3D) genome organization from individual cells. Computational methods have been developed to reveal single-cell 3D genome features based on scHi-C, including A/B compartments, topologically associating domains and chromatin loops. However, no method exists for annotating single-cell subcompartments, which is important for understanding chromosome spatial localization in single cells. Here we present scGHOST, a single-cell subcompartment annotation method using graph embedding with constrained random walk sampling. Applications of scGHOST to scHi-C data and contact maps derived from single-cell 3D genome imaging demonstrate reliable identification of single-cell subcompartments, offering insights into cell-to-cell variability of nuclear subcompartments. Using scHi-C data from complex tissues, scGHOST identifies cell-type-specific or allele-specific subcompartments linked to gene transcription across various cell types and developmental stages, suggesting functional implications of single-cell subcompartments. scGHOST is an effective method for annotating single-cell 3D genome subcompartments in a broad range of biological contexts.

Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut.

Lysosome-mediated macroautophagy, including lipophagy, is activated under nutrient deprivation but is repressed after feeding. We show that, unexpectedly, feeding activates intestinal autophagy/lipophagy in a manner dependent on both the orphan nuclear receptor, small heterodimer partner (SHP/NR0B2), and the gut hormone, fibroblast growth factor-15/19 (FGF15/19). Furthermore, postprandial intestinal triglycerides (TGs) and apolipoprotein-B48 (ApoB48), the TG-rich chylomicron marker, were elevated in SHP-knockout and FGF15-knockout mice. Genomic analyses of the mouse intestine indicated that SHP partners with the key lysosomal activator, transcription factor-EB (TFEB) to upregulate the transcription of autophagy/lipolysis network genes after feeding. FGF19 treatment activated lipophagy, reducing TG and ApoB48 levels in HT29 intestinal cells, which was dependent on TFEB. Mechanistically, feeding-induced FGF15/19 signaling increased the nuclear localization of TFEB and SHP via PKC beta/zeta-mediated phosphorylation, leading to increased transcription of the TFEB/SHP target lipophagy genes, Ulk1 and Atgl. Collectively, these results demonstrate that paradoxically after feeding, FGF15/19-activated SHP and TFEB activate gut lipophagy, limiting postprandial TGs. As excess postprandial lipids cause dyslipidemia and obesity, the FGF15/19-SHP-TFEB axis that reduces intestinal TGs via lipophagic activation provides promising therapeutic targets for obesity-associated metabolic disease.

Polar localization of a rice silicon transporter requires isoleucine at both C- and N-termini as well as positively charged residues.

Silicon (Si) is important for stable and high yields in rice (Oryza sativa), a typical Si hyperaccumulator. The high Si accumulation is achieved by the cooperation of 2 Si transporters, LOW SILICON 1 (OsLsi1) and OsLsi2, which are polarly localized in cells of the root exodermis and endodermis. However, the mechanism underlying their polar localization is unknown. Here, we identified amino acid residues critical for the polar localization of OsLsi1. Deletion of both N- and C-terminal regions resulted in the loss of its polar localization. Furthermore, the deletion of the C-terminus inhibited its trafficking from the endoplasmic reticulum to the plasma membrane. Detailed site-directed mutagenesis analysis showed that Ile18 at the N-terminal region and Ile285 at the C-terminal region were essential for the polar localization of OsLsi1. Moreover, a cluster of positively charged residues at the C-terminal region is also required for polar localization. Phosphorylation and Lys modifications of OsLsi1 are unlikely to be involved in its polar localization. Finally, we showed that the polar localization of OsLsi1 is required for the efficient uptake of Si. Our study not only identified critical residues required for the polar localization of OsLsi1, but also provided experimental evidence for the importance of transporter polarity for efficient nutrient uptake.

Peroxisome proliferator-activated receptor-α (PPARα) regulates wound healing and mitochondrial metabolism in the cornea.

Diabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-α (PPARα) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARα knockout mice and corneal epithelium-specific PPARα conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARα agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARα transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARα agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARα, suggesting a PPARα-dependent mechanism. These findings suggest that downregulation of PPARα plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes.

Interleukin-17-mediated protective cytokine signaling against degeneration of the retinal pigment epithelium.

Injuries to the retinal pigment epithelium (RPE) and outer retina often result in the accumulation of retinal microglia within the subretinal space. These subretinal microglia play crucial roles in inflammation and resolution, but the mechanisms governing their functions are still largely unknown. Our previous research highlighted the protective functions of choroidal γδ T cells in response to RPE injury. In the current study, we employed single-cell RNA sequencing approach to characterize the profiles of immune cells in mouse choroid. We found that γδ T cells were the primary producer of interleukin-17 (IL-17) in the choroid. IL-17 signaled through its receptor on the RPE, subsequently triggering the production of interleukin-6. This cascade of cytokines initiated a metabolic reprogramming of subretinal microglia, enhancing their capacity for lipid metabolism. RPE-specific knockout of IL-17 receptor A led to the dysfunction of subretinal microglia and RPE pathology. Collectively, our findings suggest that responding to RPE injury, the choroidal γδ T cells can initiate a protective signaling cascade that ensures the proper functioning of subretinal microglia.

Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion.

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.

Genomic and Phenotypic Adaptations of Rattus tanezumi to Cold Limit Its Further Northward Expansion and Range Overlap with R. norvegicus.

Global climate change has led to shifts in the distribution ranges of many terrestrial species, promoting their migration from lower altitudes or latitudes to higher ones. Meanwhile, successful invaders have developed genetic adaptations enabling the colonization of new environments. Over the past 40 years, Rattus tanezumi (RT) has expanded into northern China (Northwest and North China) from its southern origins. We studied the cold adaptation of RT and its potential for northward expansion by comparing it with sympatric Rattus norvegicus (RN), which is well adapted to cold regions. Through population genomic analysis, we revealed that the invading RT rats have split into three distinct populations: the North, Northwest, and Tibetan populations. The first two populations exhibited high genetic diversity, while the latter population showed remarkably low genetic diversity. These rats have developed various genetic adaptations to cold, arid, hypoxic, and high-UV conditions. Cold acclimation tests revealed divergent thermoregulation between RT and RN. Specifically, RT exhibited higher brown adipose tissue activity and metabolic rates than did RN. Transcriptome analysis highlighted changes in genes regulating triglyceride catabolic processes in RT, including Apoa1 and Apoa4, which were upregulated, under selection and associated with local adaptation. In contrast, RN showed changes in carbohydrate metabolism genes. Despite the cold adaptation of RT, we observed genotypic and phenotypic constraints that may limit its ability to cope with severe low temperatures farther north. Consequently, it is less likely that RT rats will invade and overlap with RN rats in farther northern regions.

Tissue-specific deposition, speciation and transport of antimony in rice.

Rice (Oryza sativa) as a staple food is a potential intake source of antimony (Sb), a toxic metalloid. However, how rice accumulates this element is still poorly understood. Here, we investigated tissue-specific deposition, speciation, and transport of Sb in rice. We found that Sb(III) is the preferential form of Sb uptake in rice, but most Sb accumulates in the roots, resulting in a very low root-to-shoot translocation (less than 2%). Analysis of Sb deposition with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) showed that most Sb deposits at the root exodermis. Furthermore, we found that Sb is mainly present as Sb(III) in the root cell sap after uptake. Further characterization showed that Sb(III) uptake is mediated by Low silicon rice 1 (Lsi1), a Si permeable transporter. Lsi1 showed transport activity for Sb(III) rather than Sb(V) in yeast (Saccharomyces cerevisiae). Knockout of Lsi1 resulted in a significant decrease in Sb accumulation in both roots and shoots. Sb concentration in the root cell sap of two independent lsi1 mutants decreased to less than 3% of that in wild-type rice, indicating that Lsi1 is a major transporter for Sb(III) uptake. Knockout of Lsi1 also enhanced rice tolerance to Sb toxicity. However, knockout of Si efflux transporter genes, including Lsi2 and Lsi3, did not affect Sb accumulation. Taken together, our results showed that Sb(III) is taken up by Lsi1 localized at the root exodermis and is deposited at this cell layer due to lack of Sb efflux transporters in rice.

The interplay of environmental luminance and genetics in the retinal dystrophy induced by the dominant RPE65 mutation.

SignificanceIn humans, genetic mutations in the retinal pigment epithelium (RPE) 65 are associated with blinding diseases, for which there is no effective therapy alleviating progressive retinal degeneration in affected patients. Our findings uncovered that the increased free opsin caused by enhancing the ambient light intensity increased retinal activation, and when compounded with the RPE visual cycle dysfunction caused by the heterozygous D477G mutation and aggregation, led to the onset of retinal degeneration.

Modulation of cGAS-STING signaling by PPARα in a mouse model of ischemia-induced retinopathy.

In ischemic retinopathy, overactivated retinal myeloid cells are a crucial driving force of pathological angiogenesis and inflammation. The cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) signaling are key regulators of inflammation. This study aims to investigate the association of cGAS-STING signaling with ischemic retinopathy and the regulation of its activation. We found that protein levels of cGAS and STING were markedly up-regulated in retinal myeloid cells isolated from mice with oxygen-induced retinopathy (OIR). Knockout of Sting and pharmacological inhibition of STING both alleviated retinal neovascularization (NV) and reduced retinal vascular leakage in OIR. Further, Sting knockout and STING inhibitor also alleviated leukocyte adhesion to retinal vasculature and infiltration into the retina as well as microglial activation in OIR. These results suggest that cGAS-STING signaling played a pathogenic role in retinal myeloid cell activation and NV in ischemic retinopathy. To identify the regulation of cGAS-STING signaling in OIR, we evaluated the role of transcription factor peroxisome proliferator-activated receptor α (PPARα). The results demonstrated that PPARα was down-regulated in OIR retinas, primarily in myeloid cells. Furthermore, Pparα knockout significantly up-regulated cGAS and STING levels in retinal CD11b+ cells, while PPARα agonist inhibited cGAS-STING signaling and cytosolic mitochondrial DNA (mtDNA) release, a causative feature for cGAS activation. Knockout of Sting ameliorated retinal NV, hyperpermeability, and leukostasis in Pparα-/- mice with OIR. These observations suggest that PPARα regulates cGAS-STING signaling, likely through mtDNA release, and thus, is a potential therapeutic target for ischemic retinopathy.

DARPP32, a target of hyperactive mTORC1 in the retinal pigment epithelium.

The mechanistic target of rapamycin (mTOR) is assembled into signaling complexes of mTORC1 or mTORC2, and plays key roles in cell metabolism, stress response, and nutrient and growth factor sensing. Accumulating evidence from human and animal model studies has demonstrated a pathogenic role of hyperactive mTORC1 in age-related macular degeneration (AMD). The retinal pigment epithelium (RPE) is a primary injury site in AMD. In mouse models of RPE-specific deletion of Tuberous sclerosis 1 (Tsc1), which encodes an upstream suppressor of mTORC1, the hyperactivated mTORC1 metabolically reprogrammed the RPE and led to the degeneration of the outer retina and choroid (CH). In the current study, we use single-cell RNA sequencing (scRNA-seq) to identify an RPE mTORC1 downstream protein, dopamine- and cyclic AMP-regulated phosphoprotein of molecular weight 32,000 (DARPP-32). DARPP-32 was not found in healthy RPE but localized to drusen and basal linear deposits in human AMD eyes. In animal models, overexpressing DARPP-32 by adeno-associated virus (AAV) led to abnormal RPE structure and function. The data indicate that DARPP-32 is a previously unidentified signaling protein subjected to mTORC1 regulation and may contribute to RPE degeneration in AMD.

The neutralizing breadth of antibodies targeting diverse conserved epitopes between SARS-CoV and SARS-CoV-2.

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.

Epstein-Barr virus microRNA miR-BART2-5p accelerates nasopharyngeal carcinoma metastasis by suppressing RNase â ¢ endonuclease DICER1.

The development and progression of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. NPC is usually asymptomatic until it spreads to other sites, and more than 70% of cases are classified as locally advanced disease at diagnosis. EBV-positive nasopharyngeal cancer tissues express only limited viral latent proteins, but express high levels of the EBV-encoded BamHI-A rightward transcript (BART) miRNA molecules. Here, we report that EBV-miRNA-BART2-5p (BART2-5p) promotes NPC cell invasion and metastasis in vivo and in vitro but has no effect on NPC cell proliferation and apoptosis. In addition, BART2-5p altered the mRNA and miRNA expression profiles of NPC cells. The development of human tumors has been reported to be associated with altered miRNAs expression, and overall miRNAs expression is reduced in many types of tumors. We found that BART2-5p downregulated the expression of several miRNAs that could exert oncogenic functions. Mechanistically, BART2-5p directly targets the RNase III endonuclease DICER1, inhibiting its function of cleaving double-stranded stem-loop RNA into short double-stranded RNA, which in turn causes altered expression of a series of key epithelial-mesenchymal transition molecules, and reverting DICER1 expression can rescue this phenotype. Furthermore, analysis from clinical samples showed a negative correlation between BART2-5p and DICER1 expression. According to our study, high expression of BART2-5p in tissues and plasma of patients with NPC is associated with poor prognosis. Our results suggest that, BART2-5p can accelerate NPC metastasis through modulating miRNA profiles which are mediated by DICER1, implying a novel role of EBV miRNAs in the pathogenesis of NPC.

Multi-omics analysis revealed that the protein kinase MoKin1 affected the cellular response to endoplasmic reticulum stress in the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Previous studies have shown that protein kinase MoKin1 played an important role in the growth, conidiation, germination and pathogenicity in rice blast fungus, Magnaporthe oryzae. ΔMokin1 mutant showed significant phenotypic defects and significantly reduced pathogenicity. However, the internal mechanism of how MoKin1 affected the development of physiology and biochemistry remained unclear in M. oryzae. RESULT: This study adopted a multi-omics approach to comprehensively analyze MoKin1 function, and the results showed that MoKin1 affected the cellular response to endoplasmic reticulum stress (ER stress). Proteomic analysis revealed that the downregulated proteins in ΔMokin1 mutant were enriched mainly in the response to ER stress triggered by the unfolded protein. Loss of MoKin1 prevented the ER stress signal from reaching the nucleus. Therefore, the phosphorylation of various proteins regulating the transcription of ER stress-related genes and mRNA translation was significantly downregulated. The insensitivity to ER stress led to metabolic disorders, resulting in a significant shortage of carbohydrates and a low energy supply, which also resulted in severe phenotypic defects in ΔMokin1 mutant. Analysis of MoKin1-interacting proteins indicated that MoKin1 really took participate in the response to ER stress. CONCLUSION: Our results showed the important role of protein kinase MoKin1 in regulating cellular response to ER stress, providing a new research direction to reveal the mechanism of MoKin1 affecting pathogenic formation, and to provide theoretical support for the new biological target sites searching and bio-pesticides developing.